{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001143","ANALYSIS_ID":"AN001882","VERSION":"1","CREATED_ON":"March 4, 2019, 8:33 pm"},

"PROJECT":{"PROJECT_TITLE":"Microbial depletion and ozone exposure","PROJECT_SUMMARY":"Ozone is an asthma trigger. In mice, the gut microbiome contributes to ozone-induced airway hyperresponsiveness, a defining feature of asthma. The purpose of this study was to identify metabolites that could be mediating this role. Gut bacterial enzymes modify ingested substances producing metabolites that enter the blood and circulate to host tissues where they may exert a variety of effects. Therefore, we performed global metabolomic profiling on serum of mice after acute ozone exposure. To identify the role of the microbiome in mediating ozone-induced metabolomic changes, mice were treated for two weeks with a cocktail of antibiotics in the drinking water or with control water and then exposed to air or ozone (2 ppm for 3 hours). Twenty four hours later, blood was harvested and serum analyzed via liquid-chromatography or gas-chromatography coupled to mass spectrometry. We observed marked effects of both ozone exposure and antibiotics on the serum metabolome. Known bacterially-derived metabolites were reduced in antibiotic-treated mice. Importantly, our data also indicated that ozone-induced changes in serum lipids, including long chain fatty acids and bile acids, as well as ozone-induced changes in polyamines were different in control and antibiotic-treated mice. Each of these metabolites has the capacity to alter airway responsiveness and may account for the role of the microbiome in pulmonary responses to ozone.","INSTITUTE":"Harvard T.H. Chan School of Public Health","DEPARTMENT":"Molecular and Integrative Physiological Sciences","LAST_NAME":"Shore","FIRST_NAME":"Stephanie","ADDRESS":"677 Huntington Ave","EMAIL":"sshore@hsph.harvard.edu","PHONE":"6174320989"},

"STUDY":{"STUDY_TITLE":"Microbial depletion and ozone exposure - Lung tissue (part I)","STUDY_SUMMARY":"Global biochemical profiles were determined in lung tissue collected from untreated control mice and mice treated for two weeks with untreated drinking water or water containing an antibiotic cocktail (ampicillin, neomycin, metronidazole, and vancomycin), followed by a three hour exposure to ambient air or ozone (2ppm). Sample collection occurred 24 hours post-ozone exposure.","INSTITUTE":"Harvard T.H. Chan School of Public Health","LAST_NAME":"Shore","FIRST_NAME":"Stephanie","ADDRESS":"677 Huntington Ave","EMAIL":"sshore@hsph.harvard.edu","PHONE":"6174320989"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL/6J","AGE_OR_AGE_RANGE":"8 weeks","GENDER":"Male","ANIMAL_ANIMAL_SUPPLIER":"Taconic Farms (New York)"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Mouse 1",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 2",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 3",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 4",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 5",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 6",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 7",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 8",
"Factors":{"Treament":"Water","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 9",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 10",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 11",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 12",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 13",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 14",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 15",
"Factors":{"Treament":"Antibiotics","Exposure":"Air"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 16",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 17",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 18",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 19",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 20",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 21",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 22",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 23",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 24",
"Factors":{"Treament":"Water","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 25",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 26",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 27",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 28",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 29",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 30",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 31",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
},
{
"Subject ID":"-",
"Sample ID":"Mouse 32",
"Factors":{"Treament":"Antibiotics","Exposure":"Ozone"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Global biochemical profiles were determined in lung tissue and serum collected from untreated control mice and mice treated for two weeks with untreated drinking water or water containing an antibiotic cocktail (ampicillin, neomycin, metronidazole, and vancomycin), followed by a three hour exposure to ambient air or ozone (2ppm). Sample collection occurred 24 hours post-ozone exposure.","COLLECTION_PROTOCOL_FILENAME":"PROTOCOL_AND_REPORT_150324.docx","SAMPLE_TYPE":"Lung"},

"TREATMENT":{"TREATMENT_SUMMARY":"Global biochemical profiles were determined in lung tissue and serum collected from untreated control mice and mice treated for two weeks with untreated drinking water or water containing an antibiotic cocktail (ampicillin, neomycin, metronidazole, and vancomycin), followed by a three hour exposure to ambient air or ozone (2ppm). Sample collection occurred 24 hours post-ozone exposure.","TREATMENT_PROTOCOL_FILENAME":"PROTOCOL_AND_REPORT_150324.docx"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Sample preparation: Samples were shipped to Metabolon for processing and prepared for metabolomics as previously described. Briefly, an equivalent amount serum (100µl) on a per sample basis was prepared using the automated MicroLab STAR® system from Hamilton Company. For QC purposes, a recovery standard was added prior to the first step in the extraction process. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were first precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions as follows. One fraction was used for analysis by UPLC-MS/MS with positive ion mode electrospray ionization. Another fraction was used for analysis by UPLC-MS/MS with negative ion mode electrospray ionization. The third and fourth fractions were used for LC polar platform, and for analysis by GC-MS. The final fraction was reserved as a backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"The LC/MS portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Each sample extract was first dried, then reconstituted in acidic or basic LC-compatible solvents. To ensure injection and chromatographic consistency, these solvents each contained 8 or more injection standards at fixed concentrations. One aliquot was analyzed using acidic positive ion optimized conditions and the other using basic negative ion optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm). Extracts reconstituted in acidic conditions were gradient eluted from a C18 column using water and methanol containing 0.1% formic acid. The basic extracts were similarly eluted from C18 using methanol and water, however with 6.5mM Ammonium Bicarbonate. The third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80-1000 m/z.","CHROMATOGRAPHY_TYPE":"Reversed phase/HILIC","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity BEH C18 (100 x 2mm, 1.7um)/Waters UPLC BEH Amide (2.1x150 mm, 1.7 um)","METHODS_FILENAME":"PROTOCOL_AND_REPORT_150324.docx"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. A recovery standard was added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: one for analysis by UPLC-MS/MS with positive ion mode electrospray ionization, one for analysis by UPLC-MS/MS with negative ion mode electrospray ionization, one for LC polar platform, one for analysis by GC-MS, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis."},

"MS_METABOLITE_DATA":{
"Units":"area under the curve",

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