#METABOLOMICS WORKBENCH rgn5011_20180713_123912 DATATRACK_ID:1444 STUDY_ID:ST001007 ANALYSIS_ID:AN001649 PROJECT_ID:PR000681
VERSION             	1
CREATED_ON             	July 13, 2018, 1:32 pm
#PROJECT
PR:PROJECT_TITLE                 	Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity
PR:PROJECT_SUMMARY               	The gut microbiota are susceptible to modulation by environmental stimuli and
PR:PROJECT_SUMMARY               	therefore can serve as biological sensors. Recent evidence suggests that
PR:PROJECT_SUMMARY               	xenobiotics can disrupt the interaction between the microbiota and host. Here,
PR:PROJECT_SUMMARY               	we describe an approach that combines in vitro microbial incubation (isolated
PR:PROJECT_SUMMARY               	cecal contents from mice), flow cytometry, and mass spectrometry- and 1H
PR:PROJECT_SUMMARY               	NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity.
PR:PROJECT_SUMMARY               	Tempol, a stabilized free radical scavenger known to remodel the microbial
PR:PROJECT_SUMMARY               	community structure and function in vivo, was studied to assess its direct
PR:PROJECT_SUMMARY               	effects on the gut microbiota. Microbiota were isolated from mouse cecum and
PR:PROJECT_SUMMARY               	were exposed to tempol for 4 h under strict anaerobic condition. The flow
PR:PROJECT_SUMMARY               	cytometry data suggested short term exposure of the microbiota to tempol is
PR:PROJECT_SUMMARY               	associated with disrupted membrane physiology as well as compromised metabolic
PR:PROJECT_SUMMARY               	activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure
PR:PROJECT_SUMMARY               	significantly disrupted microbial metabolic activity, specifically indicated by
PR:PROJECT_SUMMARY               	changes in short chain fatty acids, branched chain amino acids, amino acids,
PR:PROJECT_SUMMARY               	nucleotides, glucose, and oligosaccharides. In addition, a mouse study with
PR:PROJECT_SUMMARY               	tempol (5 days gavage) showed similar microbial physiologic and metabolic
PR:PROJECT_SUMMARY               	changes, indicating the in vitro approach reflected in vivo conditions. Our
PR:PROJECT_SUMMARY               	results, through evaluation of microbial viability, physiology and metabolism,
PR:PROJECT_SUMMARY               	and comparison of in vitro and in vivo exposures with tempol, suggests that
PR:PROJECT_SUMMARY               	physiologic and metabolic phenotyping provides unique insight into gut
PR:PROJECT_SUMMARY               	microbiota toxicity.
PR:INSTITUTE                     	The Pennsylvania State University (Penn State)
PR:LAST_NAME                     	Nichols
PR:FIRST_NAME                    	Robert
PR:ADDRESS                       	650 toftrees ave Apt #108, State College, Pa 16802
PR:EMAIL                         	rgn5011@psu.edu
PR:PHONE                         	7247662694
#STUDY
ST:STUDY_TITLE                   	Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity
ST:STUDY_SUMMARY                 	The gut microbiota are susceptible to modulation by environmental stimuli and
ST:STUDY_SUMMARY                 	therefore can serve as biological sensors. Recent evidence suggests that
ST:STUDY_SUMMARY                 	xenobiotics can disrupt the interaction between the microbiota and host. Here,
ST:STUDY_SUMMARY                 	we describe an approach that combines in vitro microbial incubation (isolated
ST:STUDY_SUMMARY                 	cecal contents from mice), flow cytometry, and mass spectrometry- and 1H
ST:STUDY_SUMMARY                 	NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity.
ST:STUDY_SUMMARY                 	Tempol, a stabilized free radical scavenger known to remodel the microbial
ST:STUDY_SUMMARY                 	community structure and function in vivo, was studied to assess its direct
ST:STUDY_SUMMARY                 	effects on the gut microbiota. Microbiota were isolated from mouse cecum and
ST:STUDY_SUMMARY                 	were exposed to tempol for 4 h under strict anaerobic condition. The flow
ST:STUDY_SUMMARY                 	cytometry data suggested short term exposure of the microbiota to tempol is
ST:STUDY_SUMMARY                 	associated with disrupted membrane physiology as well as compromised metabolic
ST:STUDY_SUMMARY                 	activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure
ST:STUDY_SUMMARY                 	significantly disrupted microbial metabolic activity, specifically indicated by
ST:STUDY_SUMMARY                 	changes in short chain fatty acids, branched chain amino acids, amino acids,
ST:STUDY_SUMMARY                 	nucleotides, glucose, and oligosaccharides. In addition, a mouse study with
ST:STUDY_SUMMARY                 	tempol (5 days gavage) showed similar microbial physiologic and metabolic
ST:STUDY_SUMMARY                 	changes, indicating the in vitro approach reflected in vivo conditions. Our
ST:STUDY_SUMMARY                 	results, through evaluation of microbial viability, physiology and metabolism,
ST:STUDY_SUMMARY                 	and comparison of in vitro and in vivo exposures with tempol, suggests that
ST:STUDY_SUMMARY                 	physiologic and metabolic phenotyping provides unique insight into gut
ST:STUDY_SUMMARY                 	microbiota toxicity.
ST:INSTITUTE                     	Pennsylvania State University
ST:LAST_NAME                     	Nichols
ST:FIRST_NAME                    	Robert
ST:ADDRESS                       	650 Toftrees ave
ST:EMAIL                         	rgn5011@psu.edu
ST:PHONE                         	7247662694
#SUBJECT
SU:SUBJECT_TYPE                  	Other
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:SPECIES_GROUP                 	Microbiome
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	C1	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	C2	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	C3	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	C4	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	C5	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	C6	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	L1	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	L2	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	L3	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	L4	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	L5	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	L6	Treatment:Low-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M1	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M2	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M3	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M4	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M5	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	M6	Treatment:Medium-Dose-Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H1	Treatment:High-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H2	Treatment:High-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H3	Treatment:High-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H4	Treatment:High-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H5	Treatment:High-Dose Tempol	
SUBJECT_SAMPLE_FACTORS           	-	H6	Treatment:High-Dose Tempol	
#COLLECTION
CO:COLLECTION_SUMMARY            	6-week-old wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine)
CO:COLLECTION_SUMMARY            	were transferred into anaerobic chamber (Coy Laboratory Products, Inc., Grass
CO:COLLECTION_SUMMARY            	Lake, MI) following CO2 asphyxiation. All the following procedures were
CO:COLLECTION_SUMMARY            	performed under strict anaerobic conditions with an oxygen level below 20 ppm
CO:COLLECTION_SUMMARY            	and cecal contents were collected
CO:SAMPLE_TYPE                   	Cecum
#TREATMENT
TR:TREATMENT_SUMMARY             	The cecal content suspension was treated with tempol at a final concentration
TR:TREATMENT_SUMMARY             	0.01 mg/mL, 0.1 mg/mL and 1 mg/mL, following a brief centrifugation and
TR:TREATMENT_SUMMARY             	incubation at 37 °C for 4 h in dark.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The microbiota suspension saved after 4h incubation was used for 1H NMR
SP:SAMPLEPREP_SUMMARY            	spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g,
SP:SAMPLEPREP_SUMMARY            	4 °C for 1 min) to pellet down large particles. The maximum supernatant volume
SP:SAMPLEPREP_SUMMARY            	was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3
SP:SAMPLEPREP_SUMMARY            	min) to pellet down bacteria. The microbial pellet was washed two times with
SP:SAMPLEPREP_SUMMARY            	PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0
SP:SAMPLEPREP_SUMMARY            	mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the
SP:SAMPLEPREP_SUMMARY            	microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the
SP:SAMPLEPREP_SUMMARY            	Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The
SP:SAMPLEPREP_SUMMARY            	homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C
SP:SAMPLEPREP_SUMMARY            	water bath, then was homogenized again and sonicated for 15 min at 250W with
SP:SAMPLEPREP_SUMMARY            	Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture
SP:SAMPLEPREP_SUMMARY            	microbial cell walls and release intracellular metabolites. The sample was
SP:SAMPLEPREP_SUMMARY            	centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to
SP:SAMPLEPREP_SUMMARY            	a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets
SP:SAMPLEPREP_SUMMARY            	and the extraction procedure was repeated. All supernatants were combined, dried
SP:SAMPLEPREP_SUMMARY            	down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4,
SP:SAMPLEPREP_SUMMARY            	containing 50% D2O and 0.005% TSP-d4 as internal standard). Following
SP:SAMPLEPREP_SUMMARY            	centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred
SP:SAMPLEPREP_SUMMARY            	into a 5 mm NMR tube for analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	-
CH:INSTRUMENT_NAME               	-
CH:COLUMN_NAME                   	-
#ANALYSIS
AN:ANALYSIS_TYPE                 	NMR
#NMR
NM:INSTRUMENT_NAME               	Bruker
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:SPECTROMETER_FREQUENCY        	600 Mhz
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS        	ppm
NMR_METABOLITE_DATA_START
Samples	C1	C2	C3	C4	C5	C6	L1	L2	L3	L4	L5	L6	M1	M2	M3	M4	M5	M6	H1	H2	H3	H4	H5	H6
Factors	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Low-Dose Tempol	Treatment:Low-Dose Tempol	Treatment:Low-Dose Tempol	Treatment:Low-Dose Tempol	Treatment:Low-Dose Tempol	Treatment:Low-Dose Tempol	Treatment:Medium-Dose-Tempol	Treatment:Medium-Dose-Tempol	Treatment:Medium-Dose-Tempol	Treatment:Medium-Dose-Tempol	Treatment:Medium-Dose-Tempol	Treatment:Medium-Dose-Tempol	Treatment:High-Dose Tempol	Treatment:High-Dose Tempol	Treatment:High-Dose Tempol	Treatment:High-Dose Tempol	Treatment:High-Dose Tempol	Treatment:High-Dose Tempol
Acetate	0.0602802	0.06344303	0.06635165	0.06031483	0.06654005	0.0651507	0.06120056	0.06585579	0.0603067	0.06333344	0.06180664	0.06295734	0.06191963	0.05962733	0.06332089	0.06381048	0.0656252	0.0611167	0.04489098	0.0431432	0.04200419	0.04283736	0.04333221	0.0427448
Propionate	0.0041192	0.00374944	0.00483217	0.00334176	0.00459513	0.00436015	0.00399124	0.00389302	0.00414445	0.00390493	0.00414203	0.00385406	0.00428864	0.0041491	0.00377386	0.00422545	0.00430192	0.00365311	0.00294052	0.00282232	0.00259972	0.00269264	0.0028849	0.00274657
Butyrate	0.01429423	0.01366463	0.01631619	0.01298833	0.01570116	0.01543256	0.01388818	0.01406942	0.0139213	0.01362712	0.01432263	0.0137545	0.01521234	0.01417828	0.01397004	0.01504416	0.01499379	0.01348895	0.01021884	0.01020082	0.00961994	0.00980473	0.00949746	0.00906464
oligosaccharides	0.10830785	0.11216735	0.10253293	0.10444561	0.10755919	0.11249062	0.11347433	0.10915296	0.10778433	0.11373085	0.10295846	0.10525577	0.11004292	0.11018482	0.11082823	0.11351202	0.11088463	0.11729258	0.15325968	0.15561917	0.15067204	0.14902115	0.16377345	0.15486385
Glucose	0.00149962	0.0019109	0.00193634	0.00345666	0.00200746	0.0011655	0.00176267	0.0022656	0.00277414	0.00179029	0.00213022	0.00294689	0.00147031	0.00173468	0.00240206	0.00174258	0.00183813	0.00209764	0.00275073	0.00276349	0.00319735	0.00317321	0.00252329	0.00359056
Tyrosine	0.00307275	0.0036514	0.00310543	0.00388437	0.0032333	0.00312439	0.00316279	0.00366711	0.00319671	0.00339703	0.00307187	0.00364892	0.00325793	0.0032168	0.00356024	0.003271	0.00331903	0.0036222	0.00373271	0.00376511	0.00470783	0.0043634	0.00429923	0.00452801
Phenylalanine	0.00261271	0.0032225	0.00260119	0.00323726	0.00280957	0.00265425	0.00279429	0.00324641	0.00252053	0.00287706	0.0025243	0.00312602	0.00270279	0.00264386	0.00308836	0.00280957	0.00287538	0.00313303	0.00292739	0.0028276	0.00350572	0.00305189	0.00325091	0.00322877
Inosine	0.00029536	0.00032476	0.00036898	0.00056717	0.00045451	0.00044804	0.00037122	0.00038262	0.00028693	0.00029197	0.00043347	0.00053468	0.00039782	0.00043335	0.00049009	0.00035372	0.00041968	0.00034176	0.00062123	0.00061321	0.00068961	0.0006472	0.00065402	0.0007074
Phenylacetate	0.00352646	0.00351406	0.00356391	0.00361141	0.00348436	0.00344697	0.00348107	0.00355059	0.00366447	0.0034223	0.00357715	0.00354615	0.00355874	0.00347011	0.00357068	0.00345764	0.00332416	0.00346872	0.00326342	0.00319555	0.00308747	0.00339111	0.0030684	0.00313117
Threonine	0.01077446	0.01038354	0.01058738	0.01010755	0.01006972	0.01056412	0.01087761	0.01045744	0.01019785	0.01011897	0.01058292	0.01068798	0.01084691	0.01074473	0.01053036	0.01053381	0.01061822	0.01049831	0.01363742	0.01333931	0.01278469	0.0121164	0.01396104	0.01331836
Valine	0.01384914	0.0150892	0.01387873	0.01432405	0.01418874	0.01400109	0.01502536	0.01501019	0.01331191	0.01449427	0.01365743	0.01458743	0.01423183	0.01404887	0.01477198	0.01402186	0.01433328	0.01466053	0.01227719	0.01202237	0.01236134	0.01211538	0.01254111	0.01244286
Isoleucine	0.00423895	0.00456226	0.00428432	0.00432125	0.00433886	0.00429687	0.00457461	0.00457103	0.00409553	0.00439398	0.00415218	0.00442901	0.00438486	0.00438256	0.00449873	0.00428379	0.00442178	0.00443937	0.00392053	0.00383536	0.0039591	0.00385432	0.00402052	0.00399867
Leucine	0.02973827	0.03042505	0.02899553	0.02957718	0.02927885	0.02929377	0.02951433	0.02989464	0.02839364	0.02963049	0.02921022	0.02908758	0.02957726	0.02955283	0.02999518	0.02900304	0.02961164	0.02953873	0.02759861	0.02725167	0.02867998	0.02812079	0.02844542	0.02834353
Glycine	0.00636615	0.00747371	0.00637384	0.00668373	0.00677745	0.00683696	0.00770049	0.00757826	0.00644477	0.00719917	0.00615941	0.00714021	0.00690119	0.00648988	0.00728033	0.00693051	0.00702642	0.00734309	0.00646069	0.00628792	0.00574162	0.00593263	0.00679085	0.00595029
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Type
Acetate	SCFA
Propionate	SCFA
Butyrate	SCFA
oligosaccharides	Sugars
Glucose	Sugars
Tyrosine	Amino Acids
Phenylalanine	Amino Acids
Inosine	Amino Acids
Phenylacetate	Amino Acids
Threonine	Amino Acids
Valine	Amino Acids
Isoleucine	Amino Acids
Leucine	Amino Acids
Glycine	Amino Acids
METABOLITES_END
#END