Summary of Study ST003131

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001945. The data can be accessed directly via it's Project DOI: 10.21228/M89M7J This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003131
Study Title	Untargeted Metabolomics on mouse caecal contents
Study Type	Mouse
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
Phone	+353-21-4901408
Submit Date	2024-03-19
Num Groups	9
Total Subjects	63
Num Males	63
Analysis Type Detail	Other
Release Date	2024-03-22
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001945
Project DOI:	doi: 10.21228/M89M7J
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.

Subject:

Subject ID:	SU003248
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 Animal Supplier:	Taconic
Animal Housing:	Gnotobiotic isolators/conventional housing
Animal Light Cycle:	12/12
Animal Feed:	RM1A (P) and RM3A (P) from Special Diet Services

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Factor
SA339176	ColGF10	Caecal Contents	ColGF_0min
SA339177	ColGF11	Caecal Contents	ColGF_0min
SA339178	ColGF8	Caecal Contents	ColGF_0min
SA339179	ColGF6	Caecal Contents	ColGF_0min
SA339180	ColGF5	Caecal Contents	ColGF_0min
SA339181	ColGF12	Caecal Contents	ColGF_0min
SA339182	ColGF7	Caecal Contents	ColGF_0min
SA339183	ColGF20	Caecal Contents	ColGF_45min
SA339184	ColGF21	Caecal Contents	ColGF_45min
SA339185	ColGF19	Caecal Contents	ColGF_45min
SA339186	ColGF18	Caecal Contents	ColGF_45min
SA339187	ColGF17	Caecal Contents	ColGF_45min
SA339188	ColGF9	Caecal Contents	ColGF_45min
SA339189	ColGF16	Caecal Contents	ColGF_45min
SA339190	ColGF3	Caecal Contents	ColGF_Pre
SA339191	ColGF1	Caecal Contents	ColGF_Pre
SA339192	ColGF4	Caecal Contents	ColGF_Pre
SA339193	ColGF2	Caecal Contents	ColGF_Pre
SA339194	ColGF14	Caecal Contents	ColGF_Pre
SA339195	ColGF13	Caecal Contents	ColGF_Pre
SA339196	ColGF15	Caecal Contents	ColGF_Pre
SA339197	Conv17	Caecal Contents	Conv_0min
SA339198	Conv16	Caecal Contents	Conv_0min
SA339199	Conv19	Caecal Contents	Conv_0min
SA339200	Conv22	Caecal Contents	Conv_0min
SA339201	Conv13	Caecal Contents	Conv_0min
SA339202	Conv21	Caecal Contents	Conv_0min
SA339203	Conv18	Caecal Contents	Conv_0min
SA339204	Conv8	Caecal Contents	Conv_45min
SA339205	Conv7	Caecal Contents	Conv_45min
SA339206	Conv9	Caecal Contents	Conv_45min
SA339207	Conv14	Caecal Contents	Conv_45min
SA339208	Conv15	Caecal Contents	Conv_45min
SA339209	Conv6	Caecal Contents	Conv_45min
SA339210	Conv10	Caecal Contents	Conv_45min
SA339211	Conv3	Caecal Contents	Conv_Pre
SA339212	Conv1	Caecal Contents	Conv_Pre
SA339213	Conv20	Caecal Contents	Conv_Pre
SA339214	Conv12	Caecal Contents	Conv_Pre
SA339215	Conv2	Caecal Contents	Conv_Pre
SA339216	Conv4	Caecal Contents	Conv_Pre
SA339217	Conv5	Caecal Contents	Conv_Pre
SA339218	GF7	Caecal Contents	GF_0min
SA339219	GF15	Caecal Contents	GF_0min
SA339220	GF16	Caecal Contents	GF_0min
SA339221	GF6	Caecal Contents	GF_0min
SA339222	GF17	Caecal Contents	GF_0min
SA339223	GF5	Caecal Contents	GF_0min
SA339224	GF4	Caecal Contents	GF_0min
SA339225	GF18	Caecal Contents	GF_45min
SA339226	GF20	Caecal Contents	GF_45min
SA339227	GF8	Caecal Contents	GF_45min
SA339228	GF11	Caecal Contents	GF_45min
SA339229	GF19	Caecal Contents	GF_45min
SA339230	GF10	Caecal Contents	GF_45min
SA339231	GF9	Caecal Contents	GF_45min
SA339232	GF3	Caecal Contents	GF_Pre
SA339233	GF2	Caecal Contents	GF_Pre
SA339234	GF12	Caecal Contents	GF_Pre
SA339235	GF1	Caecal Contents	GF_Pre
SA339236	GF21	Caecal Contents	GF_Pre
SA339237	GF14	Caecal Contents	GF_Pre
SA339238	GF13	Caecal Contents	GF_Pre

Showing results 1 to 63 of 63

Showing results 1 to 63 of 63

Collection:

Collection ID:	CO003241
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 ID:	TR003257
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.

Treatment ID:

TR003257

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.

Sample Preparation:

Sampleprep ID:	SP003255
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.

Sampleprep ID:

SP003255

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.

Combined analysis:

Analysis ID	AN005135	AN005136	AN005137	AN005138
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase	HILIC
Chromatography system	Waters Acquity	Waters Acquity	Waters Acquity	Waters Acquity
Column	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type	Other	Other	Other	Other
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	Raw Peak Area	Raw Peak Area	Raw Peak Area	Raw Peak Area

Chromatography:

Chromatography ID:	CH003885
Chromatography Summary:	Low pH polar (LC/MS Pos early)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 5% B to 80% B over 3.35 minutes
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:	100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:	Reversed phase

Chromatography ID:	CH003886
Chromatography Summary:	Low pH Lipophilic (LC/MS Pos late)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes.
Flow Rate:	0.60 mL/min
Solvent A:	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:	50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:	Reversed phase

Chromatography ID:	CH003887
Chromatography Summary:	High pH (LC/MS Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes.
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 6.5 mM ammonium bicarbonate, pH 8
Solvent B:	95% methanol/5% water; 6.5 mM ammonium bicarbonate
Chromatography Type:	Reversed phase

Chromatography ID:	CH003888
Chromatography Summary:	HILIC (LC/MS Polar Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Temperature:	40-50
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
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)
Chromatography Type:	HILIC

MS:

MS ID:	MS004871
Analysis ID:	AN005135
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Pos early)
Ion Mode:	POSITIVE

MS ID:	MS004872
Analysis ID:	AN005136
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Pos late)
Ion Mode:	POSITIVE

MS ID:	MS004873
Analysis ID:	AN005137
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Neg)
Ion Mode:	NEGATIVE

MS ID:	MS004874
Analysis ID:	AN005138
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Polar)#_1 MS_METABOLITE_DATA
Ion Mode:	NEGATIVE