{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002737","ANALYSIS_ID":"AN004439","VERSION":"1","CREATED_ON":"June 16, 2023, 8:24 am"},

"PROJECT":{"PROJECT_TITLE":"2’-fucosyllactose modulates gut microbial metabolism for the prevention of colitis","PROJECT_TYPE":"Untargeted Metabolomics analysis","PROJECT_SUMMARY":"Human milk-derived 2’-fucosyllactose (2’-FL) consumption is associated with health benefits in infancy that extend into adulthood. However, the exact biological functions of 2’-FL and corresponding mechanisms of action remain largely unknown. Here, we investigated the impact of 2’-FL on gut microbial metabolism for the prevention of colitis in adulthood. The gut microbiota from adult mice treated with 2’-FL showed an increase in abundance of several health-associated genera, including Bifidobacterium, and exhibited preventive effects on colitis. Microbial metabolic analysis demonstrated that 26 pathways that are significantly different between non-inflammatory bowel disease individuals and patients with ulcerative colitis (UC) are significantly regulated by 2’-FL in mice, indicating that 2’-FL has the potential to directly regulate dysregulated microbial metabolism in UC. Exploratory metabolomics of Bifidobacterium infantis identified novel secreted metabolites significantly enriched by 2’-FL consumption, including pantothenol. Remarkably, pantothenate significantly protects mucosal barrier and mitigates colitis in adult mice. Thus 2’-FL-modulated gut microbial metabolism may contribute to the prevention of intestinal inflammation in adulthood.","INSTITUTE":"Vanderbilt University","DEPARTMENT":"Chemistry","LABORATORY":"Center for Innovative Technology","LAST_NAME":"CODREANU","FIRST_NAME":"SIMONA","ADDRESS":"1234 STEVENSON CENTER LANE","EMAIL":"SIMONA.CODREANU@VANDERBILT.EDU","PHONE":"6158758422"},

"STUDY":{"STUDY_TITLE":"2’-fucosyllactose prevents colitis","STUDY_TYPE":"untargeted metabolomics analysis","STUDY_SUMMARY":"Human milk-derived 2’-fucosyllactose (2’-FL) consumption is associated with health benefits in infancy that extend into adulthood. However, the exact biological functions of 2’-FL and corresponding mechanisms of action remain largely unknown. Here, we investigated the impact of 2’-FL on gut microbial metabolism for the prevention of colitis in adulthood. The gut microbiota from adult mice treated with 2’-FL showed an increase in abundance of several health-associated genera, including Bifidobacterium, and exhibited preventive effects on colitis. Microbial metabolic analysis demonstrated that 26 pathways that are significantly different between non-inflammatory bowel disease individuals and patients with ulcerative colitis (UC) are significantly regulated by 2’-FL in mice, indicating that 2’-FL has the potential to directly regulate dysregulated microbial metabolism in UC. Exploratory metabolomics of Bifidobacterium infantis identified novel secreted metabolites significantly enriched by 2’-FL consumption, including pantothenol. Remarkably, pantothenate significantly protects mucosal barrier and mitigates colitis in adult mice. Thus 2’-FL-modulated gut microbial metabolism may contribute to the prevention of intestinal inflammation in adulthood.","INSTITUTE":"Vanderbilt University","DEPARTMENT":"Chemistry","LABORATORY":"Center for Innovative Technology","LAST_NAME":"CODREANU","FIRST_NAME":"SIMONA","ADDRESS":"1234 STEVENSON CENTER LANE","EMAIL":"SIMONA.CODREANU@VANDERBILT.EDU","PHONE":"6158758422","NUM_GROUPS":"2","TOTAL_SUBJECTS":"10"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Bifidobacterium longum subspecies infantis (B. infantis)","GENOTYPE_STRAIN":"ATCC 15702 and ATCC 15697","CELL_BIOSOURCE_OR_SUPPLIER":"ATCC"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Control-1",
"Sample ID":"C_1",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_C01"}
},
{
"Subject ID":"Control-2",
"Sample ID":"C_2",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_C02"}
},
{
"Subject ID":"Control-3",
"Sample ID":"C_3",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_C03"}
},
{
"Subject ID":"Control-4",
"Sample ID":"C_4",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_C04"}
},
{
"Subject ID":"Control-5",
"Sample ID":"C_5",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_C05"}
},
{
"Subject ID":"2FL-1",
"Sample ID":"F_1",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_F01"}
},
{
"Subject ID":"2FL-2",
"Sample ID":"F_2",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_F02"}
},
{
"Subject ID":"2FL-3",
"Sample ID":"F_3",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_F03"}
},
{
"Subject ID":"2FL-4",
"Sample ID":"F_4",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_F04"}
},
{
"Subject ID":"2FL-5",
"Sample ID":"F_5",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_F05"}
},
{
"Subject ID":"Media only",
"Sample ID":"MC_01",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MC01"}
},
{
"Subject ID":"Media only",
"Sample ID":"MC_02",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MC02"}
},
{
"Subject ID":"Media only",
"Sample ID":"MC_03",
"Factors":{"treatment":"0"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MC03"}
},
{
"Subject ID":"Media_2FL",
"Sample ID":"MF_01",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MF01"}
},
{
"Subject ID":"Media_2FL",
"Sample ID":"MF_02",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MF02"}
},
{
"Subject ID":"Media_2FL",
"Sample ID":"MF_03",
"Factors":{"treatment":"2'FL"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20211207_RPLCp_FMS_Yan_MF03"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"B. infantis 15702 were inoculated in RCM at 37C with or without supplementation with 10 mg/mL of 2’-FL (1.0% w/v) until OD600≈1. Bacteria were pelleted and supernatants were filtered (0.2 micron) and stored at -80ºC for untargeted metabolomics. Cultured RCM medium and 10 mg/mL 2’-FL supplemented RCM medium without bacteria inoculation served as controls. Experiments were repeated 2-3 times for collecting five biological replicates from each group.","SAMPLE_TYPE":"Bacterial cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"B. infantis 15702 were inoculated in RCM at 37C with or without supplementation with 10 mg/mL of 2’-FL (1.0% w/v) until OD600≈1. Bacteria were pelleted and supernatants were filtered (0.2 micron) and stored at -80ºC for untargeted metabolomics. Cultured RCM medium and 10 mg/mL 2’-FL supplemented RCM medium without bacteria inoculation served as controls. Experiments were repeated 2-3 times for collecting five biological replicates from each group.","TREATMENT":"2’-fucosyllactose","TREATMENT_DOSE":"10mg/mL"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Bifidobacteria infantis were cultured in Reinforced Clostridial Medium (RCM) with or without 2’-fucosyllactose (2’-FL), a human milk oligosaccharide. Bacteria were pelleted by centrifugation and supernatants were then collected, snap frozen, and stored at -80°C until analyzed via Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS and LC-HRMS/MS)-based metabolomics in the Vanderbilt Center for Innovative Technology using previously described methods. Briefly, equal volumes (200µL) of previously frozen culture medium were prepared. Isotopically labeled standards, biotin-D2 and phenylalanine-D8, were added to individual samples to assess the sample preparation steps. Samples were subjected to protein precipitation by addition of 800µL of ice-cold methanol, then incubated at -80C overnight. Following protein precipitation, samples were centrifuged at 10,000 rpm for 10 min to remove insoluble material. Supernatant(s) were transferred and dried in vacuo, and stored at -80C prior to MS characterization.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACTION_METHOD":"Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum.","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"For the RPLC analysis metabolite extracts (5μL injection volume) were separated on a Hypersil Gold, 1.9 µm, 2.1mm x 100 mm column (Thermo Fisher) held at 40°C. Liquid chromatography was performed at a 250μL min-1 using solvent A (0.1% formic acid (FA) in water) and solvent B (0.1% FA in acetonitrile:water 80:20) over a 30 min gradient. Mass spectrometry analyses were performed in positive ion mode with the parameters as previously published except for the following changes. First, tandem mass spectra were acquired using a data dependent scanning mode in which one full MS scan (m/z 70-1050) was followed by 2 or 10 MS/MS scans. MS/MS scans are acquired in profile mode using an isolation width of 1.3 m/z, stepped collision energy (NCE 20, 40), and a dynamic exclusion of 6 s.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Vanquish UHPLC binary system","COLUMN_NAME":"Thermo Hypersil Gold (100 x 2.1mm, 1.9um)","SOLVENT_A":"100% water, 0.1% Formic Acid","SOLVENT_B":"80:20 acetonitrile:water, 0.1% Formic Acid","FLOW_GRADIENT":"30 min","FLOW_RATE":"0.25 mL/min","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The acquired RPLC-HRMS raw data from five (5) biological replicates from each sample type were imported, processed, normalized and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS sample runs were aligned against a pooled QC reference run. Unique ions (retention time and m/z pairs) were de-adducted and de-isotoped to generate unique “features” (or retention time and m/z pairs). Data were normalized to all features and cleaned by removing spectral features >25% CV in the pooled QC samples. Sample process and instrument variability were also assessed to determine sample acceptance. Briefly, QA metrics for sample process variability and instrument variability are ≤10% CV and ≤5% CV, respectively. In these studies, no samples were identified as outliers. Statistical analyses were performed in Progensis QI using variance stabilized measurements achieved through log normalization to calculate p-values by one-way analysis of variance (ANOVA) test. Significantly changed metabolites were chosen with the criteria p-value <0.05 and |FC| > 2.","MS_RESULTS_FILE":"ST002737_AN004439_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes"}

}