{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003068","ANALYSIS_ID":"AN005025","VERSION":"1","CREATED_ON":"February 7, 2024, 10:06 am"},

"PROJECT":{"PROJECT_TITLE":"Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation of intracellular taurine metabolism - Study #2","PROJECT_TYPE":"Untargeted Metabolomics analysis","PROJECT_SUMMARY":"Colonization of the human stomach with H. pylori strains producing active forms of a secreted toxin (VacA) is associated with an increased risk of peptic ulcer disease and gastric cancer, compared to colonization with strains producing hypoactive forms of VacA. Previous studies have shown that VacA causes cell vacuolation and mitochondrial dysfunction. In this study, we sought to define the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared to control cells. Pathway analysis suggested that VacA caused alterations in taurine and hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of VacA, but not hypoactive s2m1 or 6-27 VacA mutant proteins (defective in membrane channel formation), caused reductions in taurine and hypotaurine levels. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Untargeted global metabolomics of AZ-521 cells or AGS cells intoxicated with VacA in the presence or absence of extracellular taurine showed that taurine was the main intracellular metabolite significantly altered by extracellular taurine supplementation. These results indicate that VacA causes alterations in cellular taurine metabolism and indicate that repletion of taurine is sufficient to attenuate VacA-induced cell death.","INSTITUTE":"Vanderbilt University","DEPARTMENT":"Chemistry","LABORATORY":"Center for Innovative Technology","LAST_NAME":"CODREANU","FIRST_NAME":"SIMONA","ADDRESS":"1234 STEVENSON CENTER LANE","EMAIL":"SIMONA.CODREANU@VANDERBILT.EDU","PHONE":"6158758422"},

"STUDY":{"STUDY_TITLE":"Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation of intracellular taurine metabolism _ Study #2","STUDY_TYPE":"untargeted metabolomics analysis","STUDY_SUMMARY":"Colonization of the human stomach with H. pylori strains producing active forms of a secreted toxin (VacA) is associated with an increased risk of peptic ulcer disease and gastric cancer, compared to colonization with strains producing hypoactive forms of VacA. Previous studies have shown that VacA causes cell vacuolation and mitochondrial dysfunction. In this study, we sought to define the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared to control cells. Pathway analysis suggested that VacA caused alterations in taurine and hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of VacA, but not hypoactive s2m1 or 6-27 VacA mutant proteins (defective in membrane channel formation), caused reductions in taurine and hypotaurine levels. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Untargeted global metabolomics of AZ-521 cells or AGS cells intoxicated with VacA in the presence or absence of extracellular taurine showed that taurine was the main intracellular metabolite significantly altered by extracellular taurine supplementation. These results indicate that VacA causes alterations in cellular taurine metabolism and indicate that repletion of taurine is sufficient to attenuate VacA-induced cell death.","INSTITUTE":"Vanderbilt University","DEPARTMENT":"Chemistry","LABORATORY":"Center for Innovative Technology","LAST_NAME":"CODREANU","FIRST_NAME":"SIMONA","ADDRESS":"1234 STEVENSON CENTER LANE","EMAIL":"SIMONA.CODREANU@VANDERBILT.EDU","PHONE":"6158758422"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENOTYPE_STRAIN":"AGS and AZ-521","SPECIES_GROUP":"AGS and AZ-521"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"A1",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_A1"}
},
{
"Subject ID":"-",
"Sample ID":"A2",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_A2"}
},
{
"Subject ID":"-",
"Sample ID":"A3",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_A3"}
},
{
"Subject ID":"-",
"Sample ID":"A4",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_A4"}
},
{
"Subject ID":"-",
"Sample ID":"A5",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_A5"}
},
{
"Subject ID":"-",
"Sample ID":"B1",
"Factors":{"Treatment":"VacA_WT_Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_B1"}
},
{
"Subject ID":"-",
"Sample ID":"B2",
"Factors":{"Treatment":"VacA_WT_Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_B2"}
},
{
"Subject ID":"-",
"Sample ID":"B3",
"Factors":{"Treatment":"VacA_WT_Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_B3"}
},
{
"Subject ID":"-",
"Sample ID":"B4",
"Factors":{"Treatment":"VacA_WT_Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_B4"}
},
{
"Subject ID":"-",
"Sample ID":"B5",
"Factors":{"Treatment":"VacA_WT_Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_B5"}
},
{
"Subject ID":"-",
"Sample ID":"C1",
"Factors":{"Treatment":"VacA_Δ6-27 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_C1"}
},
{
"Subject ID":"-",
"Sample ID":"C2",
"Factors":{"Treatment":"VacA_Δ6-27 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_C2"}
},
{
"Subject ID":"-",
"Sample ID":"C3",
"Factors":{"Treatment":"VacA_Δ6-27 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_C3"}
},
{
"Subject ID":"-",
"Sample ID":"C4",
"Factors":{"Treatment":"VacA_Δ6-27 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_C4"}
},
{
"Subject ID":"-",
"Sample ID":"C5",
"Factors":{"Treatment":"VacA_Δ6-27 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_C5"}
},
{
"Subject ID":"-",
"Sample ID":"D1",
"Factors":{"Treatment":"VacA_s2m1 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_D1"}
},
{
"Subject ID":"-",
"Sample ID":"D2",
"Factors":{"Treatment":"VacA_s2m1 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_D2"}
},
{
"Subject ID":"-",
"Sample ID":"D3",
"Factors":{"Treatment":"VacA_s2m1 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_D3"}
},
{
"Subject ID":"-",
"Sample ID":"D4",
"Factors":{"Treatment":"VacA_s2m1 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_D4"}
},
{
"Subject ID":"-",
"Sample ID":"D5",
"Factors":{"Treatment":"VacA_s2m1 Toxin"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20230107_RPLCp_FMS_Cover_D5"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"AGS cells or AZ-521 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, or minimal essential medium (MEM) containing 10% fetal bovine serum and 5% nonessential amino acids, respectively. AGS cells were seeded at 2x104 cells/well into 96-well plates and incubated overnight. Cultured cells were then incubated with 20 ug/mL purified VacA [activated with by addition of HCl to a pH of 3] in medium supplemented with 5 mM NH4Cl. Following intoxication, the media was removed, and cells were washed with PBS. Cells were detached by incubation with trypsin for 5 minutes and collected via centrifugation at 4°C at 1,000 rpm for 4 minutes. Trypsin was removed, cells were once again washed with PBS, and the cells were then flash-frozen in liquid nitrogen and stored at -70C.","SAMPLE_TYPE":"Cultured cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were incubated in media containing 20 ug/mL of purified VacA toxin and 5 mM ammonium chloride."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Briefly, cell pellets were normalized by total protein (200 ug) and the corresponding cell supernatants were normalized by volume (200 uL). Metabolites were extracted with methanol/water 80:20. Heavy labeled phenylalanine-D8 and biotin-D2 were added to individual samples prior to protein precipitation. Following overnight incubation at -80°C, precipitated proteins were pelleted by centrifugation at 10,000 rpm for 10 min and metabolite extracts were transferred into two Eppendorf tubes in equal amounts and dried down in vacuo and stored at -80°C.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)","SOLVENT_A":"100% water, 0.1% Formic Acid","SOLVENT_B":"80:20 acetonitrile:water, 0.1% Formic Acid","FLOW_GRADIENT":"30 min; 95%A, 5%B","FLOW_RATE":"0.25 mL/min","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Mass spectrometry raw data was imported, processed, normalized, and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK).","MS_RESULTS_FILE":"ST003068_AN005025_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes"}

}