#METABOLOMICS WORKBENCH judgemt_20181127_084939 DATATRACK_ID:1574 STUDY_ID:ST001103 ANALYSIS_ID:AN001793 PROJECT_ID:PR000738 VERSION 1 CREATED_ON December 3, 2018, 6:30 pm #PROJECT PR:PROJECT_TITLE Continuous in vivo metabolism by NMR PR:PROJECT_SUMMARY Metabolomics relies on analytical methods to provide holistic information about PR:PROJECT_SUMMARY metabolites, their distributions across samples, and their underlying dynamic PR:PROJECT_SUMMARY properties. The latter is gaining increasing attention due to advances in PR:PROJECT_SUMMARY modeling and new analytical methods that provide dense time-series data. We PR:PROJECT_SUMMARY extended high-resolution-magic angle spinning (HR-MAS) NMR—an established PR:PROJECT_SUMMARY technique to measure metabolites from tissues and live organisms—into a PR:PROJECT_SUMMARY flexible, untargeted, and continuous recording of in vivo metabolism. We call PR:PROJECT_SUMMARY this technique “continuous in vivo metabolism by NMR” (CIVM-NMR). We used PR:PROJECT_SUMMARY isotope-edited CIVM-NMR to reproduce a recent amino acid flux result in chronic PR:PROJECT_SUMMARY lymphoid leukemia cells. We then collected untargeted CIVM-NMR datasets for PR:PROJECT_SUMMARY Neurospora crassa, a classic multicellular model of biochemistry, genetics, and PR:PROJECT_SUMMARY metabolism. CIVM-NMR requires virtually no sample preparation and allows for PR:PROJECT_SUMMARY continuous collection of data over hours to days at ~4-min temporal resolution PR:PROJECT_SUMMARY with little noise. CIVM-NMR provided real-time measurements that unambiguously PR:PROJECT_SUMMARY reproduced the direction of flux of branched-chain amino acid accumulation in PR:PROJECT_SUMMARY leukemia cells. It also revealed the dynamics of central carbon metabolism, PR:PROJECT_SUMMARY amino acid metabolism, energy storage molecules, and lipid and cell wall PR:PROJECT_SUMMARY precursors in N. crassa. CIVM-NMR is simple and readily adapted to different PR:PROJECT_SUMMARY types of cells and microorganisms, making it ideally suited to experimentally PR:PROJECT_SUMMARY complement kinetic models of metabolism for diverse biological systems. PR:INSTITUTE University of Georgia PR:DEPARTMENT Genetics; Biochemistry and Molecular Biology PR:LABORATORY Arthur S. Edison PR:LAST_NAME Judge PR:FIRST_NAME Michael PR:ADDRESS 315 Riverbend Rd., Edison Lab, Athens, GA, 30605, USA PR:EMAIL judgemt@uga.edu PR:PHONE 7046771037 PR:FUNDING_SOURCE NSF 1713746; NSF ERC 1648035 (CMaT); Georgia Research Alliance PR:CONTRIBUTORS Michael T. Judge, Yue Wu, Fariba Tayyari, John Glushka, Ayuna Hattori, Takahiro PR:CONTRIBUTORS Ito, Jonathan Arnold, Arthur S. Edison #STUDY ST:STUDY_TITLE Continuous in vivo metabolism by NMR ST:STUDY_SUMMARY Metabolomics relies on analytical methods to provide holistic information about ST:STUDY_SUMMARY metabolites, their distributions across samples, and their underlying dynamic ST:STUDY_SUMMARY properties. The latter is gaining increasing attention due to advances in ST:STUDY_SUMMARY modeling and new analytical methods that provide dense time-series data. We ST:STUDY_SUMMARY extended high-resolution-magic angle spinning (HR-MAS) NMR—an established ST:STUDY_SUMMARY technique to measure metabolites from tissues and live organisms—into a ST:STUDY_SUMMARY flexible, untargeted, and continuous recording of in vivo metabolism. We call ST:STUDY_SUMMARY this technique “continuous in vivo metabolism by NMR” (CIVM-NMR). We used ST:STUDY_SUMMARY isotope-edited CIVM-NMR to reproduce a recent amino acid flux result in chronic ST:STUDY_SUMMARY lymphoid leukemia cells. We then collected untargeted CIVM-NMR datasets for ST:STUDY_SUMMARY Neurospora crassa, a classic multicellular model of biochemistry, genetics, and ST:STUDY_SUMMARY metabolism. CIVM-NMR requires virtually no sample preparation and allows for ST:STUDY_SUMMARY continuous collection of data over hours to days at ~4-min temporal resolution ST:STUDY_SUMMARY with little noise. CIVM-NMR provided real-time measurements that unambiguously ST:STUDY_SUMMARY reproduced the direction of flux of branched-chain amino acid accumulation in ST:STUDY_SUMMARY leukemia cells. It also revealed the dynamics of central carbon metabolism, ST:STUDY_SUMMARY amino acid metabolism, energy storage molecules, and lipid and cell wall ST:STUDY_SUMMARY precursors in N. crassa. CIVM-NMR is simple and readily adapted to different ST:STUDY_SUMMARY types of cells and microorganisms, making it ideally suited to experimentally ST:STUDY_SUMMARY complement kinetic models of metabolism for diverse biological systems. ST:INSTITUTE University of Georgia ST:LAST_NAME Judge ST:FIRST_NAME Michael ST:ADDRESS 315 Riverbend Rd., Edison Lab, Athens, GA, 30605, USA ST:EMAIL judgemt@uga.edu ST:PHONE 7046771037 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 6 #SUBJECT SU:SUBJECT_TYPE Fungi SU:SUBJECT_SPECIES Neurospora crassa SU:TAXONOMY_ID 5141 SU:GENOTYPE_STRAIN bd1859 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - aer_4 Condition:aerobic SUBJECT_SAMPLE_FACTORS - aer_5 Condition:aerobic SUBJECT_SAMPLE_FACTORS - aer_6 Condition:aerobic SUBJECT_SAMPLE_FACTORS - anaer_10 Condition:anaerobic SUBJECT_SAMPLE_FACTORS - anaer_11 Condition:anaerobic SUBJECT_SAMPLE_FACTORS - anaer_12 Condition:anaerobic #COLLECTION CO:COLLECTION_SUMMARY Mycelia were added with fresh media to a 4mm zirconia HR-MAS rotor with a sealed CO:COLLECTION_SUMMARY cap (anaerobic) or a cap with a drilled hole (aerobic). Sample was spun at 6KHz, CO:COLLECTION_SUMMARY and spectra were collected continuously over the course of 12h (4.2 min CO:COLLECTION_SUMMARY resolution). CO:SAMPLE_TYPE mycelia in media #TREATMENT TR:TREATMENT_SUMMARY Time series were taken on aerobic and anaerobic cultures #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Mycelia were added to the HR-MAS rotor with fresh media containing 1mM DSS as a SP:SAMPLEPREP_SUMMARY reference. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker NEO NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 4mm CMP HR-MAS probe NM:NMR_SOLVENT Vogel's Media containing 5% D2O and 1mM DSS NM:NMR_TUBE_SIZE 4mm #END