{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003131","ANALYSIS_ID":"AN005138","VERSION":"1","CREATED_ON":"March 19, 2024, 11:19 am"},

"PROJECT":{"PROJECT_TITLE":"The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut","PROJECT_TYPE":"Untargeted metabolomics by Metabolon","PROJECT_SUMMARY":"Adult male C57Bl/6 conventional, germ-free and colonized germ-free mice underwent a 15-minute restraint stress exposure. Caecal contents and colonic mucosal scrapings were collected from naïve mice and stressed mice, either immediately or 45 minutes following stress. Caecal contents and colonic mucosal scrapings underwent untargeted metabolomics analysis.","INSTITUTE":"University College Cork","DEPARTMENT":"Psychiatry","LABORATORY":"Microbiota-Gut-Brain Axis Group","LAST_NAME":"Clarke","FIRST_NAME":"Gerard","ADDRESS":"Gaol Walk, Cork","EMAIL":"G.Clarke@ucc.ie","PHONE":"+353-21-4901408","FUNDING_SOURCE":"The research was conducted in the APC Microbiome Ireland which is funded by Science Foundation Ireland (SFI/12/RC/2273). This project in part is a collaborative agreement (FA9550-17-1-0016) funded by European Office of Aerospace Research and Development, Air Force Office of Scientific Research and 711 Human Performance Wing, Air Force Research Laboratory."},

"STUDY":{"STUDY_TITLE":"Untargeted Metabolomics on mouse caecal contents","STUDY_SUMMARY":"While there is strong evidence for interactions between the microbiota-gut-brain axis and host physiology in the context of chronic stress, limited research has investigated the role of the microbiome in host response to acute stress. Determining the underlying mechanisms by which stress-induced microbiota changes may provoke functional changes in the gut and brain is critical for developing future therapeutics to alleviate the adverse consequences of traumatic stress. Here, we aimed to identify a biological signature of gut metabolites that are significantly altered following exposure to acute restraint stress. Adult male C57Bl/6 conventional, germ-free and colonized germ-free mice underwent a 15-minute restraint stress exposure. Caecal contents were collected from naïve mice and stressed mice, either immediately or 45 minutes following stress. Caecal contents underwent untargeted metabolomics analysis.","INSTITUTE":"University College Cork","DEPARTMENT":"Psychiatry","LABORATORY":"Microbiota-Gut-Brain Axis Group","LAST_NAME":"Clarke","FIRST_NAME":"Gerard","ADDRESS":"Gaol Walk, Cork","EMAIL":"G.Clarke@ucc.ie","NUM_GROUPS":"9","TOTAL_SUBJECTS":"63","NUM_MALES":"63","STUDY_TYPE":"Mouse","PHONE":"+353-21-4901408"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57Bl/6J","AGE_OR_AGE_RANGE":"8-10 weeks","WEIGHT_OR_WEIGHT_RANGE":"20-29g","HEIGHT_OR_HEIGHT_RANGE":"-","GENDER":"Male","ANIMAL_HOUSING":"Gnotobiotic isolators/conventional housing","ANIMAL_LIGHT_CYCLE":"12/12","ANIMAL_FEED":"RM1A (P) and RM3A (P) from Special Diet Services","ANIMAL_ANIMAL_SUPPLIER":"Taconic"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"GF1",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF2",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF3",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF12",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF13",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF14",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF21",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"GF4",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF5",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF6",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF7",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF15",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF16",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF17",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"GF8",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF9",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF10",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF11",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF18",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF19",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"GF20",
"Factors":{"Sample source":"Caecal Contents","Factor":"GF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv1",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv2",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv3",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv4",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv5",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv12",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv20",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"Conv13",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv16",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv17",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv18",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv19",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv21",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv22",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_0min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv6",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv7",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv8",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv9",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv10",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv14",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"Conv15",
"Factors":{"Sample source":"Caecal Contents","Factor":"Conv_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF1",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF2",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF3",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF4",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF13",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF14",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF15",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_Pre"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF5",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF6",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF7",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF8",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF10",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF11",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF12",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_0min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF9",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF16",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF17",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF18",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF19",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF20",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
},
{
"Subject ID":"-",
"Sample ID":"ColGF21",
"Factors":{"Sample source":"Caecal Contents","Factor":"ColGF_45min"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes. After 15 minutes of restraint stress, mice were removed from the restrainer and either transported immediately to the cull room or returned to their home cage and left undisturbed for 45 minutes. To control for the variable of transport stress, mice from the non-stress control group were placed into new cages containing fresh bedding and transported the same distance before entering the cull room. Upon entering the cull room, mice were immediately decapitated, trunk blood collected, and tissues harvested. Caecal contents and intestinal samples were manually dissected and stored in PCR-grade microfuge tubes at -80°C until analyses.","SAMPLE_TYPE":"Cecum","STORAGE_CONDITIONS":"-80?"},

"TREATMENT":{"TREATMENT_SUMMARY":"All animal work carried was approved by the Animal Experimentation Ethics Committee of University College Cork and Health Products Regulatory Authority (HPRA) before beginning this study. All experimentation was carried out in accordance with European Directive 2010/63/EU and was approved by both the Animal Experimentation Ethics Committee of University College Cork (Project Authorization AE19130/P160) and United States Air Force Surgeon General''s Office of Research Oversight and Compliance. C57/BL6 mice breeding pairs were acquired from Taconic Biosciences, and F1-generation male and female offspring were used in all experiments. Germ-free, ex-germ-free, and conventional mice were housed 2-4 mice/cage under a 12-hour light/dark cycle and maintained on ad libitum autoclaved water and autoclaved, pelleted diet (Special Diet Services). Housing conditions for germ-free, conventional, and colonized germ-free adhered to the same environmental conditions of temperature (21 ± 1°C) and humidity (55%-60%). Germ-free mice were housed in gnotobiotic flexible-film isolators. Colonized germ-free mice were born and maintained as germ-free mice in gnotobiotic flexible-film isolators until postnatal day 21 when they were removed from the isolators and, for the remaining duration of this study, re-located to the standard animal facility and housed in wire-top cages that contained used-bedding from age- and sex-matched conventional mice. The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were 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: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"HILIC (LC/MS Polar Neg)","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)","SOLVENT_A":"15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH)","SOLVENT_B":"50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH)","FLOW_GRADIENT":"Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes.","FLOW_RATE":"0.50 mL/min","COLUMN_TEMPERATURE":"40-50"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"Other","ION_MODE":"NEGATIVE","MS_COMMENTS":"Metabolon (LC/MS Polar)#_1 MS_METABOLITE_DATA"},

"MS_METABOLITE_DATA":{
"Units":"Raw Peak Area",

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}

}