Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here we demonstrate that cells respond to HSV-1 infection by promoting filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum-dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.

Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.

Herpes simplex virus 1 (HSV‐1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV‐1 invasion of human RPE cells. High‐resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)‐tagged virions with filopodia‐like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH‐dependent HSV‐1 entry and replication. Use of RT‐PCR indicated expression of nectin‐1, herpes virus entry mediator (HVEM) and 3‐O‐sulfotransferase‐3 (as a surrogate marker for 3‐O‐sulfated heparan sulfate). HVEM and nectin‐1 expression was subsequently verified by flow cytometry. Nectin‐1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against nectin‐1, but not HVEM, were able to block HSV‐1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against nectin‐1, which also blocked RPE cell fusion with HSV‐1 glycoprotein‐expressing Chinese hamster ovary (CHO‐K1) cells. Anti‐nectin‐1 antibodies and F‐actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV‐1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.

Multiple studies have shown that oxidative modifi cations of LDL (oxLDL) are a major factor in the development of atherosclerosis ( 1-6 ). The level of oxLDL increases dramatically with hypercholesterolemia in animal models of atherosclerosis ( 7,8 ), and in humans ( 9, 10 ), it is found in atherosclerotic lesions ( 11 ). It is also well established that dyslipidemia-induced dysfunction of vascular endothelial cells is a critical step in the early stage of atherosclerosis (e.g., Refs. 12-14 ) and a strong predictor of cardiovascular disease (CVD) development ( 15-17 ). The mechanisms, however, of dyslipidemia-induced endothelial dysfunction are still poorly understood. Our recent studies have shown that exposure to oxLDL in vitro or to plasma dyslipidemia in vivo signifi cantly increases the stiffness of aortic endothelial cells, which in turn is associated with an increase in endothelial contractility, enhanced angiogenic potential, and sensitivity to shear stress (18)(19)(20). Furthermore, unexpectedly, our studies showed that dyslipidemia-induced endothelial stiffening is caused not by cholesterol loading but by disruption of lipid packing of cholesterol-rich membrane domains in endothelial cells ( 18,19,21 ). Consistent with these observations, oxLDLinduced endothelial stiffness could be fully reversed by enriching the cells with cholesterol, even though oxLDL had no effect on the cholesterol content of endothelial membranes ( 19 ). These studies led us to the hypothesis that oxLDL induces endothelial dysfunction by inserting oxysterols into the plasma membrane, resulting in the disruption of cholesterol-rich membrane domains and endothelial stiffening. The goal of this study, therefore, was to determine the impact of oxysterols on endothelial stiffness.Abstract Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specifi c oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7 ␣ -hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. ox-LDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We fi nd signif...

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This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.

Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

Background and Aims: Clinical evidence for the benefits of branched-chain amino acids (BCAAs) is lacking in advanced liver disease. We evaluated the potential benefits of long-term oral BCAA supplementation in patients with advanced liver disease. Methods: Liver cirrhosis patients with Child–Pugh (CP) scores from 8 to 10 were prospectively recruited from 13 medical centers. Patients supplemented with 12.45 g of daily BCAA granules over 6 months, and patients consuming a regular diet were assigned to the BCAA and control groups, respectively. The effects of BCAA supplementation were evaluated using the model for end-stage liver disease (MELD) score, CP score, serum albumin, serum bilirubin, incidence of cirrhosis-related events, and event-free survival for 24 months. Results: A total of 124 patients was analyzed: 63 in the BCAA group and 61 in the control group. The MELD score (p = 0.009) and CP score (p = 0.011) significantly improved in the BCAA group compared to the control group over time. However, the levels of serum albumin and bilirubin in the BCAA group did not improve during the study period. The cumulative event-free survival was significantly improved in the BCAA group compared to the control group (HR = 0.389, 95% CI = 0.221–0.684, p < 0.001). Conclusion: Long-term supplementation with oral BCAAs can potentially improve liver function and reduce major complications of cirrhosis in patients with advanced liver disease.