#METABOLOMICS WORKBENCH lai_yunjia_20210428_165013 DATATRACK_ID:2607 STUDY_ID:ST001757 ANALYSIS_ID:AN002863 PROJECT_ID:000000 VERSION 1 CREATED_ON April 28, 2021, 7:00 pm #PROJECT PR:PROJECT_TITLE High-coverage metabolomics annotation uncovers microbiota-driven biochemical PR:PROJECT_TITLE landscape of interorgan transport and gut-brain communication PR:PROJECT_TYPE untargeted metabolomics PR:PROJECT_SUMMARY The mammalian gut harbors a complex and dynamic microbial ecosystem: the PR:PROJECT_SUMMARY microbiota. While emerging studies support that microbiota regulates brain PR:PROJECT_SUMMARY function with a few molecular cues suggested, the overall biochemical landscape PR:PROJECT_SUMMARY of the “microbiota-gut-brain axis” remains largely unclear. Using high PR:PROJECT_SUMMARY coverage metabolomics, we profiled feces, blood sera and cerebral cortical brain PR:PROJECT_SUMMARY tissues of germ-free C57BL/6 mice and their age-matched conventionally raised PR:PROJECT_SUMMARY counterparts. Results revealed for all three sample matrices metabolomic PR:PROJECT_SUMMARY signatures owing to microbiota, yielding hundreds of identified metabolites PR:PROJECT_SUMMARY including 533 altered for feces, 231 for sera and 58 for brain tissues with PR:PROJECT_SUMMARY numerous significantly enriched pathways involving aromatic amino acids and PR:PROJECT_SUMMARY neurotransmitters. Multicompartmental comparative analyses singled out PR:PROJECT_SUMMARY microbiota-derived metabolites potentially implicated in interorgan transport PR:PROJECT_SUMMARY and the gut-brain axis, exemplified by indoxyl sulfate and PR:PROJECT_SUMMARY trimethylamine-N-oxide. Gender-specific characteristics of these landscapes were PR:PROJECT_SUMMARY discussed. Our findings may be valuable for future research probing microbial PR:PROJECT_SUMMARY influences on host metabolism and gut-brain communication. PR:INSTITUTE University of North Carolina at Chapel Hill PR:DEPARTMENT Department of Environmental Sciences and Engineering PR:LAST_NAME Lai PR:FIRST_NAME Yunjia PR:ADDRESS 1104 MHRC, 135 Dauer Dr., Chapel Hill, NC, 27599, USA PR:EMAIL yunjia.lai@outlook.com PR:PHONE 9194805489 PR:FUNDING_SOURCE NIEHS #STUDY ST:STUDY_TITLE Metabolomics of mouse blood sera comparing GF and CONV-R mice ST:STUDY_TYPE untargeted metabolomics ST:STUDY_SUMMARY Using high coverage metabolomics, we profiled feces, blood sera and cerebral ST:STUDY_SUMMARY cortical brain tissues of germ-free C57BL/6 mice and their age-matched ST:STUDY_SUMMARY conventionally raised counterparts. Results revealed for all three sample ST:STUDY_SUMMARY matrices metabolomic signatures owing to microbiota, yielding hundreds of ST:STUDY_SUMMARY identified metabolites including 533 altered for feces, 231 for sera and 58 for ST:STUDY_SUMMARY brain tissues with numerous significantly enriched pathways involving aromatic ST:STUDY_SUMMARY amino acids and neurotransmitters. ST:INSTITUTE University of North Carolina at Chapel Hill ST:DEPARTMENT Department of Environmental Sciences and Engineering ST:LAST_NAME Lai ST:FIRST_NAME Yunjia ST:ADDRESS 1104 MHRC, 135 Dauer Dr., Chapel Hill, NC 27599 ST:EMAIL yunjia.lai@outlook.com ST:PHONE 9194805489 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 24 ST:NUM_MALES 12 ST:NUM_FEMALES 12 ST:PUBLICATIONS Nature Communications #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 7-8 week old SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A1A_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A1A_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A1B_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A1B_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A1C_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A1C_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A2A_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A2A_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A2B_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A2B_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_30A2C_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_CN_30A2C_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A1C_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A1C_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A2A_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A2A_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A2B_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A2B_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A2C_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A2C_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A2D_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A2D_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_CN_9A2E_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_CN_9A2E_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F2 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F2.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F3 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F3.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F4 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F4.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F5 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F5.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F6 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F6.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27F7 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Pos_GF_27F7.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M1 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M1.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M2 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M2.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M5 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M5.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M7 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M7.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M8 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M8.mzXML SUBJECT_SAMPLE_FACTORS - serum_Pos_GF_27M9 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Pos_GF_27M9.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A1A_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A1A_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A1B_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A1B_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A1C_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A1C_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A2A_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A2A_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A2B_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A2B_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_30A2C_M Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_CN_30A2C_M.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A1C_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A1C_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A2A_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A2A_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A2B_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A2B_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A2C_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A2C_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A2D_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A2D_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_CN_9A2E_F Experimental factor:Conventionally-raised (CONV-R) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_CN_9A2E_F.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F2 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F2.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F3 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F3.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F4 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F4.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F5 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F5.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F6 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F6.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27F7 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Female; RAW_FILE_NAME=serum_Neg_GF_27F7.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M1 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M1.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M2 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M2.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M5 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M5.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M7 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M7.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M8 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M8.mzXML SUBJECT_SAMPLE_FACTORS - serum_Neg_GF_27M9 Experimental factor:Germ-free (GF) Genotype=C57BL/6; Gender=Male; RAW_FILE_NAME=serum_Neg_GF_27M9.mzXML #COLLECTION CO:COLLECTION_SUMMARY Prior to sacrifice and sample harvest, all mice were observed under their CO:COLLECTION_SUMMARY original housing conditions for one week; animals were not considered if they CO:COLLECTION_SUMMARY exhibited significant signs of serious injury or morbidity (e.g. malocclusion or CO:COLLECTION_SUMMARY fight wounds). Upon euthanization in CO2 chambers, feces, bulk brain, and heart CO:COLLECTION_SUMMARY blood sera were harvested, snap-frozen, and stored in -80 ºC freezer before CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Steady states only: C57BL/6 mouse littermates of ~7 weeks old raised under GF TR:TREATMENT_SUMMARY and conventional conditions were age-matched upon selection for sacrifice and TR:TREATMENT_SUMMARY analyses, resulting in a final total of 24 mice, including 12 GF mice and 12 SPF TR:TREATMENT_SUMMARY CONV-R mice with 6 males and 6 females included in each. The animals were raised TR:TREATMENT_SUMMARY under the following conditions: 22ºC, 40-70% humidity, and a daily 12:12 hour TR:TREATMENT_SUMMARY light-dark cycle. For dietary administration, we consistently fed all mice the TR:TREATMENT_SUMMARY same purified (and sterile) standard rodent diets and provided tap water ad TR:TREATMENT_SUMMARY libitum. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fecal matter, blood sera, and brain tissues (of the cerebral cortical region) SP:SAMPLEPREP_SUMMARY were aliquoted and extracted for metabolome analyses. To maximize coverage and SP:SAMPLEPREP_SUMMARY throughput, extraction procedures were kept as simple and non-selective as can SP:SAMPLEPREP_SUMMARY be. SP:SAMPLEPREP_PROTOCOL_FILENAME Protocol_sample_extraction.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY UHPLC reverse phase C18 chromatography CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_GRADIENT 2% B, 0-1 min; 2% to 15% B, 1-3 min; 15% to 50% B, 3-6 min; 50% to 98% B, 6-7.5 CH:FLOW_GRADIENT min; 98% B, 7.5-11.5 min; 98% to 2% B, 11.5-11.6 min; 2% B, 11.6-15 min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS fullscan XCMS Online for data processing MS:MS_RESULTS_FILE ST001757_AN002863_Results.txt UNITS:Peak intensity (area) Has m/z:Yes Has RT:Yes RT units:Minutes #END