Project description:Geographical location influences the composition of the gut microbiota in wild house mice (Mus musculus domesticus) at a fine spatial scale
Project description:We employed RNA-sequencing to identify differentially expressed genes between two wild-derived inbred mouse strains. These strains exhibit high collateral blood vessel densities, but differ in widely in infarct volume following middle artery occlusion. This experiment helped facilitate identification of candidate loci underlying this differential response.
Project description:Accurate and reproducible quantitation of target genes depends on correct normalization. Historically, genes with variable tissue transcription e.g. GAPDH, have been used as normalization factors which is problematic, particularly in clinical samples which often are derived from different tissue sources. Using a large-scale gene database (GeneChip (Affymetrix U133A) dataset of 36 gastrointestinal tumors and normal tissues), we identified 8 candidate reference genes that were highly expressed with low variability and established expression levels by real-time RT-PCR in an independent set of GI tissue samples (n=42). Experiment Overall Design: (GeneChip (Affymetrix U133A) dataset of 36 gastrointestinal tumors and Affymetrix protocol: http://keck.med.yale.edu/affymetrix/analysis.htm
Project description:Accurate and reproducible quantitation of target genes depends on correct normalization. Historically, genes with variable tissue transcription e.g. GAPDH, have been used as normalization factors which is problematic, particularly in clinical samples which often are derived from different tissue sources. Using a large-scale gene database (GeneChip (Affymetrix U133A) dataset of 36 gastrointestinal tumors and normal tissues), we identified 8 candidate reference genes that were highly expressed with low variability and established expression levels by real-time RT-PCR in an independent set of GI tissue samples (n=42). Keywords: house-keeping, intestine, carcinoid
Project description:Comparison of mucus proteins from 6 different segments (from stomach to distal colon). 6 biological replicates, 2 MS replicates. The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the tract. We combined collection of mucus from explant tissues with FASP processing and single-shot mass spectrometry in an LTQ-Orbitrap system, to characterize the proteome of the murine mucus from stomach to distal colon. We identified ~1,300 proteins in the mucus, and found no differences in the protein composition or abundance between genders, but clear differences in the different gastrointestinal locations. Qualitatively, there was a relatively stable core proteome (~80% of the total proteins identified). Quantitatively, we found significant differences (~40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of proteins that correlated with the main intestinal mucin, Muc2 (e.g. Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system. Bioinformatics pipeline: Data was acquired in a hybrid LTQ-Orbitrap XL instrument (Thermo Scientific) in dependent mode, measuring full MS in the Orbitrap and selecting the 8 most abundant multiply charged ions for collision (CID, 30% normalized collision energy) and acquisition in the LTQ. Full MS scans were performed in the m/z 350-2,000 range, with internal calibration by lock mass (m/z 371.1012, siloxane), and resolution of 60,000. MS/MS scans were set at a target value of 100,000, with isolation width of 3 amu. The raw files obtained were analyzed in the MaxQuant 1.2.2.5 environment. Data were searched with the Andromeda search engine integrated in MaxQuant against an in-house database containing all the mucin sequences available (http://www.medkem.gu.se/mucinbiology/databases/), the UniProt-SwissProt mouse database (version 1203, reviewed sequences), and the standard MaxQuant contaminant database. Oxidation of methionines and acetylation of the protein N-terminus were set as variable modifications, and carbamidomethylation of cysteines as fixed; enzyme cleavage rules were defined for trypsin/P, with a maximum of 2 missed cleavages. Tolerances were limited as maximum 5 modifications per peptide, 20 ppm error for the first search and 6 ppm for the main search. Isoleucine and leucine were considered indistinguishable. The false discovery rate was calculated from searches against a reversed database, and set to 0.01 for proteins, peptides and modified sites. The identification rate was improved by matching between runs through remapped retention time (window of 2 min). The resulting data were loaded as protein groups into Perseus 1.3.0.4. Protein quantities were calculated as intensity values (from the extracted ion current: XIC) and normalized in ppm to the total XIC for the standard labeled peptides (AQUA) in each run.
Project description:Exposure to dogs in early infancy has been shown to reduce the risk of childhood allergic disease development and dog-ownership is associated with a distinct house dust microbial exposure. Here we demonstrate, using murine models, that exposure of mice to dog-associated house dust protects against ovalbumin or cockroach allergen mediated airway pathology. Protected animals exhibited significant reductions in the total number of airway T cells, down-regulation of Th2-related airway responses as well as mucin secretion. Following house dust exposure, the cecal microbiome of protected animals was extensively restructured with significant enrichment of, amongst others, Lactobacillus johnsonii. Supplementation of wild type animals with L. johnsonii protected them against both airway allergen challenge or infection with respiratory syncytial virus. L. johnsonii mediated protection was associated with significant reductions in the total number and proportion of activated CD11c+/CD11b+ and CD11c+/CD8+ cells, as well as significantly reduced airway Th2 cytokine expression. Our results reveal that exposure to dog-associated household dust results in protection against airway allergen challenge and a distinct GI microbiome composition. Moreover the study identifies L. johnsonii as a pivotal species within the gastrointestinal tract capable of influencing adaptive immunity at remote mucosal surfaces in a manner that is protective against a variety of respiratory insults.