A mechanism for the inhibition of DNA-PK-mediated DNA sensing by a virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses

Improving the in silico assessment of pathogenicity for compensated variants

Understanding the functional sequelae of amino-acid replacements is of fundamental importance in medical genetics. Perhaps, the most intuitive way to assess the potential pathogenicity of a given human missense variant is by measuring the degree of evolutionary conservation of the substituted amino-acid residue, a feature that generally serves as a good proxy metric for the functional/structural importance of that residue. However, the presence of putatively compensated variants as the wild-type alleles in orthologous proteins of other mammalian species not only challenges this classical view of amino-acid essentiality but also precludes the accurate evaluation of the functional impact of this type of missense variant using currently available bioinformatic prediction tools. Compensated variants constitute at least 4% of all known missense variants causing human-inherited disease and hence represent an important potential source of error in that they are likely to be disproportionately misclassified as benign variants. The consequent under-reporting of compensated variants is exacerbated in the context of next-generation sequencing where their inappropriate exclusion constitutes an unfortunate natural consequence of the filtering and prioritization of the very large number of variants generated. Here we demonstrate the reduced performance of currently available pathogenicity prediction tools when applied to compensated variants and propose an alternative machine-learning approach to assess likely pathogenicity for this particular type of variant

Variability of indication criteria in knee and hip replacement: an observational study

<p>Abstract</p> <p>Background</p> <p>Total knee (TKR) and hip (THR) replacement (arthroplasty) are effective surgical procedures that relieve pain, improve patients' quality of life and increase functional capacity. Studies on variations in medical practice usually place the indications for performing these procedures to be highly variable, because surgeons appear to follow different criteria when recommending surgery in patients with different severity levels. We therefore proposed a study to evaluate inter-hospital variability in arthroplasty indication.</p> <p>Methods</p> <p>The pre-surgical condition of 1603 patients included was compared by their personal characteristics, clinical situation and self-perceived health status. Patients were asked to complete two health-related quality of life questionnaires: the generic SF-12 (Short Form) and the specific WOMAC (Western Ontario and Mcmaster Universities) scale. The type of patient undergoing primary arthroplasty was similar in the 15 different hospitals evaluated.</p> <p>The variability in baseline WOMAC score between hospitals in THR and TKR indication was described by range, mean and standard deviation (SD), mean and standard deviation weighted by the number of procedures at each hospital, high/low ratio or extremal quotient (EQ_5-95), variation coefficient (CV_5-95) and weighted variation coefficient (WCV_5-95) for 5-95 percentile range. The variability in subjective and objective signs was evaluated using median, range and WCV_5-95. The appropriateness of the procedures performed was calculated using a specific threshold proposed by Quintana et al for assessing pain and functional capacity.</p> <p>Results</p> <p>The variability expressed as WCV_5-95was very low, between 0.05 and 0.11 for all three dimensions on WOMAC scale for both types of procedure in all participating hospitals. The variability in the physical and mental SF-12 components was very low for both types of procedure (0.08 and 0.07 for hip and 0.03 and 0.07 for knee surgery patients). However, a moderate-high variability was detected in subjective-objective signs. Among all the surgeries performed, approximately a quarter of them could be considered to be inappropriate.</p> <p>Conclusions</p> <p>A greater inter-hospital variability was observed for objective than for subjective signs for both procedures, suggesting that the differences in clinical criteria followed by surgeons when indicating arthroplasty are the main responsible factors for the variation in surgery rates.</p

Immunoregulatory Mechanisms Underlying Prevention of Colitis-Associated Colorectal Cancer by Probiotic Bacteria

Background: Inflammatory bowel disease (IBD) increases the risk of colorectal cancer. Probiotic bacteria produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anticarcinogenic effects. This study aimed to investigate the cellular and molecular mechanisms underlying the efficacy of probiotic bacteria in mouse models of cancer. Methodology/Principal Findings: The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in mouse models of inflammation-driven colorectal cancer. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen and colonic lamina propria lymphocytes (LPL) were phenotypically and functionally characterized. Mice treated with CLA or VSL#3 recovered faster from the acute inflammatory phase of disease and had lower disease severity in the chronic, tumor-bearing phase of disease. Adenoma and adenocarcinoma formation was also diminished by both treatments. VSL#3 increased the mRNA expression of TNF-a, angiostatin and PPAR c whereas CLA decreased COX-2 levels. Moreover, VSL#3-treated mice had increased IL-17 expression in MLN CD4+ T cells and accumulation of Treg LPL and memory CD4+ T cells. Conclusions/Significance: Both CLA and VSL#3 suppressed colon carcinogenesis, although VSL#3 showed greater anticarcinogeni

Variation in the CXCR1 gene (IL8RA) is not associated with susceptibility to chronic periodontitis

<p>Abstract</p> <p>Background</p> <p>The chemokine receptor 1 CXCR-1 (or IL8R-alpha) is a specific receptor for the interleukin 8 (IL-8), which is chemoattractant for neutrophils and has an important role in the inflammatory response. The polymorphism rs2234671 at position Ex2+860G > C of the <it>CXCR1 </it>gene causes a conservative amino acid substitution (S276T). This single nucleotide polymorphism (SNP) seemed to be functional as it was associated with decreased lung cancer risk. Previous studies of our group found association of haplotypes in the <it>IL8 </it>and in the <it>CXCR2 </it>genes with the multifactorial disease chronic periodontitis. In this study we investigated the polymorphism rs2234671 in 395 Brazilian subjects with and without chronic periodontitis.</p> <p>Findings</p> <p>Similar distribution of the allelic and genotypic frequencies were observed between the groups (p > 0.05).</p> <p>Conclusions</p> <p>The polymorphism rs2234671 in the <it>CXCR1 </it>gene was not associated with the susceptibility to chronic periodontitis in the studied Brazilian population.</p

Probiotic Bacteria Produce Conjugated Linoleic Acid Locally in the Gut That Targets Macrophage PPAR γ to Suppress Colitis

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis. The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis. Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis

Three endo-β-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seeds

Mannans are hemicellulosic polysaccharides in the plant primary cell wall (CW). Mature seeds, specially their endosperm cells, have CWs rich in mannan-based polymers that confer a strong mechanical resistance for the radicle protrusion upon germination. The rupture of the seed coat and endosperm are two sequential events during the germination of Arabidopsis thaliana. Endo-β-mannanases (MAN; EC. 3.2.1.78) are hydrolytic enzymes that catalyze cleavage of β1 → 4 bonds in the mannan-polymer. In the genome of Arabidopsis, the endo-β-mannanase (MAN) family is represented by eight members. The expression of these eight MAN genes has been systematically explored in different organs of this plant and only four of them (AtMAN7, AtMAN6, AtMAN2 and AtMAN5) are expressed in the germinating seeds. Moreover, in situ hybridization analysis shows that their transcript accumulation is restricted to the micropylar endosperm and to the radicle and this expression disappears soon after radicle emergence. T-DNA insertion mutants in these genes (K.O. MAN7, K.O. MAN6, K.O. MAN5), except that corresponding to AtMAN2 (K.O. MAN2), germinate later than the wild type (Wt). K.O. MAN6 is the most affected in the germination time course with a t 50 almost double than that of the Wt. These data suggest that AtMAN7, AtMAN5 and specially AtMAN6 are important for the germination of A. thaliana seeds by facilitating the hydrolysis of the mannan-rich endosperm cell walls

Genes involved in ethylene and gibberellins metabolism are required for endosperm-limited germiantion of Sisymbrium officinales L. Seeds

The rupture of the seed coat and that of the endosperm were found to be two sequential events in the germination of Sisymbrium officinale L. seeds, and radicle protrusion did not occur exactly in the micropylar area but in the neighboring zone. The germination patterns were similar both in the presence of gibberellins (GA4+7) and in presence of ethrel. The analysis of genes involved in GAs synthesis and breakdown demonstrated that (1) SoGA2ox6 expression peaked just prior to radicle protrusion (20–22 h), while SoGA3ox2 and SoGA20ox2 expression was high at early imbibition (6 h) diminishing sharply thereafter; (2) the accumulation of SoGA20ox2 transcript was strongly inhibited by paclobutrazol (PB) as well as by inhibitors of ET synthesis and signaling (IESS) early after imbibition (6 h), while SoGA3ox2 and SoGA2ox6 expression was slowly depressed as germination progressed; (3) ethrel and GA4+7 positively or negatively affected expression of SoGA3ox2, SoGA20ox2, and SoGA2ox6, depending on the germination period studied. Regarding genes involved in ET synthesis, our results showed that SoACS7 was expressed, just prior to radicle emergence while SoACO2 expression slowly increased as germination progressed. Both genes were strongly inhibited by PB but were almost unaffected by externally added ethrel or GA4+7. These results suggest that GAs are more important than ET during the early stages of imbibition, while ET is more important at the late phases of germination of S. officinale L. seed

Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor