Global transcription of CRISPR loci in the human oral cavity

© 2015 Lum et al.; licensee BioMed Central. Background: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are active in acquired resistance against bacteriophage and plasmids in a number of environments. In the human mouth, CRISPR loci evolve to counteract oral phage, but the expression of these CRISPR loci has not previously been investigated. We sequenced cDNA from CRISPR loci found in numerous different oral bacteria and compared with oral phage communities to determine whether the transcription of CRISPR loci is specifically targeted towards highly abundant phage present in the oral environment. Results: We found that of the 529,027 CRISPR spacer groups studied, 88 % could be identified in transcripts, indicating that the vast majority of CRISPR loci in the oral cavity were transcribed. There were no strong associations between CRISPR spacer repertoires and oral health status or nucleic acid type. We also compared CRISPR repertoires with oral bacteriophage communities, and found that there was no significant association between CRISPR transcripts and oral phage, regardless of the CRISPR type being evaluated. We characterized highly expressed CRISPR spacers and found that they were no more likely than other spacers to match oral phage. By reassembling the CRISPR-bearing reads into longer CRISPR loci, we found that the majority of the loci did not have spacers matching viruses found in the oral cavities of the subjects studied. For some CRISPR types, loci containing spacers matching oral phage were significantly more likely to have multiple spacers rather than a single spacer matching oral phage. Conclusions: These data suggest that the transcription of oral CRISPR loci is relatively ubiquitous and that highly expressed CRISPR spacers do not necessarily target the most abundant oral phage

Oral chondroitin sulfate and prebiotics for the treatment of canine Inflammatory Bowel Disease: a randomized, controlled clinical trial

BACKGROUND Canine inflammatory bowel disease (IBD) is a chronic enteropathy of unknown etiology, although microbiome dysbiosis, genetic susceptibility, and dietary and/or environmental factors are hypothesized to be involved in its pathogenesis. Since some of the current therapies are associated with severe side effects, novel therapeutic modalities are needed. A new oral supplement for long-term management of canine IBD containing chondroitin sulfate (CS) and prebiotics (resistant starch, β-glucans and mannaoligosaccharides) was developed to target intestinal inflammation and oxidative stress, and restore normobiosis, without exhibiting any side effects. This double-blinded, randomized, placebo-controlled trial in dogs with IBD aims to evaluate the effects of 180 days administration of this supplement together with a hydrolyzed diet on clinical signs, intestinal histology, gut microbiota, and serum biomarkers of inflammation and oxidative stress. RESULTS Twenty-seven client-owned biopsy-confirmed IBD dogs were included in the study, switched to the same hydrolyzed diet and classified into one of two groups: supplement and placebo. Initially, there were no significant differences between groups (p > 0.05) for any of the studied parameters. Final data analysis (supplement: n = 9; placebo: n = 10) showed a significant decrease in canine IBD activity index (CIBDAI) score in both groups after treatment (p < 0.001). After treatment, a significant decrease (1.53-fold; p < 0.01) in histologic score was seen only in the supplement group. When groups were compared, the supplement group showed significantly higher serum cholesterol (p < 0.05) and paraoxonase-1 (PON1) levels after 60 days of treatment (p < 0.01), and the placebo group showed significantly reduced serum total antioxidant capacity (TAC) levels after 120 days (p < 0.05). No significant differences were found between groups at any time point for CIBDAI, WSAVA histologic score and fecal microbiota evaluated by PCR-restriction fragment length polymorphism (PCR-RFLP). No side effects were reported in any group. CONCLUSIONS The combined administration of the supplement with hydrolyzed diet over 180 days was safe and induced improvements in selected serum biomarkers, possibly suggesting a reduction in disease activity. This study was likely underpowered, therefore larger studies are warranted in order to demonstrate a supplemental effect to dietary treatment of this supplement on intestinal histology and CIBDAI

Evaluation of Bovine Feces-Associated Microbial Source Tracking Markers and Their Correlations with Fecal Indicators and Zoonotic Pathogens in a Brisbane, Australia, Reservoir

This study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral {left open bracket}bovine adenovirus (B-AVs){right open bracket} microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB), Campylobacter spp., Escherichia coli O157, and Salmonella spp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for the Campylobacter 16S rRNA and E. coli O157 rfbE genes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens

Organic pollutants in sea-surface microlayer and aerosol in thecoastal environment of Leghorn—(Tyrrhenian Sea)

The levels of dissolved and particle-associated n-alkanes, alkylbenzenes, phthalates, PAHs, anionic surfactants and surfactant fluorescent organic matter ŽSFOM. were measured in sea-surface microlayer ŽSML. and sub-surface water ŽSSL. samples collected in the Leghorn marine environment in September and October 1999. Nine stations, located in the Leghorn harbour and at increasing distances from the Port, were sampled three times on the same day. At all the stations, SML concentrations of the selected organic compounds were significantly higher than SSL values and the enrichment factors ŽEFsSML concentrationrSSL concentration. were greater in the particulate phase than in the dissolved phase. SML concentrations varied greatly among the sampling sites, the highest levels Žn-alkanes 3674 mgrl, phthalates 177 mgrl, total PAHs 226 mgrl. being found in the particulate phase in the Leghorn harbour. To improve the knowledge on pollutant exchanges between sea-surface waters and atmosphere, the validity of spray drop adsorption model ŽSDAM. was verified for SFOM, surface-active agents, such as phthalates, and compounds which can interact with SFOM, such as n-alkanes and PAHs. q2001 Elsevier Science B.V. All rights reserved

Malignant germ cell tumours of the testis express interferon-γ, but are resistant to endogenous interferon-γ

Cytokines possess discrepant effects on tumour cells varying from anti- to proapoptotic activities. We recently reported that testicular germ cell tumours (TGCT) express a functional form of the proinflammatory cytokine interferon-gamma (IFNgamma). The present study asked whether TGCT-derived IFNgamma influences survival or death of neoplastic germ cells. Analysis of TGCT cell lines demonstrated that they expressed and secreted IFNgamma, but were resistant to the endogenous IFNgamma since neutralisation of IFNgamma by a specific blocking antibody had no influence on the proliferation and/or the degree of apoptosis of tumour cells. To study mechanisms providing tumour resistance to endogenous IFNgamma, we analysed primary TGCT and two human TGCT cell lines (NTERA and NCCIT) for the expression of IFNgamma receptor and for the level of phosphorylation of the signal transducer and activator of transcription ( STAT)-1. In situ hybridisation, immunocytochemistry, Western blot analysis and flow cytometry indicated that primary TGCT as well as NCCIT and NTERA cell lines expressed the heterodimeric cell surface IFNg receptor which consists of both 90-kDa alpha- and the 85-kDa beta-chains. However, the downstream transcription factor STAT-1 was not phosphorylated constitutively, indicating that STAT-1 is not activated by the endogenous IFNgamma. Upon application of recombinant human IFNgamma in excess, however, STAT-1 was phosphorylated and the interferon regulatory factor-1 (IRF-1) was induced, suggesting that both IFNgammaR and STAT-1 are functionally intact in TGCT. Altogether our results suggest that despite secreting biologically active IFNgamma, the concentration of the endogenous IFNgamma is too low to stimulate the IFNgammaR/STAT signalling pathway in TGCT in an autocrine and/or paracrine manner

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

<p>Abstract</p> <p>Background</p> <p>Invariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity <it>in vivo </it>has so far been reported.</p> <p>Results</p> <p>We used an interferon (IFN)-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice.</p> <p>Conclusions</p> <p>This is the first report that supplies direct evidence for explicit activation events of NKT cells <it>in vivo </it>and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.</p