Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague

Following inhalation, Yersinia pestis rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in Yersinia spp., the factors mediating early Y. pestis adherence in the lung remain unknown. To identify genes important for Y. pestis adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δcaf1) Y. pestis transposon mutant libraries were serially passaged in vivo to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δcaf1 Y. pestis backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for YPO3903 in Y. pestis adherence in the lung and showed that YPO3903 regulated transcript levels of psaA, which encodes a fimbria previously implicated in Y. pestis adherence in vitro. Deletion of psaA had a minor effect on Y. pes-tis adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to psaA. By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early Y. pestis adherence during primary pneumonic plague

Epstein-Barr Virus-Positive Cancers Show Altered B-Cell Clonality

Epstein-Barr virus (EBV) is convincingly associated with gastric cancer, nasopharyngeal carcinoma, and certain lymphomas, but its role in other cancer types remains controversial. To test the hypothesis that there are additional cancer types with high prevalence of EBV, we determined EBV viral expression in all the Cancer Genome Atlas Project (TCGA) mRNA sequencing (mRNA-seq) samples (n 10,396) from 32 different tumor types. We found that EBV was present in gastric adenocarcinoma and lymphoma, as expected, and was also present in 5% of samples in 10 additional tumor types. For most samples, EBV transcript levels were low, which suggests that EBV was likely present due to infected infiltrating B cells. In order to determine if there was a difference in the B-cell populations, we assembled B-cell receptors for each sample and found B-cell receptor abundance (P 1.4 1020) and diversity (P 8.3 1027) were significantly higher in EBV-positive samples. Moreover, diversity was independent of B-cell abundance, suggesting that the presence of EBV was associated with an increased and altered B-cell population. IMPORTANCE Around 20% of human cancers are associated with viruses. Epstein-Barr virus (EBV) contributes to gastric cancer, nasopharyngeal carcinoma, and certain lymphomas, but its role in other cancer types remains controversial. We assessed the prevalence of EBV in RNA-seq from 32 tumor types in the Cancer Genome Atlas Project (TCGA) and found EBV to be present in 5% of samples in 12 tumor types. EBV infects epithelial cells and B cells and in B cells causes proliferation. We hypothesized that the low expression of EBV in most of the tumor types was due to infiltration of B cells into the tumor. The increase in B-cell abundance and diversity in subjects where EBV was detected in the tumors strengthens this hypothesis. Overall, we found that EBV was associated with an increased and altered immune response. This result is not evidence of causality, but a potential novel biomarker for tumor immune status

Gene expression-phenotype associations in adults with eosinophilic esophagitis

Background: Gene expression patterns have not been extensively examined in the context of clinical features of eosinophilic esophagitis (EoE). Aims: To assess whether gene expression is associated with clinically defined phenotypes in adults with EoE. Methods: This was an analysis of prospectively collected esophageal biopsies in newly diagnosed EoE patients. We determined differential gene expression with a 94 gene panel in relation to clinical features and phenotypes. These included: endoscopic findings of esophageal rings, stricture, narrowing, linear furrows, exudates, edema, and dilation; an allergic phenotype; an inflammatory phenotype, and a fibrostenotic phenotype. Results: In 89 EoE cases analyzed, patients with exudates on endoscopy had multiple differences in gene expression compared to patients without exudates, though patients with exudates also had higher eosinophil counts (172 vs 106 eos/hpf; p =.01). Genes associated with esophageal narrowing included CCL26 (q-value = 0.028), ALOX15 (q = 0.011), GRK5 (q = 0.029), CPA3 (q = 0.012), and TRIM2 (q = 0.0027). TRIM2 was also associated with the fibrostenotic phenotype (q = 0.0051). No genes were associated with the inflammatory or atopic phenotypes, or with dilation. Conclusions: Multiple genes are associated with exudates, possibly related to higher eosinophil counts. However, a number of genes, including those related to both inflammation and remodelling, are associated with esophageal narrowing. In particular, TRIM2 is associated with clinical fibrotic phenotypes

Identification of Germline Variants in Tumor Genomic Sequencing Analysis

This Correspondence relates to the article by Li et al (Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and the College of American Pathologists. J Mol Diagn 2017, 19:4–23)

Prospective assessment of serum periostin as a biomarker for diagnosis and monitoring of eosinophilic oesophagitis

Background Periostin is highly expressed in eosinophilic oesophagitis (EoE), but has not been extensively studied as a non-invasive biomarker. Aim To assess whether serum periostin distinguished EoE from controls at baseline, had utility for monitoring treatment response, or was associated with IL-13 levels. Methods This was a sub-analysis of a prospective cohort study of adults undergoing out-patient upper endoscopy. Incident cases of EoE were diagnosed per consensus guidelines. Controls were subjects with either GERD or dysphagia without EoE. EoE patients were treated with swallowed/topical steroids and had repeat endoscopy/biopsy. Serum periostin levels for cases and controls were compared at baseline, and pre/post-treatment levels were compared for cases. Serum IL-13 and tissue expression of periostin were also assessed. Results A total of 61 incident EoE cases and 87 controls were analysed. Despite a marked increase in tissue periostin expression in cases, the median baseline serum periostin level was only slightly higher in cases than controls (22.1 ng/mL vs. 20.7; P = 0.04); there was no change in post-treatment levels. There was also no difference in serum periostin for cases by histologic response or atopic status. There was a strong trend towards higher serum IL-13 levels in cases in the highest periostin quartile (57.1 pg/mL vs. 2.6; P = 0.07). Conclusions Serum periostin levels were similar in cases and controls, and there were no changes post-treatment. Given elevated IL-13 levels in the EoE patients with the highest periostin levels, future studies could explore periostin as a biomarker in EoE, perhaps in the setting of anti-IL-13 therapy

A comparison of limited-stretch models of rubber elasticity

In this paper we describe various limited-stretch models of non-linear rubber elasticity, each dependent on only the first invariant of the left Cauchy-Green strain tensor and having only two independent material constants. The models are described as limited-stretch, or restricted elastic, because the strain energy and stress response become infinite at a finite value of the first invariant. These models describe well the limited stretch of the polymer chains of which rubber is composed. We discuss Gent's model which is the simplest limited-stretch model and agrees well with experiment. Various statistical models are then described: the one-chain, three-chain, four-chain and Arruda-Boyce eight-chain models, all of which involve the inverse Langevin function. A numerical comparison between the three-chain and eight-chain models is provided. Next, we compare various models which involve approximations to the inverse Langevin function with the exact inverse Langevin function of the eight-chain model. A new approximate model is proposed that is as simple as Cohen's original model but significantly more accurate. We show that effectively the eight-chain model may be regarded as a linear combination of the neo-Hookean and Gent models. Treloar's model is shown to have about half the percentage error of our new model but it is much more complicated. For completeness a modified Treloar model is introduced but this is only slightly more accurate than Treloar's original model. For the deformations of uniaxial tension, biaxial tension, pure shear and simple shear we compare the accuracy of these models, and that of Puso, with the eight-chain model by means of graphs and a table. Our approximations compare extremely well with models frequently used and described in the literature, having the smallest mean percentage error over most of the range of the argument

Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V'DJer

Motivation: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not. Results: We present here V'DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V'DJer's ability to accurately reconstruct BCR repertoires from short read mRNA-seq data

Virus expression detection reveals RNA-sequencing contamination in TCGA

Background: Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequences. To detect viruses with high specificity, we developed the publicly available workflow, VirDetect, that detects virus and laboratory vector sequences in RNA-seq samples. We applied VirDetect to 9143 RNA-seq samples sequenced at one TCGA sequencing center (28/33 cancer types) over 5 years. Results: We confirmed that H-HPV18 was present in many samples and determined that viral transcripts from H-HPV18 significantly co-occurred with those from xenotropic mouse leukemia virus-related virus (XMRV). Using laboratory metadata and viral transcription, we determined that the likely contaminant was a pool of cell lines known as the "common reference", which was sequenced alongside TCGA RNA-seq samples as a control to monitor quality across technology transitions (i.e. microarray to GAII to HiSeq), and to link RNA-seq to previous generation microarrays that standardly used the "common reference". One of the cell lines in the pool was a laboratory isolate of MCF-7, which we discovered was infected with XMRV; another constituent of the pool was likely HeLa cells. Conclusions: Altogether, this indicates a multi-step contamination process. First, MCF-7 was infected with an XMRV. Second, this infected cell line was added to a pool of cell lines, which contained HeLa. Finally, RNA from this pool of cell lines contaminated several TCGA tumor samples most-likely during library construction. Thus, these human tumors with H-HPV or XMRV reads were likely not infected with H-HPV 18 or XMRV

Prognostic value of B cells in cutaneous melanoma

Background: Measures of the adaptive immune response have prognostic and predictive associations in melanoma and other cancer types. Specifically, intratumoral T cell density and function have considerable prognostic and predictive value in skin cutaneous melanoma (SKCM). Less is known about the significance of tumor-infiltrating B cells in SKCM. Our goal was to understand the prognostic and predictive value of B cell phenotypic subsets in SKCM using RNA sequencing. Methods: We used our previously published algorithm, V'DJer, to assemble B cell receptor (BCR) repertoires and estimate diversity from short-read RNA sequencing (RNA-seq). We applied machine learning-based cellular phenotype classifiers to measure relative similarity of bulk tumor sample gene expression profiles and different B cell phenotypes. We assessed these aspects of B cell biology in 473 SKCM from the Cancer Genome Atlas Project (TCGA) as well as in RNA-seq data corresponding to tumor samples procured from patients who received CTLA-4 and PD-1 inhibitors for metastatic SKCM. Results: We found that the BCR repertoire was associated with different clinical factors, such as tumor tissue site and sex. However, increased clonality of the BCR repertoire was favorably prognostic in SKCM and was prognostic even after first conditioning on various clinical factors. Mutation burden was not correlated with any BCR measurement, and no specific mutation had an altered BCR repertoire. Lack of an assembled BCR in pre-treatment tumor tissues was associated with a lack of anti-tumor response to a CTLA-4 inhibitor in metastatic SKCM. Conclusions: These findings suggest an important prognostic and predictive role for B cell characteristics in SKCM. This has implications for melanoma immunobiology and potential development of immunogenomics features to predict survival and response to immunotherapy