Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes

Prefrontal cortex gyrification index in twins: an MRI study

Cortical development and folding seems to be under environmental as well as genetic control. The aim of our study was to estimate the genetic influence on gyrification and cortical volumes, comparing prefrontal gyrification index (GI) in monozygotic (MZ) and dizygotic (DZ) twin pairs, and unrelated pairs. Twenty-four subjects (6 pairs of MZ and 6 pairs of DZ twins) were included in this study. Prefrontal cortical folding (gyrification) was measured by an automated and manual version of the gyrification index (A-GI, M-GI) according to previously published protocols. MR-imaging was performed and 3 representative slices were selected from coronar MR-imaging scans. The volumes of the total brain, temporal lobes, prefrontal lobes, and cerebellum were analyzed, too. To evaluate similarity in GI, absolute differences in GI, and brain volumes as well as intraclass correlations of twin pairs were compared with regard to twin status. Finally, a control group of unrelated pairs was assembled from the first two study groups and analyzed. Compared to unrelated pairs, twin pairs exhibited more similarity concerning different brain volumes and a trend to more similarity concerning A-GI. MZ twins did not present more similarity concerning GI (automatically and manually measured) and volume measurements compared to DZ twins. Different factors, like intrauterine factors, postnatal development conditions, and especially environmental factors might account for the differences between related and unrelated pairs. The nonexistence of a pronounced similarity in MZ twins compared to DZ twins concerning prefrontal GI raises questions about the extent of genetic influence on GI

An orally available, small-molecule interferon inhibits viral replication

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute

SiteSeek: Post-translational modification analysis using adaptive locality-effective kernel methods and new profiles

<p>Abstract</p> <p>Background</p> <p>Post-translational modifications have a substantial influence on the structure and functions of protein. Post-translational phosphorylation is one of the most common modification that occur in intracellular proteins. Accurate prediction of protein phosphorylation sites is of great importance for the understanding of diverse cellular signalling processes in both the human body and in animals. In this study, we propose a new machine learning based protein phosphorylation site predictor, SiteSeek. SiteSeek is trained using a novel compact evolutionary and hydrophobicity profile to detect possible protein phosphorylation sites for a target sequence. The newly proposed method proves to be more accurate and exhibits a much stable predictive performance than currently existing phosphorylation site predictors.</p> <p>Results</p> <p>The performance of the proposed model was compared to nine existing different machine learning models and four widely known phosphorylation site predictors with the newly proposed PS-Benchmark_1 dataset to contrast their accuracy, sensitivity, specificity and correlation coefficient. SiteSeek showed better predictive performance with 86.6% accuracy, 83.8% sensitivity, 92.5% specificity and 0.77 correlation-coefficient on the four main kinase families (CDK, CK2, PKA, and PKC).</p> <p>Conclusion</p> <p>Our newly proposed methods used in SiteSeek were shown to be useful for the identification of protein phosphorylation sites as it performed much better than widely known predictors on the newly built PS-Benchmark_1 dataset.</p

ApoE−/− PGC-1α−/− Mice Display Reduced IL-18 Levels and Do Not Develop Enhanced Atherosclerosis

BACKGROUND: Atherosclerosis is a chronic inflammatory disease that evolves from the interaction of activated endothelial cells, macrophages, lymphocytes and modified lipoproteins (LDLs). In the last years many molecules with crucial metabolic functions have been shown to prevent important steps in the progression of atherogenesis, including peroxisome proliferator activated receptors (PPARs) and the class III histone deacetylase (HDAC) SIRT1. The PPARγ coactivator 1 alpha (Ppargc1a or PGC-1α) was identified as an important transcriptional cofactor of PPARγ and is activated by SIRT1. The aim of this study was to analyze total PGC-1α deficiency in an atherosclerotic mouse model. METHODOLOGY/PRINCIPAL FINDINGS: To investigate if total PGC-1α deficiency affects atherosclerosis, we compared ApoE(-/-) PGC-1α(-/-) and ApoE(-/-) PGC-1α(+/+) mice kept on a high cholesterol diet. Despite having more macrophages and a higher ICAM-1 expression in plaques, ApoE(-/-) PGC-1α(-/-) did not display more or larger atherosclerotic plaques than their ApoE(-/-) PGC-1α(+/+) littermates. In line with the previously published phenotype of PGC-1α(-/-) mice, ApoE(-/-) PGC-1α(-/-) mice had marked reduced body, liver and epididymal white adipose tissue (WAT) weight. VLDL/LDL-cholesterol and triglyceride contents were also reduced. Aortic expression of PPARα and PPARγ, two crucial regulators for adipocyte differentiation and glucose and lipid metabolism, as well as the expression of some PPAR target genes was significantly reduced in ApoE(-/-) PGC-1α(-/-) mice. Importantly, the epididymal WAT and aortic expression of IL-18 and IL-18 plasma levels, a pro-atherosclerotic cytokine, was markedly reduced in ApoE(-/-) PGC-1α(-/-) mice. CONCLUSIONS/SIGNIFICANCE: ApoE(-/-) PGC-1α(-/-) mice, similar as PGC-1α(-/-) mice exhibit markedly reduced total body and visceral fat weight. Since inflammation of visceral fat is a crucial trigger of atherogenesis, decreased visceral fat in PGC-1α-deficient mice may explain why these mice do not develop enhanced atherosclerosis

Stable Cytotoxic T Cell Escape Mutation in Hepatitis C Virus Is Linked to Maintenance of Viral Fitness

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8+ T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS31629-1637, displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication

Establishment of epigenetic patterns in development

The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic “memory” of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation

Human cell types important for Hepatitis C Virus replication in vivo and in vitro. Old assertions and current evidence

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro

Climate simulations for 1880-2003 with GISS modelE

We carry out climate simulations for 1880-2003 with GISS modelE driven by ten measured or estimated climate forcings. An ensemble of climate model runs is carried out for each forcing acting individually and for all forcing mechanisms acting together. We compare side-by-side simulated climate change for each forcing, all forcings, observations, unforced variability among model ensemble members, and, if available, observed variability. Discrepancies between observations and simulations with all forcings are due to model deficiencies, inaccurate or incomplete forcings, and imperfect observations. Although there are notable discrepancies between model and observations, the fidelity is sufficient to encourage use of the model for simulations of future climate change. By using a fixed well-documented model and accurately defining the 1880-2003 forcings, we aim to provide a benchmark against which the effect of improvements in the model, climate forcings, and observations can be tested. Principal model deficiencies include unrealistically weak tropical El Nino-like variability and a poor distribution of sea ice, with too much sea ice in the Northern Hemisphere and too little in the Southern Hemisphere. The greatest uncertainties in the forcings are the temporal and spatial variations of anthropogenic aerosols and their indirect effects on clouds.Comment: 44 pages; 19 figures; Final text accepted by Climate Dynamic