FastViromeExplorer-Novel: Recovering Draft Genomes of Novel Viruses and Phages in Metagenomic Data.

Despite the recent surge of viral metagenomic studies, recovering complete virus/phage genomes from metagenomic data is still extremely difficult and most viral contigs generated from de novo assembly programs are highly fragmented, posing serious challenges to downstream analysis and inference. In this study, we develop FastViromeExplorer (FVE)-novel, a computational pipeline for reconstructing complete or near-complete viral draft genomes from metagenomic data. The FVE-novel deploys FVE to efficiently map metagenomic reads to viral reference genomes, performs de novo assembly of the mapped reads to generate contigs, and extends the contigs through iterative assembly to produce final viral scaffolds. We applied FVE-novel to an ocean metagenomic sample and obtained 268 viral scaffolds that potentially come from novel viruses. Through manual examination and validation of the 10 longest scaffolds, we successfully recovered 4 complete viral genomes, 2 are novel as they cannot be found in the existing databases and the other 2 are related to known phages. This hybrid reference-based and de novo assembly approach used by FVE-novel represents a powerful new approach for uncovering near-complete viral genomes in metagenomic data.

Complete Genome Sequence of Providencia stuartii CMC-4104, Isolated from a Human Splenic Abscess, Containing Multiple Copies of NDM-1 and PER-1 Carbapenem Resistance Genes.

We report the complete genome sequence of a clinical isolate of Providencia stuartii strain CMC-4104, isolated from a splenic abscess. Oxford Nanopore Technologies (ONT) and Illumina sequencing reads were assembled using Geneious to generate a 4,504,925-bp circular chromosome containing multiple copies of the NDM-1 and PER-1 genes in a genomic resistance island.

Complete Genome Sequence of Curtobacterium sp. Isolated from Surface-Sterilized Germinating Alfalfa Seeds.

We reported the complete genome sequence of a member of the pathogenic Curtobacterium genus. The sample includes a circular 3,955-kb chromosome, a 164-kb megaplasmid and a 42-kb plasmid. This strain was isolated from surface-sterilized alfalfa seeds.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-097, Isolated from a Ventilator-Associated Pneumonia Patient, Containing a Novel Carbapenem Resistance Class 1 Integron.

We report the complete genome of a clinical strain of Pseudomonas aeruginosa CMC-097, which was isolated from a ventilator-associated pneumonia patient with a chronic infection. Illumina sequence reads were assembled using Geneious to yield a 7,044,064-bp circular chromosome containing a carbapenem resistance integron, In2020.

Association of RERG Expression with Female Survival Advantage in Malignant Pleural Mesothelioma.

Sex differences in incidence, prognosis, and treatment response have been described for many cancers. In malignant pleural mesothelioma (MPM), a lethal disease associated with asbestos exposure, men outnumber women 4 to 1, but women consistently live longer than men following surgery-based therapy. This study investigated whether tumor expression of genes associated with estrogen signaling could potentially explain observed survival differences. Two microarray datasets of MPM tumors were analyzed to discover estrogen-related genes associated with survival. A validation cohort of MPM tumors was selected to balance the numbers of men and women and control for competing prognostic influences. The RAS like estrogen regulated growth inhibitor (RERG) gene was identified as the most differentially-expressed estrogen-related gene in these tumors and predicted prognosis in discovery datasets. In the sex-matched validation cohort, low RERG expression was significantly associated with increased risk of death among women. No association between RERG expression and survival was found among men, and no relationship between estrogen receptor protein or gene expression and survival was found for either sex. Additional investigations are needed to elucidate the molecular mechanisms underlying this association and its sex specificity.

Large-scale analysis of BAP1 expression reveals novel associations with clinical and molecular features of malignant pleural mesothelioma.

BRCA1-associated protein-1 (BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact of BAP1 mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA-sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT-qPCR on 483 cases to estimate the relative proportions of epithelial-like and mesenchymal-like components in each tumor. Four BAP1 staining patterns were observed: single-pattern nuclear staining (36%), single-pattern cytoplasmic staining (25%), single-pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild-type BAP1 and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) with BAP1 gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial-to-mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra-tumoral heterogeneity of BAP1 status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role of BAP1 in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM, potentially addressing a critical need in clinical decision-making in this disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming.

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-115, a Clinical Strain from an Acute Ventilator-Associated Pneumonia Patient.

We report the complete genome of clinical strain Pseudomonas aeruginosa CMC-115, which was isolated from an acute ventilator-associated pneumonia patient. Illumina sequencing reads were assembled using Geneious to yield a 6,375,262-bp circular chromosome that exhibited an unusual ferrichrome receptor in the pyoverdine synthesis locus and the absence of type 3 secretion system genes.

Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces.

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 ß-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. ß-glucuronidase expression was then measured in cells grown in liquid or on plates. ß-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.

Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct.

A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion.

Differential expression patterns of housekeeping genes increase diagnostic and prognostic value in lung cancer.

BACKGROUND: Using DNA microarrays, we previously identified 451 genes expressed in 19 different human tissues. Although ubiquitously expressed, the variable expression patterns of these "housekeeping genes" (HKGs) could separate one normal human tissue type from another. Current focus on identifying "specific disease markers" is problematic as single gene expression in a given sample represents the specific cellular states of the sample at the time of collection. In this study, we examine the diagnostic and prognostic potential of the variable expressions of HKGs in lung cancers. METHODS: Microarray and RNA-seq data for normal lungs, lung adenocarcinomas (AD), squamous cell carcinomas of the lung (SQCLC), and small cell carcinomas of the lung (SCLC) were collected from online databases. Using 374 of 451 HKGs, differentially expressed genes between pairs of sample types were determined via two-sided, homoscedastic t-test. Principal component analysis and hierarchical clustering classified normal lung and lung cancers subtypes according to relative gene expression variations. We used uni- and multi-variate cox-regressions to identify significant predictors of overall survival in AD patients. Classifying genes were selected using a set of training samples and then validated using an independent test set. Gene Ontology was examined by PANTHER. RESULTS: This study showed that the differential expression patterns of 242, 245, and 99 HKGs were able to distinguish normal lung from AD, SCLC, and SQCLC, respectively. From these, 70 HKGs were common across the three lung cancer subtypes. These HKGs have low expression variation compared to current lung cancer markers (e.g., EGFR, KRAS) and were involved in the most common biological processes (e.g., metabolism, stress response). In addition, the expression pattern of 106 HKGs alone was a significant classifier of AD versus SQCLC. We further highlighted that a panel of 13 HKGs was an independent predictor of overall survival and cumulative risk in AD patients. DISCUSSION: Here we report HKG expression patterns may be an effective tool for evaluation of lung cancer states. For example, the differential expression pattern of 70 HKGs alone can separate normal lung tissue from various lung cancers while a panel of 106 HKGs was a capable class predictor of subtypes of non-small cell carcinomas. We also reported that HKGs have significantly lower variance compared to traditional cancer markers across samples, highlighting the robustness of a panel of genes over any one specific biomarker. Using RNA-seq data, we showed that the expression pattern of 13 HKGs is a significant, independent predictor of overall survival for AD patients. This reinforces the predictive power of a HKG panel across different gene expression measurement platforms. Thus, we propose the expression patterns of HKGs alone may be sufficient for the diagnosis and prognosis of individuals with lung cancer.

The Skin Microbiome of the Neotropical Frog Craugastor fitzingeri: Inferring Potential Bacterial-Host-Pathogen Interactions From Metagenomic Data.

Skin symbiotic bacteria on amphibians can play a role in protecting their host against pathogens. Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, Bd, has caused dramatic population declines and extinctions of amphibians worldwide. Anti-Bd bacteria from amphibian skin have been cultured, and skin bacterial communities have been described through 16S rRNA gene amplicon sequencing. Here, we present a shotgun metagenomic analysis of skin bacterial communities from a Neotropical frog, Craugastor fitzingeri. We sequenced the metagenome of six frogs from two different sites in Panamá: three frogs from Soberanía (Sob), a Bd-endemic site, and three frogs from Serranía del Sapo (Sapo), a Bd-naïve site. We described the taxonomic composition of skin microbiomes and found that Pseudomonas was a major component of these communities. We also identified that Sob communities were enriched in Actinobacteria while Sapo communities were enriched in Gammaproteobacteria. We described gene abundances within the main functional classes and found genes enriched either in Sapo or Sob. We then focused our study on five functional classes of genes: biosynthesis of secondary metabolites, metabolism of terpenoids and polyketides, membrane transport, cellular communication and antimicrobial drug resistance. These gene classes are potentially involved in bacterial communication, bacterial-host and bacterial-pathogen interactions among other functions. We found that C. fitzingeri metagenomes have a wide array of genes that code for secondary metabolites, including antibiotics and bacterial toxins, which may be involved in bacterial communication, but could also have a defensive role against pathogens. Several genes involved in bacterial communication and bacterial-host interactions, such as biofilm formation and bacterial secretion systems were found. We identified specific genes and pathways enriched at the different sites and determined that gene co-occurrence networks differed between sites. Our results suggest that skin microbiomes are composed of distinct bacterial taxa with a wide range of metabolic capabilities involved in bacterial defense and communication. Differences in taxonomic composition and pathway enrichments suggest that skin microbiomes from different sites have unique functional properties. This study strongly supports the need for shotgun metagenomic analyses to describe the functional capacities of skin microbiomes and to tease apart their role in host defense against pathogens.

Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn-Seq analysis.

The bacterium Pantoea stewartii ssp. stewartii causes Stewart's wilt disease in corn. Pantoea stewartii is transmitted to plants via corn flea beetles, where it first colonizes the apoplast causing water-soaked lesions, and then migrates to the xylem and forms a biofilm that blocks water transport. Bacterial quorum sensing ensures that the exopolysaccharide production necessary for biofilm formation occurs only at high cell density. A genomic-level transposon sequencing (Tn-Seq) analysis was performed to identify additional bacterial genes essential for survival in planta and to provide insights into the plant-microbe interactions occurring during wilt disease. A mariner transposon library of approximately 40 000 mutants was constructed and used to inoculate corn seedlings through a xylem infection model. Cultures of the library grown in Luria-Bertani (LB) broth served as the in vitro pre-inoculation control. Tn-Seq analysis showed that the number of transposon mutations was reduced by more than 10-fold for 486 genes in planta compared with the library that grew in LB, suggesting that they are important for xylem survival. Interestingly, a small set of genes had a higher abundance of mutants in planta versus in vitro conditions, indicating enhanced strain fitness with loss of these genes inside the host. In planta competition assays retested the trends of the Tn-Seq data for several genes, including two outer membrane proteins, Lon protease and two quorum sensing-associated transcription factors, RcsA and LrhA. Virulence assays were performed to check for correlation between growth/colonization and pathogenicity. This study demonstrates the capacity of a Tn-Seq approach to advance our understanding of P. stewartii-corn interactions.

FastViromeExplorer: a pipeline for virus and phage identification and abundance profiling in metagenomics data.

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.

Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2-/- Lung Fibroblasts.

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

Dominance-function relationships in the amphibian skin microbiome.

Some amphibian skin bacteria inhibit growth of a fungal amphibian pathogen, Batrachochytrium dendrobatidis (Bd), but it is unclear how dominant these anti-Bd bacteria are in skin communities. Using in vitro co-culture challenge assays, we quantified Bd inhibition by bacterial isolates collected from the skin of four amphibian species: bullfrogs, Eastern newts, spring peepers and American toads. The 16S rRNA sequences for each isolate were matched to culture-independent amplicon sequences from the same individuals to assess inhibitory function versus relative abundance. Dominant bacteria had higher Bd inhibition than rare bacteria in bullfrog and newt populations, in which Bd was prevalent (> 25%). Dominant and rare bacteria did not differ in Bd inhibition in spring peeper and toad populations, in which Bd was absent or at low prevalence (< 7%). In addition, over half of the relative abundance of cultured bacteria on bullfrogs and newts was comprised of inhibitory bacteria, while only 25% and 37% of the relative abundance was inhibitory on spring peepers and toads, respectively. These results suggest that the dominant members of the amphibian skin bacterial community may be functionally important in terms of disease-resistance, and that Bd prevalence and/or host species identity may impact the relative abundance and inhibitory properties of skin bacteria.

Mechanisms for Differential Protein Production in Toxin-Antitoxin Systems.

Toxin-antitoxin (TA) systems are key regulators of bacterial persistence, a multidrug-tolerant state found in bacterial species that is a major contributing factor to the growing human health crisis of antibiotic resistance. Type II TA systems consist of two proteins, a toxin and an antitoxin; the toxin is neutralized when they form a complex. The ratio of antitoxin to toxin is significantly greater than 1.0 in the susceptible population (non-persister state), but this ratio is expected to become smaller during persistence. Analysis of multiple datasets (RNA-seq, ribosome profiling) and results from translation initiation rate calculators reveal multiple mechanisms that ensure a high antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms include both translational and transcriptional regulation. We classified E. coli type II TA systems into four distinct classes based on the mechanism of differential protein production between toxin and antitoxin. We find that the most common regulation mechanism is translational regulation. This classification scheme further refines our understanding of one of the fundamental mechanisms underlying bacterial persistence, especially regarding maintenance of the antitoxin-to-toxin ratio.

Complete Genome Assembly of Pantoea stewartii subsp. stewartii DC283, a Corn Pathogen.

The phytopathogen Pantoea stewartii subsp. stewartii DC283 causes Stewart's wilt disease in corn after transmission from the corn flea beetle insect vector. Here, we report that the complete annotated genome of P. stewartii DC283 has been fully assembled into one circular chromosome, 10 circular plasmids, and one linear phage.

Analysis of the in planta transcriptome expressed by the corn pathogen Pantoea stewartii subsp. stewartii via RNA-Seq.

Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart's wilt disease in corn. It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during an in planta infection versus either a pre-inoculum in vitro liquid culture or an in vitro agar plate culture to identify genes specifically expressed in planta that may also be important for colonization and/or virulence. RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA. Fold gene expression changes in the in planta data set in comparison to the two in vitro grown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association. Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems important for oxidation reduction processes appear to play a critical role for P. stewartii as it colonizes and causes wilt disease in corn plants.