Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals

The Impact of Cross Cultural Communication on Collective Efficacy in NCAA Basketball Teams

This research contributes to the knowledge and theory on cross cultural communication by investigating the impact of cross cultural communication competence on the collective efficacy of multicultural National Collegiate Athletic Association basketball teams. Data was collected from 140 U.S. National Collegiate Athletic Association basketball coaches via the Cross Cultural Communication Competence Questionnaire and the Collective Efficacy Questionnaire for Sports. Principle component analysis was conducted on the data, revealing that the cross cultural communication competence and collective efficacy of basketball teams are multidimensional. The hypothesized relationship between cross cultural communication competence and collective efficacy was confirmed and statistically measured through regression analysis. It was found that four of the cross cultural communication competence dimensions produced by the principle component analysis exhibited a significant positive relationship with one of the two dimensions within collective efficacy. Given the well-supported relationship between collective efficacy and team performance in business, this study produces important implications for scholars and practitioners working with multicultural sporting teams

Switching Virally Suppressed, Treatment-Experienced Patients to a Raltegravir-Containing Regimen Does Not Alter Levels of HIV-1 DNA

Background: Current HIV-1 antiretroviral therapy (ART) greatly reduces virus replication but does not significantly affect the viral reservoir. Raltegravir, a recently introduced integrase inhibitor, could, at least theoretically, reduce residual viremia in patients on ART and affect the viral reservoir size. The aim of this study was to assess whether switching therapy in treatment-experienced patients that were virally suppressed to a raltegravir-containing regimen reduces the size of the viral reservoir, and if such treatment leads to a change in levels of HIV 2-LTR circles in this patient group. Methods: 14 ART experienced individuals with a suppressed viral load (,50 HIV-1 RNA copies/mL plasma) at baseline (for at least 2 months) were switched to a raltegravir-containing regimen. Blood samples were taken at baseline and at $2 timepoints up to 4866 weeks. Levels of total HIV-1 DNA and 2-LTR circles in peripheral blood mononuclear cells (PBMCs) were measured using real-time PCR assays. Results: There was no significant change in HIV-1 total DNA levels over the study duration (p = 0.808), median slope 0.24 (conservative nonparametric 95 % CI: 211.78, 26.23). Low levels of 2-LTR circles were detected in 2 patients. One had 16 copies/10 6 PBMCs at baseline and the other had 34 copies/10 6 PBMCs at week 51. Conclusions: The switch to a raltegravir containing regimen was not associated with a significant change in HIV-1 total DNA levels in this cohort. There were no observed changes in the levels of HIV-1 2-LTR circles associated with raltegravi

Persistence of viral reservoirs in multiple tissues after antiretroviral therapy suppression in a macaque RT-SHIV model

Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood. © 2013 Kline et al

Effects of Heavy Metals and Arbuscular Mycorrhiza on the Leaf Proteome of a Selected Poplar Clone: A Time Course Analysis

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein

Intensification of Antiretroviral Therapy with a CCR5 Antagonist in Patients with Chronic HIV-1 Infection: Effect on T Cells Latently Infected

Objective: The primary objective was to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy. Methods: We performed an open-label pilot phase II clinical trial involving chronically HIV-1-infected patients receiving stable antiretroviral therapy whose regimen was intensified with 48 weeks of maraviroc therapy. We analyzed the latent reservoir, the residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation. Results: Overall a non significant reduction in the size of the latent reservoir was found (p = 0.068). A mean reduction of 1.82 IUPM was observed in 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification. No effect on plasma residual viremia was observed. Unexpectedly, all the patients had detectable 2LTR DNA circles at week 24, while none of them showed those circles at the end of the study. No changes were detected in CD4+ or CD8+ counts, although a significant decrease was found in the proportion of HLA-DR+/CD38+ CD4+ and CD8+ T-cells. LPS and sCD14 levels increased. Conclusions: Intensification with MVC was associated with a trend to a decrease in the size of the latent HIV-1 reservoir in memory T cells. No impact on residual viremia was detected. Additional studies with larger samples are needed to confirm the results

The CXC-Chemokine CXCL4 Interacts with Integrins Implicated in Angiogenesis

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet α-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with αvβ3 on the surface of αvβ3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through αvβ3 integrin, and also through other integrins, such as αvβ5 and α5β1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect

Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation