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Host-microbe symbioses rely on the successful transmission or acquisition of symbionts in each new generation. Amphibians host a diverse cutaneous microbiota, and many of these symbionts appear to be mutualistic and may limit infection by the chytrid fungus, Batrachochytrium dendrobatidis , which has caused global amphibian population declines and extinctions in recent decades. Using bar-coded 454 pyrosequencing of the 16S rRNA gene, we addressed the question of symbiont transmission by examining variation in amphibian skin microbiota across species and sites and in direct relation to environmental microbes. Although acquisition of environmental microbes occurs in some host-symbiont systems, this has not been extensively examined in free-living vertebrate-microbe symbioses. Juvenile bullfrogs ( Rana catesbeiana ), adult red-spotted newts ( Notophthalmus viridescens ), pond water and pond substrate were sampled at a single pond to examine host-specificity and potential environmental transmission of microbiota. To assess population level variation in skin microbiota, adult newts from two additional sites were also sampled. Cohabiting bullfrogs and newts had distinct microbial communities, as did newts across the three sites. The microbial communities of amphibians and the environment were distinct; there was very little overlap in the amphibians’ core microbes and the most abundant environmental microbes, and the relative abundances of OTUs that were shared by amphibians and the environment were inversely related. These results suggest that, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor–normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection. Recommendations are given on optimal read coverage and selection of calling algorithm to maximize the reproducibility of cancer mutation detection in whole-genome or whole-exome sequencing.

Vibrio parahaemolyticus is an emerging world-wide human pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. Expression of virulence factors in this organism is modulated by the phenomenon known as quorum sensing, which permits differential gene regulation at low versus high cell density. The master regulator of quorum sensing in V. parahaemolyticus is OpaR. OpaR not only controls virulence factor gene expression, but also the colony and cellular morphology associated with growth on a surface and biofilm formation. Whole transcriptome Next Generation sequencing (RNA-Seq) was utilized to determine the OpaR regulon by comparing strains BB22OP (opaR+, LM5312) and BB22TR (∆opaR1, LM5674). This work, using the published V. parahaemolyticus BB22OP genome sequence, confirms and expands upon a previous microarray analysis for these two strains that used an Affymetrix GeneChip designed from the closely related V. parahaemolyticus RIMD2210633 genome sequence. Overall there was excellent correlation between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control were identified by both methods and further confirmed by quantitative reverse transcription PCR (qRT-PCR) analysis. Nine of these transcription factors were demonstrated to be direct OpaR targets via in vitro electrophoretic mobility shift assays with purified hexahistidine-tagged OpaR. Identification of the direct and indirect targets of OpaR, including small RNAs, will enable the construction of a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic states.

With the increase in the availability of metagenomic data generated by next generation sequencing, there is an urgent need for fast and accurate tools for identifying viruses in host-associated and environmental samples. In this paper, we developed a stand-alone pipeline called FastViromeExplorer for the detection and abundance quantification of viruses and phages in large metagenomic datasets by performing rapid searches of virus and phage sequence databases. Both simulated and real data from human microbiome and ocean environmental samples are used to validate FastViromeExplorer as a reliable tool to quickly and accurately identify viruses and their abundances in large datasets.

The Gram-negative proteobacterium Pantoea stewartii subsp. stewartii causes wilt disease in corn plants. Wilting is primarily due to bacterial exopolysaccharide (EPS) production that blocks water transport in the xylem during the late stages of infection. EsaR, the master quorum-sensing (QS) regulator in P. stewartii, modulates EPS levels. At low cell densities EsaR represses or activates expression of a number of genes in the absence of its acyl homoserine lactone (AHL) ligand. At high cell densities, binding of AHL inactivates EsaR leading to derepression or deactivation of its direct targets. Two of these direct targets are the key transcription regulators RcsA and LrhA, which in turn control EPS production and surface motility/adhesion, respectively. In this study, RNA-Seq was used to further examine the physiological impact of deleting the genes encoding these two second-tier regulators. Quantitative reverse transcription PCR (qRT-PCR) was used to validate the regulation observed in the RNA-Seq data. A GFP transcriptional fusion reporter confirmed the existence of a regulatory feedback loop in the system between LrhA and RcsA. Plant virulence assays carried out with rcsA and lrhA deletion and complementation strains demonstrated that both transcription factors play roles during establishment of wilt disease in corn. These efforts further define the hierarchy of the QS-regulated network controlling plant virulence in P. stewartii.

Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment. Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII. Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.

Abstract The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were present in all samples. The largest number of reads belonged to Firmicutes (43.7% of total bacterial sequences), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%). The less abundant Actinobacteria, Cyanobacteria, and TM7 phyla presented here have not been previously described in the gut contents or feces of horses. Unclassified sequences represented 38.1% of total bacterial sequences; therefore, the equine fecal microbiome diversity is likely greater than that described. This is the first study to characterize the fecal bacterial community in horses by the use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the fecal microbiota of forage-fed horses.

Background Clostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq. Results Hundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding β-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. β-glucuronidase expression was then measured in cells grown in liquid or on plates. β-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces. Conclusions This study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.

Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus somni sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in H . somni were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by in vitro transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of H . somni sRNAs that show they may have important regulatory roles in virulence and biofilm formation.

Parkinson's disease (PD) progresses relentlessly and affects five million people worldwide. Laboratory tests for PD are critically needed for developing treatments designed to slow or prevent progression of the disease. We performed a transcriptome-wide scan in 105 individuals to interrogate the molecular processes perturbed in cellular blood of patients with early-stage PD. The molecular multigene marker here identified is associated with risk of PD in 66 samples of the training set comprising healthy and disease controls [third tertile cross-validated odds ratio of 5.7 (P for trend 0.005)]. It is further validated in 39 independent test samples [third tertile odds ratio of 5.1 (P for trend 0.04)]. Insights into disease-linked processes detectable in peripheral blood are offered by 22 unique genes differentially expressed in patients with PD versus healthy individuals. These include the cochaperone ST13, which stabilizes heat-shock protein 70, a modifier of α-synuclein misfolding and toxicity. ST13 messenger RNA copies are lower in patients with PD (mean ± SE 0.59 ± 0.05) than in controls (0.96 ± 0.09) (P = 0.002) in two independent populations. Thus, gene expression signals measured in blood can facilitate the development of biomarkers for PD.