#METABOLOMICS WORKBENCH youngh_20231101_190036 DATATRACK_ID:4436 STUDY_ID:ST002961 ANALYSIS_ID:AN004862 PROJECT_ID:PR001842 VERSION 1 CREATED_ON November 3, 2023, 6:06 am #PROJECT PR:PROJECT_TITLE Mid-Old Cells are Potential Target for Anti-aging Interventions in the Elderly PR:PROJECT_SUMMARY The biological process of aging is thought to result in part from accumulation PR:PROJECT_SUMMARY of senescent cells in organs. However, the present study identified a subset of PR:PROJECT_SUMMARY fibroblasts and smooth muscle cells which are the major constituents of organ PR:PROJECT_SUMMARY stroma neither proliferative nor senescent in tissues of the elderly, which we PR:PROJECT_SUMMARY termed “mid-old status” cells. Upregulation of pro-inflammatory genes (IL1B PR:PROJECT_SUMMARY and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were PR:PROJECT_SUMMARY detected in mid-old cells. In the stroma, SAA1 promotes development of the PR:PROJECT_SUMMARY inflammatory microenvironment via upregulation of MMP9, which decreases the PR:PROJECT_SUMMARY stability of epithelial cells present on the basement membrane, decreasing PR:PROJECT_SUMMARY epithelial cell function. Remarkably, the microenvironmental change and the PR:PROJECT_SUMMARY functional decline of mid-old cells could be reversed by a young cell-originated PR:PROJECT_SUMMARY protein, SLIT2. Our data identify functional reversion of mid-old cells as a PR:PROJECT_SUMMARY potential method to prevent or ameliorate aspects of aging-related tissue PR:PROJECT_SUMMARY dysfunction. PR:INSTITUTE Ajou University Medical Center PR:LAST_NAME Kim PR:FIRST_NAME Young Hwa PR:ADDRESS 206, World cup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea PR:EMAIL skyblue32@nate.com PR:PHONE +82-10-5153-3636 #STUDY ST:STUDY_TITLE Analysis of Metabolites Secreted from Fibroblast Young Cells ST:STUDY_SUMMARY In this experimental study, we aimed to uncover the factors in young cell ST:STUDY_SUMMARY secretions that trigger the reverse aging of mid-old cells, co-culturing them ST:STUDY_SUMMARY and observing a striking transformation, although we could not identify the ST:STUDY_SUMMARY specific factors responsible for this rejuvenation ST:INSTITUTE Ajou University Medical Center ST:LAST_NAME Kim ST:FIRST_NAME Young Hwa ST:ADDRESS 206, World cup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea ST:EMAIL skyblue32@nate.com ST:PHONE +82-10-5153-3636 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 20210624_AJU_Y1 *Factor:Test sample RAW_FILE_NAME=20210624_AJU_Y1.mzML SUBJECT_SAMPLE_FACTORS - 20210624_AJU_Y2 *Factor:Test sample RAW_FILE_NAME=20210624_AJU_Y2.mzML SUBJECT_SAMPLE_FACTORS - 20210624_AJU_Y3 *Factor:Test sample RAW_FILE_NAME=20210624_AJU_Y3.mzML SUBJECT_SAMPLE_FACTORS - 20210624_AJU_M1 *Factor:Control sample RAW_FILE_NAME=20210624_AJU_M1.mzML SUBJECT_SAMPLE_FACTORS - 20210624_AJU_M2 *Factor:Control sample RAW_FILE_NAME=20210624_AJU_M2.mzML SUBJECT_SAMPLE_FACTORS - 20210624_AJU_M3 *Factor:Control sample RAW_FILE_NAME=20210624_AJU_M3.mzML SUBJECT_SAMPLE_FACTORS - blank_20210624 *Factor:Non RAW_FILE_NAME=blank_20210624.mzML #COLLECTION CO:COLLECTION_SUMMARY The experiment was initiated by seeding the cells in a 100mm cell culture dish, CO:COLLECTION_SUMMARY followed by a 2-day incubation period, during which the culture medium was CO:COLLECTION_SUMMARY carefully extracted from the cell culture dish. Use a sterile technique to CO:COLLECTION_SUMMARY minimize contamination. Transfer the harvested culture medium to a suitable CO:COLLECTION_SUMMARY container. CO:SAMPLE_TYPE Cultured fibroblasts #TREATMENT TR:TREATMENT_SUMMARY no treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted using 80% methanol. In brief, the samples were added SP:SAMPLEPREP_SUMMARY with 80% methanol. After vortexing for 1 min and centrifugation at 2000×g for SP:SAMPLEPREP_SUMMARY 10 min, supernatant was transferred to a new 1.5 mL tube and completely dried SP:SAMPLEPREP_SUMMARY using a HyperVAC-MAX VC2200 centrifugal vacuum concentrator (Hanil Scientific SP:SAMPLEPREP_SUMMARY Inc., Korea). Dried metabolite contents were reconstituted in 100 µL of 0.1% SP:SAMPLEPREP_SUMMARY formic acid in water and then subjected to LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Q Exactive™ Hybrid Quadrupole-Orbitrap MS coupled with a 1290 Infinity UHPLC CH:COLUMN_NAME ZORBAX Eclipse Plus C18 Rapid Resolution High Definition (RRHD) column CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in 80% acetonitrile CH:FLOW_GRADIENT 2.5% solvent B in 5 min, 2.5–12.5% solvent B in 29 min, 12.5–25% solvent B CH:FLOW_GRADIENT in 11 min, 25–37.5% solvent B in 11 min, 37.5-80% solvent B in 0.1 min, CH:FLOW_GRADIENT holding at 80% of solvent B in 13.9 min, 80–2.5% solvent B in 0.1 min, 2.5% CH:FLOW_GRADIENT solvent B for 19.9 min CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 320℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Q Exactive™ Hybrid Quadrupole-Orbitrap MS coupled with a 1290 Infinity UHPLC MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Obtained UHPLC-Orbitrap-MS/MS RAW files were processed using Compound Discoverer MS:MS_COMMENTS 3.1.1.12TM (Thermo Fisher Scientific, Waltham, MA, USA). Untargeted metabolomics MS:MS_COMMENTS workflow was used to perform retention time alignment and compound MS:MS_COMMENTS identification. Identification of compounds using mzCloud and ChemSpider MS:MS_RESULTS_FILE ST002961_AN004862_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END