Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

How portuguese and american teachers plan for literacy instruction

This study explored American and Portuguese elementary teachers' preferences in planning for literacy instruction using the Language Arts Activity Grid (LAAG; Cunningham, Zibulsky, Stanovich, & Stanovich, 2009), on which teachers described their preferred instructional activities for a hypothetical 2-h language arts block. Portuguese teachers (N = 186) completed Portuguese versions of a background questionnaire and LAAG electronically, in Survey Monkey; American teachers (N = 102) completed identical English measures using paper and pencil. Results showed that teachers in both groups usually addressed comprehension and reading fluency on their LAAGs and that they also allocated the most time to these two areas. However, American teachers were more likely to include teacher-directed fluency activities, whereas Portuguese teachers were more likely to include fluency activities that were not teacher directed. Significantly more American than Portuguese teachers addressed phonics in their planning, whereas significantly more Portuguese than American teachers addressed writing processes such as revision. Both groups of educators demonstrated large variability in planning, with many teachers omitting important components of literacy identified by researchers, for writing as well as reading. The study highlights the importance of providing teachers with comprehensive, research-based core literacy curricula as well as professional development on key components of literacy. Study findings also suggest significant relationships between orthographic transparency and teachers' instructional planning.This research was supported by a 2-year grant from the Foundation Francisco Manuel dos Santos in Portugal as well as by a Connecticut State University research grant in the U.S.A. We would like to express our sincere gratitude to these funding agencies as well as to the teachers and school districts who participated in the study and sent messages of interest about our research. In addition, warm thanks to our research assistants for their help with data collection, coding, and analysis, and to Anne Cunningham for providing us with inspiration as well as guidance in this work.info:eu-repo/semantics/publishedVersio

Wnt-reporter expression pattern in the mouse intestine during homeostasis

<p>Abstract</p> <p>Background</p> <p>The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells.</p> <p>Methods</p> <p>To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, <it>in situ </it>hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined.</p> <p>Results</p> <p>Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells.</p> <p>Conclusion</p> <p>We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.</p

Acanthaster planci Outbreak: Decline in Coral Health, Coral Size Structure Modification and Consequences for Obligate Decapod Assemblages

Although benthic motile invertebrate communities encompass the vast majority of coral reef diversity, their response to habitat modification has been poorly studied. A variety of benthic species, particularly decapods, provide benefits to their coral host enabling them to cope with environmental stressors, and as a result benefit the overall diversity of coral-associated species. However, little is known about how invertebrate assemblages associated with corals will be affected by global perturbations, (either directly or indirectly via their coral host) or their consequences for ecosystem resilience. Analysis of a ten year dataset reveals that the greatest perturbation at Moorea over this time was an outbreak of the corallivorous sea star Acanthaster planci from 2006 to 2009 impacting habitat health, availability and size structure of Pocillopora spp. populations and highlights a positive relationship between coral head size and survival. We then present the results of a mensurative study in 2009 conducted at the end of the perturbation (A. planci outbreak) describing how coral-decapod communities change with percent coral mortality for a selected coral species, Pocillopora eydouxi. The loss of coral tissue as a consequence of A. planci consumption led to an increase in rarefied total species diversity, but caused drastic modifications in community composition driven by a shift from coral obligate to non-obligate decapod species. Our study highlights that larger corals left with live tissue in 2009, formed a restricted habitat where coral obligate decapods, including mutualists, could subsist. We conclude that the size structure of Pocillopora populations at the time of an A. planci outbreak may greatly condition the magnitude of coral mortality as well as the persistence of local populations of obligate decapods

The influence of simulator input conditions on the wear of total knee replacements: an experimental and computational study

Advancements in knee replacement design, material and sterilisation processes have provided improved clinical results. However, surface wear of the polyethylene leading to osteolysis is still considered the longer-term risk factor. Experimental wear simulation is an established method for evaluating the wear performance of total joint replacements. The aim of this study was to investigate the influence of simulation input conditions, specifically input kinematic magnitudes, waveforms and directions of motion and position of the femoral centre of rotation, on the wear performance of a fixed-bearing total knee replacement through a combined experimental and computational approach. Studies were completed using conventional and moderately cross-linked polyethylene to determine whether the influence of these simulation input conditions varied with material. The position of the femoral centre of rotation and the input kinematics were shown to have a significant influence on the wear rates. Similar trends were shown for both the conventional and moderately cross-linked polyethylene materials, although lower wear rates were found for the moderately cross-linked polyethylene due to the higher level of cross-linking. The most important factor influencing the wear was the position of the relative contact point at the femoral component and tibial insert interface. This was dependent on the combination of input displacement magnitudes, waveforms, direction of motion and femoral centre of rotation. This study provides further evidence that in order to study variables such as design and material in total knee replacement, it is important to carefully control knee simulation conditions. This can be more effectively achieved through the use of displacement control simulation

Gut microbiota and diabetes: from pathogenesis to therapeutic perspective

More than several hundreds of millions of people will be diabetic and obese over the next decades in front of which the actual therapeutic approaches aim at treating the consequences rather than causes of the impaired metabolism. This strategy is not efficient and new paradigms should be found. The wide analysis of the genome cannot predict or explain more than 10–20% of the disease, whereas changes in feeding and social behavior have certainly a major impact. However, the molecular mechanisms linking environmental factors and genetic susceptibility were so far not envisioned until the recent discovery of a hidden source of genomic diversity, i.e., the metagenome. More than 3 million genes from several hundreds of species constitute our intestinal microbiome. First key experiments have demonstrated that this biome can by itself transfer metabolic disease. The mechanisms are unknown but could be involved in the modulation of energy harvesting capacity by the host as well as the low-grade inflammation and the corresponding immune response on adipose tissue plasticity, hepatic steatosis, insulin resistance and even the secondary cardiovascular events. Secreted bacterial factors reach the circulating blood, and even full bacteria from intestinal microbiota can reach tissues where inflammation is triggered. The last 5 years have demonstrated that intestinal microbiota, at its molecular level, is a causal factor early in the development of the diseases. Nonetheless, much more need to be uncovered in order to identify first, new predictive biomarkers so that preventive strategies based on pre- and probiotics, and second, new therapeutic strategies against the cause rather than the consequence of hyperglycemia and body weight gain

Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism