Evaluation of versions of electric power grid repair schemes from the results of structural reliability calculations

A procedure for processing repair claims using the results of an estimate of the structural reliability of the electric grid, the results of the approval of algorithms using the example of a test model, and the principles for producing short-term charts of the repairs of electric-grid equipment, is presented. © 2013 Springer Science+Business Media New York

Metagenomic profiling of viral and microbial communities from the pox lesions of lumpy skin disease virus and sheeppox virus-infected hosts

IntroductionIt has been recognized that capripoxvirus infections have a strong cutaneous tropism with the manifestation of skin lesions in the form of nodules and scabs in the respective hosts, followed by necrosis and sloughing off. Considering that the skin microbiota is a complex community of commensal bacteria, fungi and viruses that are influenced by infections leading to pathological states, there is no evidence on how the skin microbiome is affected during capripoxvirus pathogenesis.MethodsIn this study, shotgun metagenomic sequencing was used to investigate the microbiome in pox lesions from hosts infected with lumpy skin disease virus and sheep pox virus.ResultsThe analysis revealed a high degree of variability in bacterial community structures across affected skin samples, indicating the importance of specific commensal microorganisms colonizing individual hosts. The most common and abundant bacteria found in scab samples were Fusobacterium necrophorum, Streptococcus dysgalactiae, Helcococcus ovis and Trueperella pyogenes, irrespective of host. Bacterial reads belonging to the genera Moraxella, Mannheimia, Corynebacterium, Staphylococcus and Micrococcus were identified.DiscussionThis study is the first to investigate capripox virus-associated changes in the skin microbiome using whole-genome metagenomic profiling. The findings will provide a basis for further investigation into capripoxvirus pathogenesis. In addition, this study highlights the challenge of selecting an optimal bioinformatics approach for the analysis of metagenomic data in clinical and veterinary practice. For example, direct classification of reads using a kmer-based algorithm resulted in a significant number of systematic false positives, which may be attributed to the peculiarities of the algorithm and database selection. On the contrary, the process of de novo assembly requires a large number of target reads from the symbiotic microbial community. In this work, the obtained sequencing data were processed by three different approaches, including direct classification of reads based on k-mers, mapping of reads to a marker gene database, and de novo assembly and binning of metagenomic contigs. The advantages and disadvantages of these techniques and their practicality in veterinary settings are discussed in relation to the results obtained

Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood

Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

BACKGROUND: Recently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS) via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. METHODS: Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis, respectively. Activation of nuclear factor (NF)-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP) kinase and interferon regulatory factor (IRF)-3 was detected by immunoblot analysis. RESULTS: Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. CONCLUSION: Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3

Baicalin Downregulates Porphyromonas gingivalis Lipopolysaccharide-Upregulated IL-6 and IL-8 Expression in Human Oral Keratinocytes by Negative Regulation of TLR Signaling

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LPS Regulates SOCS2 Transcription in a Type I Interferon Dependent Autocrine-Paracrine Loop

Recent studies suggest that SOCS2 is involved in the regulation of TLR signaling. In this study, we found that the expression of SOCS2 is regulated in human monocyte-derived DC by ligands stimulating TLR2, 3, 4, 5, 8 and 9 signaling. SOCS2 induction by LPS was dependent on the type I IFN regulated transcription factors IRF1 and IRF3 as shown by using silencing RNAs for IRFs. Blocking endogenous type I IFN signaling, by neutralizing antibodies to the receptor IFNAR2, abolished SOCS2 mRNA expression after TLR4 stimulation. Transcription factors STAT3, 5 and 6 displayed putative binding sites in the promoter regions of the human SOCS2 gene. Subsequent silencing experiments further supported that STAT3 and STAT5 are involved in LPS induced SOCS2 regulation. In mice we show that SOCS2 mRNA induction is 45% lower in bone marrow derived macrophages derived from MyD88−/− mice, and do not increase in BMMs from IRF3−/− mice after BCG infection. In conclusion, our results suggest that TLR4 signaling indirectly increases SOCS2 in late phase mainly via the production of endogenous type I IFN, and that subsequent IFN receptor signaling activates SOCS2 via STAT3 and STAT5

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.</p

Type I Interferon Induction Is Detrimental during Infection with the Whipple's Disease Bacterium, Tropheryma whipplei

Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple's disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Toll/IL-1R resistance (TIR) domain–containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow–derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF–TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF–TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF–TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis