{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000898","ANALYSIS_ID":"AN001460","VERSION":"1","CREATED_ON":"November 13, 2017, 1:31 pm"},

"PROJECT":{"PROJECT_TITLE":"TAp73 is a marker of glutamine addiction in medulloblastoma","PROJECT_TYPE":"siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours","PROJECT_SUMMARY":"Metabolically-targeted therapies hold the promise of offering an effective and less toxic treatment for tumours including medulloblastoma, the most common malignant brain tumour of childhood. Current treatment relies on the sensitivity of these tumours to DNA damage that was discovered more than 50 years ago. Finding new tumour-specific susceptibilities to complement sensitivity to DNA damage is key to developing new more effective adjuvant therapies. The specific metabolic program of tumours is an attractive vulnerability, as restriction diet are low cost and easy to implement. Here, we present compelling pre-clinical evidence that glutamine restriction diet can be used as an adjuvant treatment for p73-expressing medulloblastoma.","INSTITUTE":"Queen Mary University of London","DEPARTMENT":"Blizard Institute","LABORATORY":"Centre for Genomics and Child Health","LAST_NAME":"Marino","FIRST_NAME":"Silvia","ADDRESS":"4 Newark Street, E1 2AT, London","EMAIL":"s.marino@qmul.ac.uk","PHONE":"+44 20 7882 2360","FUNDING_SOURCE":"Children with Cancer UK fellowship (Reference Nº2014/178)"},

"STUDY":{"STUDY_TITLE":"TAp73 is a marker of glutamine addiction in medulloblastoma","STUDY_TYPE":"siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours","STUDY_SUMMARY":"Metabolically-targeted therapies hold the promise of offering an effective and less toxic treatment for tumours including medulloblastoma, the most common malignant brain tumour of childhood. Current treatment relies on the sensitivity of these tumours to DNA damage that was discovered more than 50 years ago. Finding new tumour-specific susceptibilities to complement sensitivity to DNA damage is key to developing new more effective adjuvant therapies. The specific metabolic program of tumours is an attractive vulnerability, as restriction diet are low cost and easy to implement. Here, we present compelling pre-clinical evidence that glutamine restriction diet can be used as an adjuvant treatment for p73-expressing medulloblastoma.","INSTITUTE":"Queen Mary University of London","DEPARTMENT":"Blizard Institute","LABORATORY":"Centre for Genomics and Child Health","LAST_NAME":"Marino","FIRST_NAME":"Silvia","ADDRESS":"4 Newark Street, E1 2AT, London","EMAIL":"s.marino@qmul.ac.uk","PHONE":"+44 20 7882 2360","NUM_GROUPS":"2","TOTAL_SUBJECTS":"18","STUDY_COMMENTS":"We include 3 biological replicate with 3 technical replicates for each condition."},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_BIOSOURCE_OR_SUPPLIER":"ATCC","CELL_STRAIN_DETAILS":"Daoy (ATCC® HTB-186™)","SUBJECT_COMMENTS":"cancer cell line"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Control 1",
"Sample ID":"Control_1.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 1",
"Sample ID":"Control_1.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 1",
"Sample ID":"Control_1.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 2",
"Sample ID":"Control_2.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 2",
"Sample ID":"Control_2.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 2",
"Sample ID":"Control_2.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 3",
"Sample ID":"Control_3.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 3",
"Sample ID":"Control_3.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 3",
"Sample ID":"Control_3.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 4",
"Sample ID":"Control_4.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 4",
"Sample ID":"Control_4.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 4",
"Sample ID":"Control_4.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 5",
"Sample ID":"Control_5.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 5",
"Sample ID":"Control_5.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 5",
"Sample ID":"Control_5.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 6",
"Sample ID":"Control_6.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 6",
"Sample ID":"Control_6.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 6",
"Sample ID":"Control_6.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 7",
"Sample ID":"Control_7.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 7",
"Sample ID":"Control_7.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 7",
"Sample ID":"Control_7.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 8",
"Sample ID":"Control_8.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 8",
"Sample ID":"Control_8.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 8",
"Sample ID":"Control_8.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 9",
"Sample ID":"Control_9.1",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 9",
"Sample ID":"Control_9.2",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Control 9",
"Sample ID":"Control_9.3",
"Factors":{"treatment":"Control"}
},
{
"Subject ID":"Si p73 1",
"Sample ID":"Si p73_1.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 1",
"Sample ID":"Si p73_1.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 1",
"Sample ID":"Si p73_1.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 2",
"Sample ID":"Si p73_2.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 2",
"Sample ID":"Si p73_2.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 2",
"Sample ID":"Si p73_2.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 3",
"Sample ID":"Si p73_3.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 3",
"Sample ID":"Si p73_3.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 3",
"Sample ID":"Si p73_3.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 4",
"Sample ID":"Si p73_4.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 4",
"Sample ID":"Si p73_4.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 4",
"Sample ID":"Si p73_4.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 5",
"Sample ID":"Si p73_5.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 5",
"Sample ID":"Si p73_5.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 5",
"Sample ID":"Si p73_5.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 6",
"Sample ID":"Si p73_6.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 6",
"Sample ID":"Si p73_6.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 6",
"Sample ID":"Si p73_6.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 7",
"Sample ID":"Si p73_7.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 7",
"Sample ID":"Si p73_7.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 7",
"Sample ID":"Si p73_7.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 8",
"Sample ID":"Si p73_8.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 8",
"Sample ID":"Si p73_8.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 8",
"Sample ID":"Si p73_8.3",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 9",
"Sample ID":"Si p73_9.1",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 9",
"Sample ID":"Si p73_9.2",
"Factors":{"treatment":"Si p73"}
},
{
"Subject ID":"Si p73 9",
"Sample ID":"Si p73_9.3",
"Factors":{"treatment":"Si p73"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"After 48 hours of transfection the cells were washed three times with ice-cold PBS (3X 5mL) and the cells were collected using a cell scraper.","SAMPLE_TYPE":"Cell","COLLECTION_METHOD":"Scraper","TISSUE_CELL_IDENTIFICATION":"Daoy cell","TISSUE_CELL_QUANTITY_TAKEN":"protein normalization"},

"TREATMENT":{"TREATMENT_SUMMARY":"siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours","CELL_GROWTH_CONTAINER":"plate","CELL_GROWTH_CONFIG":"attached","CELL_GROWTH_RATE":"36 hours","CELL_INOC_PROC":"lipofectamine 3000","CELL_MEDIA":"DMEM + GlutaMAX medium (Gibco), supplemented with 10% v/v foetal bovine serum (FBS, Gibco) and penicillin/streptomycin (1 U/ml, Gibco),","CELL_ENVIR_COND":"37°C in humidified 5% CO2","CELL_HARVESTING":"After 48 h of transfection the cells were washed three times with ice-cold PBS (3x5 mL) and the celles were collected using a cell scraper. Ice-cold water (3 mL,sterile, MilliQ) was added to the samples. The suspension was transferred to a 15 mL polypropylene tube and then snap-frozen in liquid N2 and stored on ice for 5 min. The cells were lysed by two freeze-thaw cycles where the cells were thawed at 37°C in a water bath and frozen using liquid N2. Subsequently, the cells were sonicated in a","CELL_PCT_CONFLUENCE":"80-90%","CELL_MEDIA_LASTCHANGED":"48 hours"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The samples were thawed at room temperature and subjected to centrifugation for 17 min at 3000 rpm and 4°C. A quality control (QC) sample was created by pooling an equal volume from all samples. The aqueous supernatants were transferred to clean extraction tubes followed by addition of chloroform and methanol for the final proportion 2.85:4:4 water:methanol:chloroform. The extraction tubes were gently vortexed and then stored at 8°C for 20 min prior to centrifugation for 20 min at 3000 rpm and 4°C. The aqueous phases were recovered and evaporated to dryness at 40°C under N2. All samples were stored at -80°C after evaporation. Prior to analysis the samples were reconstituted in acetonitrile:Milli-Q water 90:10","PROCESSING_METHOD":"lysis, freeze thaw cycles and sonication (see cell harvesting)","PROCESSING_STORAGE_CONDITIONS":"Room temperature and 8°C during extraction, -80°C before and after extraction","EXTRACTION_METHOD":"Liquid Liquid Extraction using CHCl3:MeOH:H2O 4:4:2.85","EXTRACT_STORAGE":"Extracts were stored in -80°C after evaporation","SAMPLE_RESUSPENSION":"Acetonitrile:Milli-Q water 90:10","CELL_TYPE":"Daoy cells"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"The samples were analyzed on a Acquity UPLC I-class system from Waters. A HILIC-Amide column (1.7 μm, i.d. 2.1x50 mm) from Waters was used for sample separation and a non-linear gradient elution profile from 100 % A to 100 % B was used. Mobile phase A consisted of 90:10 acetonitrile/water with 10 mM ammonium formate and 0.1% FA while mobile phase B consisted of 50:50 acetonitrile/ water with 10 mM ammonium formate and 0.1% FA. The flow rate was 0.3 ml/min, the column temperature 40°C and the injection volume 5 µL.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Waters Acquity I-Class","COLUMN_NAME":"Waters Acquity BEH Amide (50 x 2.1mm, 1.7um)","FLOW_GRADIENT":"Non-linear gradient elution profile from 100 % A to 100 % B was used. In detail; 100% A was kept for 0.5 min then decreased non-linearly (slope-factor 8 in MassLynx) over 12.5 min to 100% B, 100% B was held for 3 min followed by 7 min at 100 % A to re-equilibrate the column for a total run-time of 23 min.","FLOW_RATE":"0.3 ml/min","COLUMN_TEMPERATURE":"40°C","SOLVENT_A":"90:10 acetonitrile/water with 10 mM ammonium formate and 0.1% FA","SOLVENT_B":"50:50 acetonitrile/ water with 10 mM ammonium formate and 0.1% FA","INJECTION_TEMPERATURE":"4°C","SAMPLE_INJECTION":"5 μl","ANALYTICAL_TIME":"23 min total sample run-time","RANDOMIZATION_ORDER":"All samples were analyzed in a randomized order with three QC injections interspaced every eleventh injection"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Analytical Pharmaceutical Chemistry, Department of Medicinal chemistry, Uppsala University","OPERATOR_NAME":"Ida Erngren"},

"MS":{"INSTRUMENT_NAME":"Waters Synapt G2 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function.","CAPILLARY_TEMPERATURE":"500°C","CAPILLARY_VOLTAGE":"1 kV","COLLISION_ENERGY":"20- 45 eV collision ramp, MSE acquistion","SOURCE_TEMPERATURE":"120°C","DATAFORMAT":"The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function. The resulting dataset was exported to Microsoft Excel and all features with a retention time less than 45 s were removed. The data was normalized using median fold change. After normalization, all features with coefficient of variance (CV) >30 % in the QC samples were excluded","SCAN_RANGE_MOVERZ":"m/z 50-800","MS_RESULTS_FILE":"ST000898_AN001460_Results.txt UNITS:Peak Area"}

}