{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001090","ANALYSIS_ID":"AN001775","VERSION":"1","CREATED_ON":"November 6, 2018, 11:12 am"},

"PROJECT":{"PROJECT_TITLE":"NMR metabolomics study of Gemcitabine resistant cancer cells","PROJECT_TYPE":"NMR metabolomics","PROJECT_SUMMARY":"1D 1H NMR based differential metabolomics study of T3M4 cells, which are gemcitabine sensitive, and gemcitabine resistant T3M4 cells (GemR) were cultured with or without gemcitabine treatment. 1D 1H NMR spectra were collected from the samples and analyzed using MVAPACK software (http://bionmr.unl.edu/mvapack.php). Using multivariate analysis, unique responses to gemcitabine treatment were revealed, which may be useful in the clinical setting for monitoring a patient’s therapeutic response.","INSTITUTE":"University of Nebraska-Lincoln","DEPARTMENT":"Chemistry","LABORATORY":"Robert Powers and Pankaj Singh labs","LAST_NAME":"Gebregiworgis","FIRST_NAME":"Teklab","ADDRESS":"552 Hamilton Hall, 639 N. 12th Street, Lincoln, NE, 68588, USA","EMAIL":"teklab@huskers.unl.edu","PHONE":"(402) 472-3039","FUNDING_SOURCE":"NCI, NIGMS, AACR","CONTRIBUTORS":"Teklab Gebregiworgis, Fatema Bhinderwala, Vinee Purohit, Nina V. Chaika, Pankaj K. Singh, and Robert Powers"},

"STUDY":{"STUDY_TITLE":"NMR metabolomics study of Gemcitabine resistant cancer cells","STUDY_TYPE":"NMR metabolomics","STUDY_SUMMARY":"1D 1H NMR spectra were collected from the samples and analyzed using MVAPACK software (http://bionmr.unl.edu/mvapack.php). Multivariate analysis was conducted to study the metabolic phenotypes of the samples.","INSTITUTE":"University of Nebraska-Lincoln","DEPARTMENT":"Chemistry","LABORATORY":"Dr. Robert Powers and Pankaj Singh labs","LAST_NAME":"Gebregiworgis","FIRST_NAME":"Teklab","ADDRESS":"552 Hamilton Hall, 639 N. 12th Street, Lincoln, NE, 68588, USA","EMAIL":"teklab@huskers.unl.edu","PHONE":"(402) 472-3039"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_BIOSOURCE_OR_SUPPLIER":"Gift from Dr. Michael Hollingsworth","CELL_STRAIN_DETAILS":"T3M4"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"T3M4_01",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_02",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_03",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_04",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_05",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_06",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_07",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_08",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_09",
"Factors":{"Treatment":"No","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_10",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_11",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_12",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_13",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_14",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_15",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_16",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_17",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_18",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_19",
"Factors":{"Treatment":"Yes","Sensitivity":"Sensitive"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_20",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_21",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_22",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_23",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_24",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_25",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_26",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_27",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_28",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_29",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_30",
"Factors":{"Treatment":"No","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_31",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_32",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_33",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_34",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_35",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_36",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_37",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_38",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_39",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
},
{
"Subject ID":"-",
"Sample ID":"T3M4_40",
"Factors":{"Treatment":"Yes","Sensitivity":"Resistant"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For each of the cell culture, 1 x 106 cells were cultured and harvested at 70-80% confluence in a 100 mm Petri dish. The media was then removed by aspiration upon harvesting, and the cells were washed twice with PBS at pH 7.2. The cells were then lysed by the addition of 1 mL of an 80% methanol:water mixture at -80oC followed by incubation for 15 minutes in a -80oC freezer. The lysed cells were then transferred to a 1 ml Eppendorf tube using a cell scraper. The cell lysate was centrifuged at 16,200 g for 5 minutes and the supernatant was collected and transferred to a clean Eppendorf tube. 250 µL of nanopure water (Nanopure, Dubuque, IA) was added to the cell debris, the sample was mixed by pipetting, and then centrifuged as before. The two supernatants were combined, and the sample was dried by vacuum evaporation (SpeedVac® Plus, Savant, Thermo Scientific, Waltham, MA) followed by freeze-drying (Labconco, Kansas City, MO).","SAMPLE_TYPE":"Cultured cells","STORAGE_CONDITIONS":"-80?"},

"TREATMENT":{"TREATMENT_SUMMARY":"T3M4 wild type (WT) pancreatic cancer cells were cultured in DMEM with increasing concentrations of gemcitabine over a period of about 6 months. The resistance status of the cells at each gemcitabine dose was determined by calculating an IC50 using the MTT cytotoxicity assays. At the end of the 6-month treatment, the cell lines generated (GemR) were approximately 500-1000 fold resistant to gemcitabine as compared to the parental (WT) cells.Wild type T3M4 cells (WT), which are gemcitabine sensitive, and gemcitabine resistant T3M4 (GemR) were cultured with or without the addition of gemcitabine for the NMR metabolomics studies. A total of four cell culture groups were prepared that consisted of: (1) untreated T3M4 (WT) cells, (2) WT cells treated with 10 nM gemcitabine for a period of 12 hours (WT+), (3) untreated GemR cells, and (4) GemR cells treated with 10 nm gemcitabine for 12 hours (GemR+)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The dried metabolite extracts were reconstituted in 600 µL of 50 mM phosphate buffer in 99.8% D2O (Isotec, St. Louis, MO) at pH 7.2 (uncorrected). 50 µM of TMSP (3-(tetramethysilane) propionic acid-2,2,3,3-d4) were added for spectral referencing. The solution was mixed by gentle votexing and centrifuged at 13000 rpm for 5 min, and then the supernatant were transferred to 5 mm NMR tube."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"-","INSTRUMENT_NAME":"-","COLUMN_NAME":"-"},

"ANALYSIS":{"ANALYSIS_TYPE":"NMR","LABORATORY_NAME":"Dr. Robert Powers","OPERATOR_NAME":"Teklab Gebregiworgis"},

"NM":{"INSTRUMENT_NAME":"Bruker AVANCE DRX","INSTRUMENT_TYPE":"FT-NMR","NMR_EXPERIMENT_TYPE":"1D-1H","SPECTROMETER_FREQUENCY":"500 MHz","TEMPERATURE":"25","NUMBER_OF_SCANS":"128","DUMMY_SCANS":"16","SPECTRAL_WIDTH":"5483 HZ","NUM_DATA_POINTS_ACQUIRED":"32000","NMR_RESULTS_FILE":"ST001090_AN001775_Results.txt UNITS:ppm"}

}