Complete Genome Sequence of Thiohalobacter sp. Strain COW1, Isolated from Activated Sludge Treating Coke Oven Wastewater.

A thiocyanate-degrading bacterium, Thiohalobacter sp. strain COW1, was isolated from activated sludge treating coke oven wastewater, and the complete genome sequence was determined. COW1 contained a single circular chromosome (3.23 Mb; G+C content, 63.4%) in which 2,788 protein-coding genes, 39 tRNA genes, and 3 rRNA genes were identified.

Virus-like insertions with sequence signatures similar to those of endogenous nonretroviral RNA viruses in the human genome.

Understanding the genetics and taxonomy of ancient viruses will give us great insights into not only the origin and evolution of viruses but also how viral infections played roles in our evolution. Endogenous viruses are remnants of ancient viral infections and are thought to retain the genetic characteristics of viruses from ancient times. In this study, we used machine learning of endogenous RNA virus sequence signatures to identify viruses in the human genome that have not been detected or are already extinct. Here, we show that the k-mer occurrence of ancient RNA viral sequences remains similar to that of extant RNA viral sequences and can be differentiated from that of other human genome sequences. Furthermore, using this characteristic, we screened RNA viral insertions in the human reference genome and found virus-like insertions with phylogenetic and evolutionary features indicative of an exogenous origin but lacking homology to previously identified sequences. Our analysis indicates that animal genomes still contain unknown virus-derived sequences and provides a glimpse into the diversity of the ancient virosphere.

Thiocyanate Degradation by a Highly Enriched Culture of the Neutrophilic Halophile Thiohalobacter sp. Strain FOKN1 from Activated Sludge and Genomic Insights into Thiocyanate Metabolism.

Thiocyanate (SCN-) is harmful to a wide range of organisms, and its removal is essential for environmental protection. A neutrophilic halophile capable of thiocyanate degradation, Thiohalobacter sp. strain FOKN1, was highly enriched (relative abundance; 98.4%) from activated sludge collected from a bioreactor receiving thiocyanate-rich wastewater. The enrichment culture degraded 3.38 mM thiocyanate within 140 h, with maximum activity at pH 8.8, 37°C, and 0.18 M sodium chloride. Thiocyanate degradation was inhibited by 30 mg L-1 phenol, but not by thiosulfate. Microbial thiocyanate degradation is catalyzed by thiocyanate dehydrogenase, while limited information is currently available on the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles. Therefore, (meta)genomic and proteomic analyses of enrichment cultures were performed to elucidate the whole genome sequence and proteome of Thiohalobacter sp. strain FOKN1. The 3.23-Mb circular Thiohalobacter sp. strain FOKN1 genome was elucidated using a PacBio RSII sequencer, and the expression of 914 proteins was identified by tandem mass spectrometry. The Thiohalobacter sp. strain FOKN1 genome had a gene encoding thiocyanate dehydrogenase, which was abundant in the proteome, suggesting that thiocyanate is degraded by thiocyanate dehydrogenase to sulfur and cyanate. The sulfur formed may be oxidized to sulfate by the sequential oxidation reactions of dissimilatory sulfite reductase, adenosine-5'-phosphosulfate reductase, and dissimilatory ATP sulfurylase. Although the Thiohalobacter sp. strain FOKN1 genome carried a gene encoding cyanate lyase, its protein expression was not detectable. The present study advances the understanding of the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles.

Tissue Factor Facilitates Wound Healing in Human Airway Epithelial Cells.

BACKGROUND: Tissue factor (TF) canonically functions as the initiator of the coagulation cascade. TF levels are increased in inflamed airways and seem to be important for tumor growth and metastasis. We hypothesized that airway epithelia release TF as part of a wound repair program. OBJECTIVES: The goal of this study was to evaluate whether airway epithelia release TF in response to pro-inflammatory stimuli and to investigate roles of TF in cell growth and repair. METHODS: Airway epithelial cells were exposed to 10 µg/mL of lipopolysaccharide or 1 ng/mL of transforming growth factor ß (TGF-ß). TF and TGF-ß messenger RNA and protein were measured in cell lysate and culture media, respectively. Signaling pathways were evaluated by using selective agonists and inhibitors. Airway epithelia were mechanically injured in the presence of TF and tissue factor pathway inhibitor to investigate their roles in wound repair. RESULTS: TF protein levels increased in cell media after exposure to lipopolysaccharide (P < .01) but only in growing cells, and this action was blocked when exposed to an extracellular signal-regulated kinase inhibitor or a "small" worm phenotype and mothers against Decapentaplegic inhibitor. TF protein also increased in the presence of TGF-ß (P < .05). Exposure to TF pathway inhibitor decreased the rate of cell growth by 60% (P < .05), and exposure to TF increased the rate of airway healing after injury by 19% (P < .05). CONCLUSIONS: Growing airway epithelia release TF when exposed to lipopolysaccharide or TGF-ß. TF reduces wound-healing time in airway epithelia and therefore may be important to airway recovery after injury.

Tiotropium inhibits mucin production stimulated by neutrophil elastase but not by IL-13.

Tiotropium, a muscarinic antagonist, is approved for the treatment of chronic obstructive pulmonary disease and poorly controlled asthma. Because mucus hypersecretion is characteristic of both of these diseases, and muscarinic agonists stimulate mucus secretion, we hypothesized that tiotropium would attenuate airway MUC5AC expression. We grew normal human bronchial epithelial (NHBE) cells to a goblet cell phenotype with 1 or 5 ng/mL of IL-13 and exposed these cells to 10 nM tiotropium or excipient for the full 14 days. Normally differentiated NHBE cells (without IL-13) were exposed to neutrophil elastase (NE) 1 × 10-7 or 5 × 10-7 M for 1 h. MUC5AC was measured by quantitative PCR and ELISA. Acetylcholine production by the epithelium was evaluated by quantitative PCR and by choline/acetylcholine quantification. Tiotropium had no effect on IL-13-stimulated MUC5AC, but attenuated MUC5AC stimulated by NE (p = 0.007 at 5 × 10-7 M). IL-13 increased CarAT mRNA (p < 0.001 at 5 ng/mL) and acetylcholine concentration in the medium (p = 0.018 at 5 ng/mL), while NE had no effect. Tiotropium had no direct effect on IL-13 or NE-induced CarAT or acetylcholine concentration. Tiotropium decreased MUC5AC stimulated by NE, but had no effect on MUC5AC stimulated by IL-13. These results may be due to IL-13, but not NE, increasing acetylcholine production.

Inhibition of IL-13-induced periostin in airway epithelium attenuates cellular protein expression of MUC5AC.

BACKGROUND AND OBJECTIVE: Serum periostin is increased in asthma and serves as a surrogate marker for IL-13 activity in the lung. Serum levels of periostin are the most robust biomarker predicting a favourable response to the anti-IL-13 drug, lebrikizumab. We investigated the mechanisms of IL-13 stimulation of periostin, the polarized secretion of periostin and whether periostin would have a direct effect on mucin secretion by airway cells. METHODS: Normal human bronchial epithelial (NHBE) cells were cultured at air-liquid interface (ALI) in the presence of IL-13, and we evaluated the effect of the specific inhibitors, leflunomide (Janus kinase (JAK)/signal transducer and activator of transcription factor 6 (STAT6) inhibitor) or PD98059 (MEK/extracellular regulated protein kinase (ERK) inhibitor), on periostin production. We examined MUC5AC secretion from NHBE cells exposed to recombinant human (rh) periostin or IL-13 in the presence and absence of OC-20, a periostin-neutralizing antibody. RESULTS: IL-13 induced periostin protein which was predominantly secreted towards the basal surface of the cells. Periostin production was much greater from goblet cells than ciliated cells (P < 0.001). Periostin production after exposure to IL-13 was attenuated by both leflunomide (P < 0.001) and PD98059 (P < 0.001). The addition of exogenous periostin modestly increased MUC5AC secretion (P < 0.01), but did not visibly change cell morphology. IL-13-induced MUC5AC secretion was attenuated by OC-20 (P < 0.01). CONCLUSION: Periostin production in differentiated airway cells is mediated by JAK/STAT6 and MEK/ERK pathways. Periostin secretion is much greater from immunologically active goblet cells. IL-13-driven mucin production is partially inhibited by OC-20.

Successful diagnosis of tuberculous lymphadenitis by loop-mediated isothermal amplification of cutaneous samples from an ulcerated surface lesion: a case report.

INTRODUCTION: Tuberculous lymphadenitis is the most frequent form of extrapulmonary tuberculous. Although nucleic acid amplification assays such as polymerase chain reaction have recently become mainstream techniques for diagnosing tuberculous lymphadenitis, they are still not routinely performed in developing countries because of their high costs and complicated procedures. CASE PRESENTATION: We describe a case of tuberculous lymphadenitis in a 79-year-old Japanese man who had been on continuous hemodialysis for end-stage renal disease. We employed loop-mediated isothermal amplification and the procedure for ultrarapid extraction to develop a fast and easy-to-perform procedure for diagnosing tuberculous lymphadenitis. CONCLUSIONS: The commercially available loop-mediated isothermal amplification assay kit and a rapid purification procedure enabled us to identify and amplify a Mycobacterium tuberculosis-specific gene within just 1.5 hours.

[A case of eosinophilic granulomatosis with polyangiitis with extra-vascular granuloma and eosinophilic vasculitis diagnosed by transbronchial lung biopsy].

A 62-year-old man was suffering from bronchial asthma and referred to our institution with dry cough and dyspnea on exertion in November, 2010. He was diagnosed with eosinophilic granulomatosis with polyangiitis (EPGA, formerly Churg-Strauss syndrome) by chest radiographic findings, blood eosinophilia, mononeuritis multiplex and cardiomyopathy. Steroid therapy was started and he was rapidly improved. Steroid therapy had been tapered off by May, 2012. After 2 months, however, progressive dyspnea, neural symptoms, deafness, re-elevation of blood eosinophils and bilateral multifocal infiltrations appeared. He was re-admitted to our institution. Transbronchial lung biopsy (TBLB) specimens revealed extra-vascular granuloma, eosinophilic vasculitis and eosinophilic pneumonia and we diagnosed him with the reccurence of EGPA. He was improved by steroid pulse therapy, then tapered. This case was the antineutrophil cytoplasmic autoantibodies negative EGPA. The case of EGPA with granuloma and vasculitis diagnosed by TBLB was rare.

Identifying gene pathways associated with cancer characteristics via sparse statistical methods.

We propose a statistical method for uncovering gene pathways that characterize cancer heterogeneity. To incorporate knowledge of the pathways into the model, we define a set of activities of pathways from microarray gene expression data based on the Sparse Probabilistic Principal Component Analysis (SPPCA). A pathway activity logistic regression model is then formulated for cancer phenotype. To select pathway activities related to binary cancer phenotypes, we use the elastic net for the parameter estimation and derive a model selection criterion for selecting tuning parameters included in the model estimation. Our proposed method can also reverse-engineer gene networks based on the identified multiple pathways that enables us to discover novel gene-gene associations relating with the cancer phenotypes. We illustrate the whole process of the proposed method through the analysis of breast cancer gene expression data.

Increased risk for CRC in diabetic patients with the nonrisk allele of SNPs at 8q24.

BACKGROUND: Colorectal cancer (CRC) oncogenesis was considered to be determined by interactions between genetic and environmental factors. Specific interacting factors that influence CRC morbidity have yet to be fully investigated. METHODS: A multi-institutional collaborative study with 1511 CRC patients and 2098 control subjects was used to compare the odds ratios for the occurrence of polymorphisms at 11 known single nucleotide polymorphisms (SNPs). TaqMan PCR and questionnaires were used to evaluate the effects of environmental exposures. RESULTS: Variants of rs6983267 on 8q24 were the most significant markers of risk for CRC (odds ratio 1.16, 95% confidence interval 1.06-1.27, P = 0.0015). Non-insulin-dependent diabetes mellitus (DM), a higher body mass index at age 20, and meat consumption were environmental risk factors, whereas a tuna-rich diet and vitamin intake were protective factors. The cohort of rs6983267 SNP major (T) allele at 8q24 and DM had a 1.66-fold higher risk ratio than the cohort of major allele patients without DM. CONCLUSIONS: We confirmed that interactions between the genetic background and environmental factors are associated with increased risk for CRC. There is a robust risk of the minor G allele at the 8q24 rs6983267 SNP; however, a major T allele SNP could more clearly reveal a correlation with CRC specifically when DM is present.

Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas.

Activation of the EGFR, KRAS, and ALK oncogenes defines 3 different pathways of molecular pathogenesis in lung adenocarcinoma. However, many tumors lack activation of any pathway (triple-negative lung adenocarcinomas) posing a challenge for prognosis and treatment. Here, we report an extensive genome-wide expression profiling of 226 primary human stage I-II lung adenocarcinomas that elucidates molecular characteristics of tumors that harbor ALK mutations or that lack EGFR, KRAS, and ALK mutations, that is, triple-negative adenocarcinomas. One hundred and seventy-four genes were selected as being upregulated specifically in 79 lung adenocarcinomas without EGFR and KRAS mutations. Unsupervised clustering using a 174-gene signature, including ALK itself, classified these 2 groups of tumors into ALK-positive cases and 2 distinct groups of triple-negative cases (groups A and B). Notably, group A triple-negative cases had a worse prognosis for relapse and death, compared with cases with EGFR, KRAS, or ALK mutations or group B triple-negative cases. In ALK-positive tumors, 30 genes, including ALK and GRIN2A, were commonly overexpressed, whereas in group A triple-negative cases, 9 genes were commonly overexpressed, including a candidate diagnostic/therapeutic target DEPDC1, that were determined to be critical for predicting a worse prognosis. Our findings are important because they provide a molecular basis of ALK-positive lung adenocarcinomas and triple-negative lung adenocarcinomas and further stratify more or less aggressive subgroups of triple-negative lung ADC, possibly helping identify patients who may gain the most benefit from adjuvant chemotherapy after surgical resection.