HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns). One of the earliest events in this process is recognition of the 3' splice site (3'ss) by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proofreading of 3'ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control and HNRNPA1 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3'ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3'ss

Reprogramming to pluripotency using designer TALE transcription factors targeting enhancers

The modular DNA recognition code of the transcription-activator-like effectors (TALEs) from plant pathogenic bacterial genus Xanthomonas provides a powerful genetic tool to create designer transcription factors (dTFs) targeting specific DNA sequences for manipulating gene expression. Previous studies have suggested critical roles of enhancers in gene regulation and reprogramming. Here, we report dTF activator targeting the distal enhancer of the Pou5f1 (Oct4) locus induces epigenetic changes, reactivates its expression, and substitutes exogenous OCT4 in reprogramming mouse embryonic fibroblast cells (MEFs) to induced pluripotent stem cells (iPSCs). Similarly, dTF activator targeting a Nanog enhancer activates Nanog expression and reprograms epiblast stem cells (EpiSCs) to iPSCs. Conversely, dTF repressors targeting the same genetic elements inhibit expression of these loci, and effectively block reprogramming. This study indicates that dTFs targeting specific enhancers can be used to study other biological processes such as transdifferentiation or directed differentiation of stem cells. © 2013 The Authors.Link_to_subscribed_fulltex

The Effects of Circumcision on the Penis Microbiome

Circumcision is associated with significant reductions in HIV, HSV-2 and HPV infections among men and significant reductions in bacterial vaginosis among their female partners.We assessed the penile (coronal sulci) microbiota in 12 HIV-negative Ugandan men before and after circumcision. Microbiota were characterized using sequence-tagged 16S rRNA gene pyrosequencing targeting the V3-V4 hypervariable regions. Taxonomic classification was performed using the RDP Naïve Bayesian Classifier. Among the 42 unique bacterial families identified, Pseudomonadaceae and Oxalobactericeae were the most abundant irrespective of circumcision status. Circumcision was associated with a significant change in the overall microbiota (PerMANOVA p = 0.007) and with a significant decrease in putative anaerobic bacterial families (Wilcoxon Signed-Rank test p = 0.014). Specifically, two families-Clostridiales Family XI (p = 0.006) and Prevotellaceae (p = 0.006)-were uniquely abundant before circumcision. Within these families we identified a number of anaerobic genera previously associated with bacterial vaginosis including: Anaerococcus spp., Finegoldia spp., Peptoniphilus spp., and Prevotella spp.The anoxic microenvironment of the subpreputial space may support pro-inflammatory anaerobes that can activate Langerhans cells to present HIV to CD4 cells in draining lymph nodes. Thus, the reduction in putative anaerobic bacteria after circumcision may play a role in protection from HIV and other sexually transmitted diseases

Global distribution of two fungal pathogens threatening endangered sea turtles

This work was supported by grants of Ministerio de Ciencia e Innovación, Spain (CGL2009-10032, CGL2012-32934). J.M.S.R was supported by PhD fellowship of the CSIC (JAEPre 0901804). The Natural Environment Research Council and the Biotechnology and Biological Sciences Research Council supported P.V.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Thanks Machalilla National Park in Ecuador, Pacuare Nature Reserve in Costa Rica, Foundations Natura 2000 in Cape Verde and Equilibrio Azul in Ecuador, Dr. Jesus Muñoz, Dr. Ian Bell, Dr. Juan Patiño for help and technical support during samplingPeer reviewedPublisher PD

Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry

We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal–specific contamination

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds-approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure-reducing some bacteria while selecting for others

Fibrosis Progression Rate in Biopsy-Proven Nonalcoholic Fatty Liver Disease Among People With Diabetes Versus People Without Diabetes: A Multicenter Study

Background & aimsThere are limited data regarding fibrosis progression in biopsy-proven nonalcoholic fatty liver disease (NAFLD) in people with type 2 diabetes mellitus (T2DM) compared with people without T2DM. We assessed the time to fibrosis progression in people with T2DM compared with people without T2DM in a large, multicenter, study of people with NAFLD who had paired liver biopsies.MethodsThis study included 447 adult participants (64% were female) with NAFLD who had paired liver biopsies more than 1 year apart. Liver histology was systematically assessed by a central pathology committee blinded to clinical data. The primary outcome was the cumulative incidence of a ≥1-stage increase in fibrosis in participants with T2DM compared with participants without T2DM.ResultsThe mean (SD) age and body mass index (calculated as weight in kilograms divided by the square of the height in meters) were 50.9 (11.5) years and 34.7 (6.3), respectively. The median time between biopsies was 3.3 years (interquartile range, 1.8-6.1 years). Participants with T2DM had a significantly higher cumulative incidence of fibrosis progression at 4 years (24% vs 20%), 8 years (60% vs 50%), and 12 years (93% vs 76%) (P = .005). Using a multivariable Cox proportional hazards model adjusted for multiple confounders, T2DM remained an independent predictor of fibrosis progression (adjusted hazard ratio, 1.69; 95% CI, 1.17-2.43; P = .005). The cumulative incidence of fibrosis regression by ≥1 stage was similar in participants with T2DM compared with participants without T2DM (P = .24).ConclusionsIn this large, multicenter cohort study of well-characterized participants with NAFLD and paired liver biopsies, we found that fibrosis progressed faster in participants with T2DM compared with participants without T2DM. These data have important implications for clinical practice and trial design

Transcriptomic convergence despite genomic divergence drive field cancerization in synchronous squamous tumors

IntroductionField cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence.MethodsWe performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors.ResultsOur data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors.DiscussionOur analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity

Delineating nuclear reprogramming

Nuclear reprogramming is described as a molecular switch, triggered by the conversion of one cell type to another. Several key experiments in the past century have provided insight into the field of nuclear reprogramming. Previously deemed impossible, this research area is now brimming with new findings and developments. In this review, we aim to give a historical perspective on how the notion of nuclear reprogramming was established, describing main experiments that were performed, including (1) somatic cell nuclear transfer, (2) exposure to cell extracts and cell fusion, and (3) transcription factor induced lineage switch. Ultimately, we focus on (4) transcription factor induced pluripotency, as initiated by a landmark discovery in 2006, where the process of converting somatic cells to a pluripotent state was narrowed down to four transcription factors. The conception that somatic cells possess the capacity to revert to an immature status brings about huge clinical implications including personalized therapy, drug screening and disease modeling. Although this technology has potential to revolutioni ze the medical field, it is still impeded by technical and biological obstacles. This review describes the effervescent changes in this field, addresses bottlenecks hindering its advancement and in conclusion, applies the latest findings to overcome these issues. © 2012 Higher Education Press and Springer-Verlag Berlin Heidelberg.Link_to_subscribed_fulltex