{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001718","ANALYSIS_ID":"AN002801","VERSION":"1","CREATED_ON":"March 12, 2021, 9:47 am"}, "PROJECT":{"PROJECT_TITLE":"Fecal Metabolomics","PROJECT_TYPE":"Untargeted MS of mice fecal samples","PROJECT_SUMMARY":"Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.","INSTITUTE":"Mayo Clinic","DEPARTMENT":"Biomedical Statistics and Informatics","LABORATORY":"ENSP","LAST_NAME":"Grover","FIRST_NAME":"Madhu","ADDRESS":"200 First Street SW, Rochester, MN, 55905, USA","EMAIL":"Dasari.Surendra@mayo.edu","PHONE":"507-284-0513"}, "STUDY":{"STUDY_TITLE":"Commensal intestinal microbiota regulates host luminal proteolytic activity and intestinal barrier integrity through β-glucuronidase activity","STUDY_TYPE":"Complex","STUDY_SUMMARY":"Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.","INSTITUTE":"Mayo Clinic","DEPARTMENT":"Gastroenterology","LAST_NAME":"Grover","FIRST_NAME":"Madhu","ADDRESS":"200 First Street SW, Rochester, MN","EMAIL":"dasari.surendra@mayo.edu","PHONE":"5072840513","NUM_GROUPS":"2","TOTAL_SUBJECTS":"16","NUM_MALES":"8","NUM_FEMALES":"8"}, "SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","AGE_OR_AGE_RANGE":"16-60","WEIGHT_OR_WEIGHT_RANGE":"NA","HEIGHT_OR_HEIGHT_RANGE":"NA","GENDER":"Male and female"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"12", "Sample ID":"ms5520-1", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_004-r001.d (Negative HILIC), 10jun15_004.d (Positive C18), 11apr15_004-r001.d (Positive HILIC)"} }, { "Subject ID":"10", "Sample ID":"ms5520-2", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_005-r001.d (Negative HILIC), 10jun15_005.d (Positive C18), 11apr15_005-r001.d (Positive HILIC)"} }, { "Subject ID":"8", "Sample ID":"ms5520-3", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_006-r001.d (Negative HILIC), 10jun15_006.d (Positive C18), 11apr15_006-r001.d (Positive HILIC)"} }, { "Subject ID":"5", "Sample ID":"ms5520-4", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_007-r001.d (Negative HILIC), 10jun15_007.d (Positive C18), 11apr15_007-r001.d (Positive HILIC)"} }, { "Subject ID":"3", "Sample ID":"ms5520-5", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_008-r001.d (Negative HILIC), 10jun15_008.d (Positive C18), 11apr15_008-r001.d (Positive HILIC)"} }, { "Subject ID":"15", "Sample ID":"ms5520-6", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_009-r001.d (Negative HILIC), 10jun15_009.d (Positive C18), 11apr15_009-r001.d (Positive HILIC)"} }, { "Subject ID":"1", "Sample ID":"ms5520-7", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_010-r001.d (Negative HILIC), 10jun15_010.d (Positive C18), 11apr15_010-r001.d (Positive HILIC)"} }, { "Subject ID":"6", "Sample ID":"ms5520-8", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_011-r001.d (Negative HILIC), 10jun15_011.d (Positive C18), 11apr15_011-r001.d (Positive HILIC)"} }, { "Subject ID":"2", "Sample ID":"ms5520-9", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_012-r001.d (Negative HILIC), 10jun15_012.d (Positive C18), 11apr15_012-r001.d (Positive HILIC)"} }, { "Subject ID":"13", "Sample ID":"ms5520-10", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_013-r001.d (Negative HILIC), 10jun15_013.d (Positive C18), 11apr15_013-r001.d (Positive HILIC)"} }, { "Subject ID":"4", "Sample ID":"ms5520-11", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_014-r001.d (Negative HILIC), 10jun15_014.d (Positive C18), 11apr15_014-r001.d (Positive HILIC)"} }, { "Subject ID":"7", "Sample ID":"ms5520-12", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_015-r001.d (Negative HILIC), 10jun15_015.d (Positive C18), 11apr15_015-r001.d (Positive HILIC)"} }, { "Subject ID":"16", "Sample ID":"ms5520-13", "Factors":{"Factor":"case"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_016-r001.d (Negative HILIC), 10jun15_016.d (Positive C18), 11apr15_016-r001.d (Positive HILIC)"} }, { "Subject ID":"17", "Sample ID":"ms5520-14", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_017-r001.d (Negative HILIC), 10jun15_017.d (Positive C18), 11apr15_017-r001.d (Positive HILIC)"} }, { "Subject ID":"11", "Sample ID":"ms5520-15", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_018-r001.d (Negative HILIC), 10jun15_018.d (Positive C18), 11apr15_018-r001.d (Positive HILIC)"} }, { "Subject ID":"9", "Sample ID":"ms5520-16", "Factors":{"Factor":"control"}, "Additional sample data":{"RAW_FILE_NAME":"16apr15_019-r001.d (Negative HILIC), 10jun15_019.d (Positive C18), 11apr15_019-r001.d (Positive HILIC)"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"Fecal supernatants (FSNs) were made fresh prior to each experiment. Feces from patients (0.1g) or mice (1 pellet) was added to 0.8mL of phosphate buffered saline (PBS) and subsequently homogenized with a pellet pestle for 5-10 seconds (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were spun twice at 5,000 g for 10 min at 4°C and then added to a 0.22 µm Spin-X tube filter (Corning Life Sciences, Durham, NC, USA). Samples were filtered at 4°C, 10,000 g for 5 min and FSN was stored on ice until use.","SAMPLE_TYPE":"Feces"}, "TREATMENT":{"TREATMENT_SUMMARY":"A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068)."}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification."}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"HILIC","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 6550","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.","MS_RESULTS_FILE":"ST001718_AN002801_Results.txt UNITS:intensity Has m/z:Neutral masses Has RT:Yes RT units:Seconds"} }