Pruritus Ani

Pruritus ani is an unpleasant cutaneous sensation that induces the desire to scratch the skin around the anal orifice. It may start insidiously and appears in 1% to 5% of the population. It is classified as primary (idiopathic) pruritus ani when no cause can be found. However, as 25% to 75% of cases have co-existing pathology, a detailed history and examination are necessary. The goal of treatment is asymptomatic, intact, dry, clean perianal skin with reversal of morphological changes. The management of pruritus ani is directed towards the underlying cause. If the diagnosis is idiopathic pruritus ani, the patients can still be managed with great success by eliminating of irritants and scratching, by giving general advice regarding hygiene and lifestyle modification and by using active treatment measures

Optimizing structural modeling for a specific protein scaffold: knottins or inhibitor cystine knots

<p>Abstract</p> <p>Background</p> <p>Knottins are small, diverse and stable proteins with important drug design potential. They can be classified in 30 families which cover a wide range of sequences (1621 sequenced), three-dimensional structures (155 solved) and functions (> 10). Inter knottin similarity lies mainly between 15% and 40% sequence identity and 1.5 to 4.5 Å backbone deviations although they all share a tightly knotted disulfide core. This important variability is likely to arise from the highly diverse loops which connect the successive knotted cysteines. The prediction of structural models for all knottin sequences would open new directions for the analysis of interaction sites and to provide a better understanding of the structural and functional organization of proteins sharing this scaffold.</p> <p>Results</p> <p>We have designed an automated modeling procedure for predicting the three-dimensionnal structure of knottins. The different steps of the homology modeling pipeline were carefully optimized relatively to a test set of knottins with known structures: template selection and alignment, extraction of structural constraints and model building, model evaluation and refinement. After optimization, the accuracy of predicted models was shown to lie between 1.50 and 1.96 Å from native structures at 50% and 10% maximum sequence identity levels, respectively. These average model deviations represent an improvement varying between 0.74 and 1.17 Å over a basic homology modeling derived from a unique template. A database of 1621 structural models for all known knottin sequences was generated and is freely accessible from our web server at <url>http://knottin.cbs.cnrs.fr</url>. Models can also be interactively constructed from any knottin sequence using the structure prediction module Knoter1D3D available from our protein analysis toolkit PAT at <url>http://pat.cbs.cnrs.fr</url>.</p> <p>Conclusions</p> <p>This work explores different directions for a systematic homology modeling of a diverse family of protein sequences. In particular, we have shown that the accuracy of the models constructed at a low level of sequence identity can be improved by 1) a careful optimization of the modeling procedure, 2) the combination of multiple structural templates and 3) the use of conserved structural features as modeling restraints.</p

Characterization of bacterial populations of 2,4,6-trinitrotoluene (TNT) contaminated soils and isolation of a Pseudomonas aeruginosa strain with TNT denitration activities

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. These differences in hybridizations between the two soil samples were likely the result from the presence of TNT. The effect of TNT on soil microbial communities was further investigated with additional uncontaminated and TNT-contaminated soil samples using 16S rRNA PCR-DGGE and cultivation-dependent techniques. In all contaminated soil samples, the amount of DNA extracted was lower than in the uncontaminated ones. Analysis of bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soil samples compared to the uncontaminated ones. Caulobacteraceae were also present in several contaminated soil samples. The culturable microflora of these soils was studied by plate counts on agar supplemented with dilute nutrient broth. The number of CFUs was lower in a TNT-contaminated soil inoculum than in an uncontaminated one. In the former, most of the CFUs belonged to Pseudomonadaceae, and to a lesser extent, to Caulobacteraceae. In addition to the above contaminated soil samples, a pristine soil was artificially contaminated with different concentrations of TNT and incubated for 4 months.The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was omitted and nitrite and ferrihydrite were provided. These results suggest that nitrite-reducing conditions were initially achieved, followed by iron-reducing conditions. When grown aerobically on a chemically defined medium, P. aeruginosa strain ESA-5 produced a greenish extracellular compound. This product was identified as phenazine-1-carboxylic acid (PCA). When purified PCA was incubated with TNT in the presence of NADH, nitrite was released. The concentration of nitrite released was dependent on the concentration of NADH and PCA. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitroaromatic parent molecule. The results obtained with the PCA mediated denitration of TNT in the presence of inhibitors of oxygen reactive species suggested the involvement of superoxide (O2.-). When exogenous PCA was added to a P. aeruginosa ESA-5 liquid culture containing TNT as sole nitrogen source, bacterial growth was significantly enhanced compared to cultures containing TNT without PCA.(AGRO 3)--UCL, 200

Denitration of 2,4,6-trinitrotoluene by Pseudomonas aeruginosa ESA-5 in the presence of ferrihydrite.

Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5

Effect of 2,4,6-trinitrotoluene on soil microbial communities

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Effect of 2,4,6-trinitrotoluene on soil bacterial communities.

To gain insight into the impact of 2,4,6-trinitrotoluene (TNT) on soil microbial communities, we characterized the bacterial community of several TNT-contaminated soils from two sites with different histories of contamination and concentrations of TNT. The amount of extracted DNA, the total cell counts and the number of CFU were lower in the TNT-contaminated soils. Analysis of soil bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soils, as well as the presence of Caulobacteraceae. CFU from TNT-contaminated soils were identified as Pseudomonadaceae, and, to a lesser extent, Caulobacteraceae. Finally, a pristine soil was spiked with different concentrations of TNT and the soil microcosms were incubated for 4 months. The amount of extracted DNA decreased in the microcosms with a high TNT concentration [1.4 and 28.5 g TNT/kg (dry wt) of soil] over the incubation period. After 7 days of incubation of these soil microcosms, there was already a clear shift of their original flora towards a community dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. These results indicate that TNT affects soil bacterial diversity by selecting a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae

High-throughput approaches to analyse waste biotreatment in confined environments

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