Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

Campylobacter jejuni is naturally transformable but is selective in the DNA used in transformation. Natural transformation allows for rapid adaptation and provides a means for DNA repair, both of which enhance bacterial fitness. Our work demonstrates that the methylation status of C. jejuni DNA is critical for transformation; methylation of a single 6-bp sequence in proximity to the transforming DNA is sufficient to allow nontransforming DNA to transform C. jejuni. Our data argue against previously described means for DNA discrimination; specifically, there is no evidence that a restriction enzyme recognizes the unmodified 6-bp sequence, and no evidence of a typical DNA uptake sequence. Therefore, we have identified a distinct DNA discrimination mechanism in Campylobacter

Characterization of the Competitive Pneumocin Peptides of Streptococcus pneumoniae

In the polymicrobial environment of the human nasopharynx, Streptococcus pneumoniae (pneumococcus) competes with other members of the microbial community for limited nutrients in part by secreting small peptide bacteriocins called pneumocins. Pneumocin production is controlled by a quorum sensing system encoded by the blp locus. Although the locus is found in all pneumococci, there is significant variability in the repertoire of pneumocins and associated immunity proteins encoded in the Bacteriocin Immunity Region (BIR) and in the presence or absence of a functional Blp transporter. Strains without an active Blp transporter are inactive in plate overlay assays and rely on a homologous transporter that is only produced during brief periods of competence to stimulate the blp locus and secrete pneumocins. The variability of the locus suggests that selective pressure is influencing the content to promote the optimal competitive environment. Much of the variability in the blp locus has been described at the genome level; the phenotypic activity attributable to the various BIR genes has not been fully described. To examine the role of the predicted pneumocin peptides in competition, 454 isolates were screened for competence independent blp pheromone secretion using plate assays. Active strains were characterized for inhibition, BIR content, BlpC pherotype and serotype. Deletion analysis on inhibitory strains demonstrated that BlpI and BlpJ peptides function as a two-peptide bacteriocin and that BlpIJ immunity is encoded by the co-transcribed blpU4/5 genes. BlpIJ secretion promotes inhibitory activity against the majority of pneumococcal isolates when expressed in a Blp transporter intact background. Intermediate levels of competition in biofilms were noted when BlpIJ containing strains carried the non-functional Blp transporter. Based on genome data, the combination of BlpIJ in a Blp transporter intact strain is surprisingly rare, despite clear advantages during colonization and biofilm growth. In contrast, we show that the blpK/pncF operon encoding the single-peptide pneumocin BlpK and its immunity protein is found in the majority of isolates. Unlike, BlpIJ and BlpK were shown to promote a limited spectrum of inhibition due in part to immunity that is independent of activation of the blp locus

Conserved Mutations in the Pneumococcal Bacteriocin Transporter Gene, blpA, Result in a Complex Population Consisting of Producers and Cheaters

All fully sequenced strains of Streptococcus pneumoniae possess a version of the blp locus, which is responsible for bacteriocin production and immunity. Activation of the blp locus is stimulated by accumulation of the peptide pheromone, BlpC, following its secretion by the ABC transporter, BlpA. The blp locus is characterized by significant diversity in blpC type and in the region of the locus containing putative bacteriocin and immunity genes. In addition, the blpA gene can represent a single large open reading frame or be divided into several smaller fragments due to the presence of frameshift mutations. In this study, we use a collection of strains with blp-dependent inhibition and immunity to define the genetic changes that bring about phenotypic differences in bacteriocin production or immunity. We demonstrate that alterations in blpA, blpC, and bacteriocin/immunity content likely play an important role in competitive interactions between pneumococcal strains. Importantly, strains with a highly conserved frameshift mutation in blpA are unable to secrete bacteriocins or BlpC, but retain the ability to respond to exogenous peptide pheromone produced by cocolonizing strains, stimulating blp-mediated immunity. These “cheater” strains can only coexist with bacteriocin-producing strains that secrete their cognate BlpC and share the same immunity proteins. The variable outcome of these interactions helps to explain the heterogeneity of the blp pheromone, bacteriocin, and immunity protein content

Differences in genotype and virulence among four multidrug-resistant <i>Streptococcus pneumoniae</i> isolates belonging to the PMEN1 clone

We report on the comparative genomics and characterization of the virulence phenotypes of four &lt;i&gt;S. pneumoniae&lt;/i&gt; strains that belong to the multidrug resistant clone PMEN1 (Spain&lt;sup&gt;23F&lt;/sup&gt; ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinant

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Optimizing the diagnostic work-up of acute uncomplicated urinary tract infections

<p>Abstract</p> <p>Background</p> <p>Most diagnostic tests for acute uncomplicated urinary tract infections (UTIs) have been previously studied in so-called single-test evaluations. In practice, however, clinicians use more than one test in the diagnostic work-up. Since test results carry overlapping information, results from single-test studies may be confounded. The primary objective of the Amsterdam Cystitis/Urinary Tract Infection Study (ACUTIS) is to determine the (additional) diagnostic value of relevant tests from patient history and laboratory investigations, taking into account their mutual dependencies. Consequently, after suitable validation, an easy to use, multivariable diagnostic rule (clinical index) will be derived.</p> <p>Methods</p> <p>Women who contact their GP with painful and/or frequent micturition undergo a series of possibly relevant tests, consisting of patient history questions and laboratory investigations. Using urine culture as the reference standard, two multivariable models (diagnostic indices) will be generated: a model which assumes that patients attend the GP surgery and a model based on telephone contact only. Models will be made more robust using the bootstrap. Discrimination will be visualized in high resolution histograms of the posterior UTI probabilities and summarized as 5^th, 10^th, 25^th50^th, 75^th, 90^th, and 95^thcentiles of these, Brier score and the area under the receiver operating characteristics curve (ROC) with 95% confidence intervals. Using the regression coefficients of the independent diagnostic indicators, a diagnostic rule will be derived, consisting of an efficient set of tests and their diagnostic values.</p> <p>The course of the presenting complaints is studied using 7-day patient diaries. To learn more about the natural history of UTIs, patients will be offered the opportunity to postpone the use of antibiotics.</p> <p>Discussion</p> <p>We expect that our diagnostic rule will allow efficient diagnosis of UTIs, necessitating the collection of diagnostic indicators with proven added value. GPs may use the rule (preferably after suitable validation) to estimate UTI probabilities for women with different combinations of test results. Finally, in a subcohort, an attempt is made to identify which indicators (including antibiotic treatment) are useful to prognosticate recovery from painful and/or frequent micturition.</p

Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types

Protein ubiquitination is a dynamic and reversibleprocess of adding single ubiquitin molecules orvarious ubiquitin chains to target proteins. Here,using multidimensional omic data of 9,125 tumorsamples across 33 cancer types from The CancerGenome Atlas, we perform comprehensive molecu-lar characterization of 929 ubiquitin-related genesand 95 deubiquitinase genes. Among them, we sys-tematically identify top somatic driver candidates,including mutatedFBXW7with cancer-type-specificpatterns and amplifiedMDM2showing a mutuallyexclusive pattern withBRAFmutations. Ubiquitinpathway genes tend to be upregulated in cancermediated by diverse mechanisms. By integratingpan-cancer multiomic data, we identify a group oftumor samples that exhibit worse prognosis. Thesesamples are consistently associated with the upre-gulation of cell-cycle and DNA repair pathways, char-acterized by mutatedTP53,MYC/TERTamplifica-tion, andAPC/PTENdeletion. Our analysishighlights the importance of the ubiquitin pathwayin cancer development and lays a foundation fordeveloping relevant therapeutic strategies