The DEAD-box RNA Helicase DDX6 is Required for Efficient Encapsidation of a Retroviral Genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging

Search for time-dependent B0s - B0s-bar oscillations using a vertex charge dipole technique

We report a search for B0s - B0s-bar oscillations using a sample of 400,000 hadronic Z0 decays collected by the SLD experiment. The analysis takes advantage of the electron beam polarization as well as information from the hemisphere opposite that of the reconstructed B decay to tag the B production flavor. The excellent resolution provided by the pixel CCD vertex detector is exploited to cleanly reconstruct both B and cascade D decay vertices, and tag the B decay flavor from the charge difference between them. We exclude the following values of the B0s - B0s-bar oscillation frequency: Delta m_s < 4.9 ps-1 and 7.9 < Delta m_s < 10.3 ps-1 at the 95% confidence level.Comment: 18 pages, 3 figures, replaced by version accepted for publication in Phys.Rev.D; results differ slightly from first versio

Genotype of metabolic enzymes and the benefit of tamoxifen in postmenopausal breast cancer patients

BACKGROUND: Tamoxifen is widely used as endocrine therapy for oestrogen-receptor-positive breast cancer. However, many of these patients experience recurrence despite tamoxifen therapy by incompletely understood mechanisms. In the present report we propose that tamoxifen resistance may be due to differences in activity of metabolic enzymes as a result of genetic polymorphism. Cytochrome P450 2D6 (CYP2D6) and sulfotransferase 1A1 (SULT1A1) are polymorphic and are involved in the metabolism of tamoxifen. The CYP2D6*4 and SULT1A1*2 genotypes result in decreased enzyme activity. We therefore investigated the genotypes of CYP2D6 and SULT1A1 in 226 breast cancer patients participating in a trial of adjuvant tamoxifen treatment in order to validate the benefit from the therapy. METHODS: The patients were genotyped using PCR followed by cleavage with restriction enzymes. RESULTS: Carriers of the CYP2D6*4 allele demonstrated a decreased risk of recurrence when treated with tamoxifen (relative risk = 0.28, 95% confidence interval = 0.11–0.74, P = 0.0089). A similar pattern was seen among the SULT1A1*1 homozygotes (relative risk = 0.48, 95% confidence interval = 0.21–1.12, P = 0.074). The combination of CYP2D6*4 and/or SULT1A1*1/*1 genotypes comprised 60% of the patients and showed a 62% decreased risk of distant recurrence with tamoxifen (relative risk = 0.38, 95% confidence interval = 0.19–0.74, P = 0.0041). CONCLUSION: The present study suggests that genotype of metabolic enzymes might be useful as a guide for adjuvant endocrine treatment of postmenopausal breast cancer patients. However, results are in contradiction to prior hypotheses and the present sample size is relatively small. Findings therefore need to be confirmed in a larger cohort

NK-, NKT-and CD8-derived IFNγ drives myeloid cell activation and erythrophagocytosis, resulting in Trypanosomosis-associated acute anemia

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia

Regulation of the let-7a-3 Promoter by NF-κB

Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp −833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp −947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation

Did Corporate Governance Compliance Have an Impact on Auditor Selection and Quality? Evidence From FTSE 350

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.This paper examines the possible effects of corporate governance (GC) on audit quality (AQ) among the FTSE 350 companies. Using a sample of 180 companies from 2012 to 2017 (i.e., 1080 firm-year observations) a binary logistic model has been employed to investigate the CG-AQ nexus. This analysis was supported by conducting a probit logistic model as a sensitivity analysis. Our findings are associative of a heterogeneous impact of CG on AQ post the implementation of the 2012 CG reforms in the UK. For example, although institutional ownership and management ownership are positively associated with auditor selection and AQ, board independence, non-executive directors and audit committee are not attributed to AQ in the UK. This implies that corporate compliance with good CG practices has a limited impact on the decision to select a Big4 auditor in the UK. Despite the limitations of our study, we hope it can motivate further investigations in this area

Learning a Prior on Regulatory Potential from eQTL Data

Genome-wide RNA expression data provide a detailed view of an organism's biological state; hence, a dataset measuring expression variation between genetically diverse individuals (eQTL data) may provide important insights into the genetics of complex traits. However, with data from a relatively small number of individuals, it is difficult to distinguish true causal polymorphisms from the large number of possibilities. The problem is particularly challenging in populations with significant linkage disequilibrium, where traits are often linked to large chromosomal regions containing many genes. Here, we present a novel method, Lirnet, that automatically learns a regulatory potential for each sequence polymorphism, estimating how likely it is to have a significant effect on gene expression. This regulatory potential is defined in terms of “regulatory features”—including the function of the gene and the conservation, type, and position of genetic polymorphisms—that are available for any organism. The extent to which the different features influence the regulatory potential is learned automatically, making Lirnet readily applicable to different datasets, organisms, and feature sets. We apply Lirnet both to the human HapMap eQTL dataset and to a yeast eQTL dataset and provide statistical and biological results demonstrating that Lirnet produces significantly better regulatory programs than other recent approaches. We demonstrate in the yeast data that Lirnet can correctly suggest a specific causal sequence variation within a large, linked chromosomal region. In one example, Lirnet uncovered a novel, experimentally validated connection between Puf3—a sequence-specific RNA binding protein—and P-bodies—cytoplasmic structures that regulate translation and RNA stability—as well as the particular causative polymorphism, a SNP in Mkt1, that induces the variation in the pathway

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress

RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (\u3c7CRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein\u2013RNA interactions in vivo on a minute time-scale. Here, using \u3c7CRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein\u2013RNA interactions within 1\u2009min following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. \u3c7CRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein\u2013RNA interactions

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions