Glycosaminoglycan Interactions in Murine Gammaherpesvirus-68 Infection

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces

2019 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations : summary from the basic life support; advanced life support; pediatric life support; neonatal life support; education, implementation, and teams; and first aid task forces

The International Liaison Committee on Resuscitation has initiated a continuous review of new, peer-reviewed, published cardiopulmonary resuscitation science. This is the third annual summary of the International Liaison Committee on Resuscitation International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations. It addresses the most recent published resuscitation evidence reviewed by International Liaison Committee on Resuscitation Task Force science experts. This summary addresses the role of cardiac arrest centers and dispatcher-assisted cardiopulmonary resuscitation, the role of extracorporeal cardiopulmonary resuscitation in adults and children, vasopressors in adults, advanced airway interventions in adults and children, targeted temperature management in children after cardiac arrest, initial oxygen concentration during resuscitation of newborns, and interventions for presyncope by first aid providers. Members from 6 International Liaison Committee on Resuscitation task forces have assessed, discussed, and debated the certainty of the evidence on the basis of the Grading of Recommendations, Assessment, Development, and Evaluation criteria, and their statements include consensus treatment recommendations. Insights into the deliberations of the task forces are provided in the Justification and Evidence to Decision Framework Highlights sections. The task forces also listed priority knowledge gaps for further research

Androgen receptor expression in metastatic adenocarcinoma in females favors a breast primary

BACKGROUND: The differential diagnosis of metastatic mammary adenocarcinoma and adenocarcinomas from other primary sites can be challenging, particularly in tumors that are poorly differentiated and negative for Estrogen/Progesterone receptors (ER/PR). With progression of disease, Androgen receptors (AR) are preserved with higher frequency than ER/PR in metastatic mammary carcinoma. This study was undertaken to evaluate the diagnostic significance of AR expression in adenocarcinoma of breast and other morphologically similar adenocarcinomas. DESIGN: Formalin-fixed paraffin-embedded tissue sections of 113 primary adenocarcinoma of various sites [breast (34, all females), lung (23, M- 6, F-17), colon (9, M-2, F-7), stomach (6, M-4, F-2), liver and bile duct (11, M-5, F-6), pancreas (7, M-2, F-5), ovary (10), endometrium (7), and cervix (6)] were immunostained with monoclonal antibody for AR. Except for well differentiated lobular carcinoma of breast (5) and bronchoalveolar carcinoma of lung (10), majority of the tumors were moderately to poorly differentiated. Tumors immunoreactive for ≥ 10% of nuclei were considered AR positive. However, AR immunoreactivity in the cytoplasm only was also recorded. RESULTS: 56% (19/34) mammary carcinoma and 20% (2/10) adenocarcinoma of ovary were positive for AR. Remaining 69 adenocarcinomas did not show nuclear immunoreactivity for AR in ≥ 10% nuclei; however, 52% (36/69) showed variable cytoplasmic immunoreactivity. CONCLUSION: Significant proportion of mammary carcinomas and some ovarian carcinomas express AR in the nuclei of more than 10% tumor cells. If metastatic tumor with unknown primary in a female is AR positive, breast and ovary are the most likely primary sites. Cytoplasmic immunoreactivity alone without nuclear immunoreactivity for AR was non-specific for this differential diagnosis

The Murid Herpesvirus-4 gH/gL Binds to Glycosaminoglycans

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is therefore likely to involve a GAG-binding virion glycoprotein. We have previously identified two such proteins, gp70 and gp150. Gp70 binds strongly to GAGs. However, deleting it makes little difference to MuHV-4 cell binding or GAG-dependence. Deleting gp150, by contrast, frees MuHV-4 from GAG dependence. This implies that GAGs normally displace gp150 to allow GAG-independent cell binding. But the gp150 GAG interaction is weak, and so would seem unlikely to make an effective first contact. Since neither gp70 nor gp150 matches the expected profile of a first contact glycoprotein, our understanding of MuHV-4 GAG interactions must be incomplete. Here we relate the seemingly disconnected gp70 and gp150 GAG interactions by showing that the MuHV-4 gH/gL also binds to GAGs. gH/gL-blocking and gp70-blocking antibodies individually had little effect on cell binding, but together were strongly inhibitory. Thus, there was redundancy in GAG binding between gp70 and gH/gL. Gp150-deficient MuHV-4 largely resisted blocks to gp70 and gH/gL binding, consistent with its GAG independence. The failure of wild-type MuHV-4 to do the same argues that gp150 is normally engaged only down-stream of gp70 or gH/gL. MuHV-4 GAG dependence is consequently two-fold: gp70 or gH/gL binding provides virions with a vital first foothold, and gp150 is then engaged to reveal GAG-independent binding

Explaining co-movements between equity and CDS bid-ask spreads

In this paper I show that the co-movements between bid-ask spreads of equities and credit default swaps vary over time and increase over crisis periods. The co-movements are strongly related to systematic risk factors and to the theoretical debt-to-equity hedge ratio. I document that hedging and asymmetric information, besides higher funding costs and market volatility risk, are driving factors of the commonality and are significantly priced in CDS bid-ask spreads

Inhibition of HCV 3a genotype entry through Host CD81 and HCV E2 antibodies

<p>Abstract</p> <p>Background</p> <p>HCV causes acute and chronic hepatitis which can eventually lead to permanent liver damage hepatocellular carcinoma and death. HCV glycoproteins play an important role in HCV entry by binding with CD81 receptors. Hence inhibition of virus at entry step is an important target to identify antiviral drugs against HCV.</p> <p>Methods and result</p> <p>The present study elaborated the role of CD81 and HCV glycoprotein E2 in HCV entry using retroviral pseudo-particles of 3a local genotype. Our results demonstrated that HCV specific antibody E2 and host antibody CD81 showed dose- dependent inhibition of HCV entry. HCV E2 antibody showed 50% reduction at a concentration of 1.5 ± 1 μg while CD81 exhibited 50% reduction at a concentration of 0.8 ± 1 μg. In addition, data obtained with HCVpp were also confirmed with the infection of whole virus of HCV genotype 3a in liver cells.</p> <p>Conclusion</p> <p>Our data suggest that HCV specific E2 and host CD81 antibodies reduce HCVpp entry and full length viral particle and combination of host and HCV specific antibodies showed synergistic effect in reducing the viral titer.</p

Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.</p> <p>Materials and methods</p> <p>The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for <it>in vitro </it>analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.</p> <p>Results</p> <p>The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.</p> <p>Conclusions</p> <p>Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.</p

A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition

siRNAs: Potential therapeutic agents against Hepatitis C Virus

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HC