#METABOLOMICS WORKBENCH youngh_20231101_183536 DATATRACK_ID:4435 STUDY_ID:ST002960 ANALYSIS_ID:AN004861 PROJECT_ID:PR001842
VERSION             	1
CREATED_ON             	November 3, 2023, 5:49 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 Lipids Secreted from Fibroblast Young Cells
ST:STUDY_SUMMARY                 	In this experimental study, we aimed to understand the potential factors within
ST:STUDY_SUMMARY                 	the secretions of young cells that could trigger the reverse aging of Mid-old
ST:STUDY_SUMMARY                 	cells. To investigate this phenomenon, we co-cultured young cells with Mid-old
ST:STUDY_SUMMARY                 	cells and observed a fascinating outcome: the Mid-old cells exhibited reverse
ST:STUDY_SUMMARY                 	aging and transformed into a more youthful state. To uncover the specific
ST:STUDY_SUMMARY                 	factors responsible for this reverse aging effect, we conducted a detailed
ST:STUDY_SUMMARY                 	analysis of the secreted factors from the young cells. Our analysis focused on a
ST:STUDY_SUMMARY                 	range of biomolecules, including lipids. However, despite our efforts, we did
ST:STUDY_SUMMARY                 	not identify any distinct factors that could be directly attributed to this
ST:STUDY_SUMMARY                 	remarkable reverse aging process.
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           	-	20210625_AJU_Y1_lipidomics	*Factor:Test sample	RAW_FILE_NAME=20210625_AJU_Y1_lipidomics.mzML
SUBJECT_SAMPLE_FACTORS           	-	20210625_AJU_Y2_lipidomics	*Factor:Test sample	RAW_FILE_NAME=20210625_AJU_Y2_lipidomics.mzML
SUBJECT_SAMPLE_FACTORS           	-	20210625_AJU_Y3_lipidomics	*Factor:Test sample	RAW_FILE_NAME=20210625_AJU_Y3_lipidomics.mzML
SUBJECT_SAMPLE_FACTORS           	-	20210625_AJU_M1_lipidomics	*Factor:Control sample	RAW_FILE_NAME=20210625_AJU_M1_lipidomics.mzML
SUBJECT_SAMPLE_FACTORS           	-	20210625_AJU_M2_lipidomics	*Factor:Control sample	RAW_FILE_NAME=20210625_AJU_M2_lipidomics.mzML
SUBJECT_SAMPLE_FACTORS           	-	20210625_AJU_M3_lipidomics	*Factor:Control sample	RAW_FILE_NAME=20210625_AJU_M3_lipidomics.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 harvested 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            	For lipid extraction from samples, a two-step method involving neutral and
SP:SAMPLEPREP_SUMMARY            	acidic extraction were used. At first, in neutral extraction, lipids from the
SP:SAMPLEPREP_SUMMARY            	samples were extracted according to the Folch method using a mixture of
SP:SAMPLEPREP_SUMMARY            	chloroform and methanol (2:1, v/v). The samples were vortexed and incubated on
SP:SAMPLEPREP_SUMMARY            	ice for 10 min. The samples were centrifuged at 13,800×g for 2 min at 4°C,
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to a new 1.5 mL tube. Next, in Acidic extraction,
SP:SAMPLEPREP_SUMMARY            	750 μL of chloroform:methanol:HCl (1N, 37%) (40:80:1, v/v/v) was added to the
SP:SAMPLEPREP_SUMMARY            	remaining samples. After incubating for 15 min at room temperature, 250 μL of
SP:SAMPLEPREP_SUMMARY            	cold chloroform and 450 μL of cold 0.1 M HCl were added to the sample. The
SP:SAMPLEPREP_SUMMARY            	mixture was vortexed for 1 min and centrifuged at 6,500×g for 2 min at 4°C.
SP:SAMPLEPREP_SUMMARY            	The lower organic phase was collected and combined with the prior extract. The
SP:SAMPLEPREP_SUMMARY            	sample was then dried using HyperVAC-MAX VC2200 centrifugal vacuum concentrator
SP:SAMPLEPREP_SUMMARY            	(Hanil Scientific Inc., Korea). Dried metabolite contents were reconstituted in
SP:SAMPLEPREP_SUMMARY            	50 µL of mobile phase solvent A:solvent B (2 :1, v/v) and then subjected to
SP:SAMPLEPREP_SUMMARY            	LC-MS/MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Q Exactive™ Hybrid Quadrupole-Orbitrap MS coupled to a 1290 Infinity UHPLC
CH:COLUMN_NAME                   	e Hypersil GOLD™ C18 HPLC column(2.1 × 100 mm; 1.9 μm particle size; Thermo
CH:COLUMN_NAME                   	Fischer Scientific)
CH:SOLVENT_A                     	Acetonitrile:Methanol:Water (19:19:2, v/v/v) + 20 mmol/L ammonium formate + 0.1%
CH:SOLVENT_A                     	formic acid (v/v)
CH:SOLVENT_B                     	2-propanol + 20 mmol/L ammonium formate + 0.1% formic acid (v/v)
CH:FLOW_GRADIENT                 	0–5 min, 5% B; 5–15 min, 5–30% B; 15–22 min, 30–90% B; 22–25 min,
CH:FLOW_GRADIENT                 	90% B; 25–26 min, 90–5% B; 26–30 min, 5% B. Parameters were set as
CH:FLOW_GRADIENT                 	follows: positive mode, spray voltage; 3.8 kV
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
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 Lipid Search 4.2TM
MS:MS_COMMENTS                   	(Thermo Fisher Scientific, Waltham, MA, USA). Lipid profiling under the
MS:MS_COMMENTS                   	following conditions: parent search; 0.2 Da, product search; 5.0 ppm, precursor
MS:MS_COMMENTS                   	ion mass tolerance; 8.0 ppm, M-score threshold; 2.0.
MS:MS_RESULTS_FILE               	ST002960_AN004861_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END