Project description:Longitudinal analysis of Salmonella typhimurium mRNA from superspeader mouse cecal content and stool compared to in vitro Salmonella typhimurium mRNA.
Project description:With the technological advances of the last decade, it is now feasible to analyze environmental samples of vast complexity, such as human stool specimen, using meta-omics techniques like metaproteomics. Still the most sophisticated, sensitive instruments can only extract information that a sample contains in the first place. This highlights the need for initial sample preparation to preserve as much unaltered information as possible. Yet little is known about the effects different processing approaches have on the final analysis results. This study analyzes human stool samples applying metaproteomics and shows that the initial sample storage has a massive effect on the taxonomic composition of proteins identified. The findings are backed up by the results of the metagenomics analysis of the same samples. This suggests, that great care should be taken in choosing storage conditions for (omics) studies, as well as in comparing the results of experiments with different initial processing.
Project description:Gut microbiome research is rapidly moving towards the functional characterization of the microbiota by means of shotgun meta-omics. Here, we selected a cohort of healthy subjects from an indigenous and monitored Sardinian population to analyze their gut microbiota using both shotgun metagenomics and shotgun metaproteomics. We found a considerable divergence between genetic potential and functional activity of the human healthy gut microbiota, in spite of a quite comparable taxonomic structure revealed by the two approaches. Investigation of inter-individual variability of taxonomic features revealed Bacteroides and Akkermansia as remarkably conserved and variable in abundance within the population, respectively. Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the functional activity with the higher expression rate and the lower inter-individual variability in the study cohort, highlighting the key importance of the biosynthesis of this microbial by-product for the gut homeostasis. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several gut microbiota members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis and short-chain fatty acid production). In conclusion, our results provide useful indications regarding the main functions actively exerted by the gut microbiota members of a healthy human cohort, and support metaproteomics as a valuable approach to investigate the functional role of the gut microbiota in health and disease.
Project description:Cardioviruses are a genus of picornaviruses that cause severe illnesses in rodents, but little is known about the prevalence, diversity, or spectrum of disease of such agents among humans. We report the identification of a group of human cardioviruses that have been detected and cloned directly from patient specimens (Chiu and DeRisi, et al, PNAS, 2008). This series includes 9 arrays (both raw and normalized data) used to detect cardioviruses in human respiratory and stool specimens. The arrays employed here are capable of pan-viral detection (Wang and DeRisi, et al., PNAS, 2002). Keywords: viral detection, cardiovirus, TMEV, gastroenteritis The series includes 3 arrays from respiratory samples and 6 arrays from stool samples. Among the 3 arrays from respiratory sample, 1 array has a signature for an adenovirus, 1 array has a signature for human metapneumovirus, and 1 array has a signature for cardiovirus UC1 (see Chiu and DeRisi, et al., PNAS, in 2008). All 6 arrays from stool samples are cardiovirus-positive; some show evidence of dual infection with other gastroenteritis viruses (i.e. norovirus, rotavirus, etc.). Data in Sample records fed to E-Predict (Urisman, et al, Genome Biology, 2005) E-Predict normalization metrics Array Normalization: Sum E-Matrix Normalization: Quadratic Distance Metric: Pearson Uncentered
Project description:Understanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from the early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. cholerae colonization factor, was significantly more highly expressed in early compared with late infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators, toxRS and tcpPH, was uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines. The V. cholerae microarray consists of 3,890 full-length PCR products representing the annotated open reading frames from the initial release of the V. cholerae N16961 genome. Each labeling and hybridization was performed in duplicate. Genomic DNA was used as a universal internal control for the quality of the microarray and to allow for the comparison of results across multiple experiments. Data were normalized using locally-weighted regression (Lowess) to obtain the relative abundance of each transcript as an intensity ratio with respect to that of genomic DNA. High correlation coefficients were observed between technical replicates (Pearsonâs correlation coefficient (r) > 0.80) and between results of separate clinical specimens of vomitus (r > 0.77) and of stool (r > 0.80). Hence, the results from the two clinical vomitus specimens and the five clinical stool specimens were pooled. Fold changes for the relative expression of a given gene between the two clinical specimens were calculated by dividing the normalized median intensity ratios with respect to genomic DNA.