#METABOLOMICS WORKBENCH hjb17_20200911_115320 DATATRACK_ID:2173 STUDY_ID:ST001492 ANALYSIS_ID:AN002474 PROJECT_ID:PR001010 VERSION 1 CREATED_ON September 29, 2020, 11:58 am #PROJECT PR:PROJECT_TITLE Global metabolomics of IFNy cued neurogenic NSCs seeded on hydrogel PR:PROJECT_SUMMARY Neural stem cells (NSCs) provide a strategy to replace damaged neurons following PR:PROJECT_SUMMARY traumatic central nervous system injuries. A major hurdle to translation of this PR:PROJECT_SUMMARY therapy is that direct application of NSCs to CNS injury does not support PR:PROJECT_SUMMARY sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial PR:PROJECT_SUMMARY cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply PR:PROJECT_SUMMARY physical and biochemical signals to instruct the encapsulated NSCs to be PR:PROJECT_SUMMARY neurogenic. However, the immobilization of factors, including IFN-γ, versus PR:PROJECT_SUMMARY soluble delivery of the same factor, has been incompletely characterized PR:PROJECT_SUMMARY especially with respect to activation of signaling and metabolism in cells over PR:PROJECT_SUMMARY longer time points. In this study, protein and metabolite changes in NSCs PR:PROJECT_SUMMARY induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble PR:PROJECT_SUMMARY IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs PR:PROJECT_SUMMARY compared to immobilized IFN-γ indicating that immobilization may not sustain PR:PROJECT_SUMMARY signaling or has altered ligand/receptor interaction and integrity. However, PR:PROJECT_SUMMARY both IFN-γ delivery types supported increased βIII tubulin expression in PR:PROJECT_SUMMARY parallel with canonical and non-canonical receptor-signaling compared to no PR:PROJECT_SUMMARY IFN-γ. Global metabolomics and pathway analysis revealed that soluble and PR:PROJECT_SUMMARY immobilized IFN-γ altered metabolic pathway activities including energy, lipid PR:PROJECT_SUMMARY and amino acid synthesis, with soluble IFN-γ having the greatest metabolic PR:PROJECT_SUMMARY impact overall. PR:INSTITUTE University of Akron PR:DEPARTMENT Chemistry PR:LABORATORY Shriver lab PR:LAST_NAME Baumann PR:FIRST_NAME Hannah PR:ADDRESS 190 E. Buchtel Common, Akron, OH, 44325, USA PR:EMAIL hjb17@zips.uakron.edu PR:PHONE 4196100269 PR:FUNDING_SOURCE NIH PR:PUBLICATIONS Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ PR:PUBLICATIONS in Neural Stem Cells #STUDY ST:STUDY_TITLE Global metabolomics of IFNy cued neurogenic NSCs seeded on hydrogel ST:STUDY_SUMMARY Neural stem cells (NSCs) provide a strategy to replace damaged neurons following ST:STUDY_SUMMARY traumatic central nervous system injuries. A major hurdle to translation of this ST:STUDY_SUMMARY therapy is that direct application of NSCs to CNS injury does not support ST:STUDY_SUMMARY sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial ST:STUDY_SUMMARY cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply ST:STUDY_SUMMARY physical and biochemical signals to instruct the encapsulated NSCs to be ST:STUDY_SUMMARY neurogenic. However, the immobilization of factors, including IFN-γ, versus ST:STUDY_SUMMARY soluble delivery of the same factor, has been incompletely characterized ST:STUDY_SUMMARY especially with respect to activation of signaling and metabolism in cells over ST:STUDY_SUMMARY longer time points. In this study, protein and metabolite changes in NSCs ST:STUDY_SUMMARY induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble ST:STUDY_SUMMARY IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs ST:STUDY_SUMMARY compared to immobilized IFN-γ indicating that immobilization may not sustain ST:STUDY_SUMMARY signaling or has altered ligand/receptor interaction and integrity. However, ST:STUDY_SUMMARY both IFN-γ delivery types supported increased βIII tubulin expression in ST:STUDY_SUMMARY parallel with canonical and non-canonical receptor-signaling compared to no ST:STUDY_SUMMARY IFN-γ. Global metabolomics and pathway analysis revealed that soluble and ST:STUDY_SUMMARY immobilized IFN-γ altered metabolic pathway activities including energy, lipid ST:STUDY_SUMMARY and amino acid synthesis, with soluble IFN-γ having the greatest metabolic ST:STUDY_SUMMARY impact overall. ST:INSTITUTE University of Akron ST:DEPARTMENT Chemistry ST:LABORATORY Shriver lab ST:LAST_NAME Baumann ST:FIRST_NAME Hannah ST:ADDRESS 190 E. Buchtel Common ST:EMAIL hjb17@zips.uakron.edu ST:PHONE 4198864033 ST:NUM_GROUPS 3 ST:PUBLICATIONS Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ ST:PUBLICATIONS in Neural Stem Cells #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN Fischer 344 SU:AGE_OR_AGE_RANGE 6 wk SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Envigo #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 - A1 Treatment group:no IFN y Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918A.wiff SUBJECT_SAMPLE_FACTORS - A2 Treatment group:no IFN y Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918B.wiff SUBJECT_SAMPLE_FACTORS - A3 Treatment group:no IFN y Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918C.wiff SUBJECT_SAMPLE_FACTORS - A4 Treatment group:no IFN y Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918D.wiff SUBJECT_SAMPLE_FACTORS - B1 Treatment group:soluble IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918E.wiff SUBJECT_SAMPLE_FACTORS - B2 Treatment group:soluble IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918F.wiff SUBJECT_SAMPLE_FACTORS - B3 Treatment group:soluble IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918G.wiff SUBJECT_SAMPLE_FACTORS - B4 Treatment group:soluble IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918H.wiff SUBJECT_SAMPLE_FACTORS - C1 Treatment group:immobilized IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918I.wiff SUBJECT_SAMPLE_FACTORS - C2 Treatment group:immobilized IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918J.wiff SUBJECT_SAMPLE_FACTORS - C3 Treatment group:immobilized IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918K.wiff SUBJECT_SAMPLE_FACTORS - C4 Treatment group:immobilized IFNy Sample Type=primary NSC; RAW_FILE_NAME=SFB pos experiment 032918L.wiff #COLLECTION CO:COLLECTION_SUMMARY Neural stem cells were collected from the subventricular zone of the brain in a CO:COLLECTION_SUMMARY 6-8 wk old rat. Papain tissue dissociation was used to isolate cells and the CO:COLLECTION_SUMMARY neurosphere culture system used to expand them for up to 7 passages. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY Neurospheres were dissociated and plated onto laminin functionalized soft TR:TREATMENT_SUMMARY chitosan hydrogel surfaces. Three groups were grown for 7 days on their TR:TREATMENT_SUMMARY substrate in basal media . One group had no IFNy, another group 300 ng/mL TR:TREATMENT_SUMMARY soluble IFNy and the final group 300 ng/mL hydrogel immobilized IFNy. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY NSC seeded gels were snap frozen and stored at -80C until extraction. Two SP:SAMPLEPREP_SUMMARY gels were combined per group and extracted using a modified Bligh and Dyer SP:SAMPLEPREP_SUMMARY extraction technique. In brief, 100 uL of methanol was added to each sample then SP:SAMPLEPREP_SUMMARY underwent a series of snap freezing, sonication and vortexing three times. 750 SP:SAMPLEPREP_SUMMARY μl of 1:2 chloroform:methanol was added to each homogenized sample, vortexed, SP:SAMPLEPREP_SUMMARY then an additional 250 μL of chloroform was added, finally 250 μL water was SP:SAMPLEPREP_SUMMARY added, all solvents used were LC grade. Samples were stored in -20 overnight and SP:SAMPLEPREP_SUMMARY centrifuged to separate the phase layers and solidify the protein precipitate SP:SAMPLEPREP_SUMMARY interface. Aqueous and organic layers were separated, dried down using Centrivap SP:SAMPLEPREP_SUMMARY (Labconco) and stored in the -80 C until use. Aqueous portions of the extract SP:SAMPLEPREP_SUMMARY were resuspended in 200 μL of 35% acetonitrile. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The mobile phases for separation consisted of water (A) and acetonitrile (B), CH:CHROMATOGRAPHY_SUMMARY both supplemented with 5 mM ammonium acetate and adjusted to pH 7.3 using CH:CHROMATOGRAPHY_SUMMARY ammonium hydroxide. The gradient proceeded at a flow rate of 30 μL/min as CH:CHROMATOGRAPHY_SUMMARY follows: 98% B at 0 min, 95% B at 1 min, 80% B at 5 min, 46% B at 6 min, 14.7% B CH:CHROMATOGRAPHY_SUMMARY at 13 min, 0% B at 17 min, 100% B at 17.1 min, and 100% B at 23 min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Eksigent microLC 200 CH:COLUMN_NAME Phenomenex Luna NH2( 150 mm × 1.0 mm, 3 µm) CH:SOLVENT_A water CH:SOLVENT_B acetonitrile CH:CHROMATOGRAPHY_COMMENTS Phenomenex (Luna 3 μ NH2 100 Å, 150 mm × 1.0 mm) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Shriver Lab #MS MS:INSTRUMENT_NAME ABI Sciex 5600+ TripleTOF MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Independent data acquisition was used with rolling collision energy for selected MS:MS_COMMENTS parent ions.Retention times and mass to charge values were aligned between MS:MS_COMMENTS samples using MarkerView software. Putative metabolite identifications were made MS:MS_COMMENTS using metaboanalyst, masstrix and HMDB softwares/databases. MS:MS_RESULTS_FILE ST001492_AN002474_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END