{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003328","ANALYSIS_ID":"AN005452","PROJECT_ID":"PR002070","VERSION":"1","CREATED_ON":"July 18, 2024, 10:16 pm"},

"PROJECT":{"PROJECT_TITLE":"Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem Cell-Derived Model of Multiple Sclerosis","PROJECT_SUMMARY":"Senescent neural progenitor cells have been identified in brain lesions of people with progressive multiple sclerosis (PMS). However, their role in disease pathobiology and contribution to the lesion environment remains unclear. By establishing directly induced neural stem/progenitor cell (iNSC) lines from PMS patient fibroblasts, we studied their senescent phenotype in vitro. Senescence was strongly associated with inflammatory signaling, hypermetabolism, and the senescence associated secretory phenotype (SASP). PMS-derived iNSCs displayed increased glucose-dependent fatty acid and cholesterol synthesis, which resulted in the accumulation of cholesteryl ester-enriched lipid droplets. An HMG-CoA reductase-mediated lipogenic state was found to induce secretion of the SASP in PMS iNSC conditioned media via transcriptional regulation by cholesterol-dependent transcription factors. SASP from PMS iNSCs induced neurotoxicity. Chemical targeting of HMG-CoA reductase using the cholesterol-lowering drug simvastatin (SV) reprogrammed the SASP and rescued neurotoxicity. Our findings suggest a disease-associated, cholesterol-related, hypermetabolic phenotype of PMS iNSCs that leads to neurotoxic signaling and is rescuable pharmacologically.","INSTITUTE":"University of Colorado Denver","LABORATORY":"Lab of Angelo D''Alessandro in collaboration with lab of Stefano Pluchino (Univ of Cambridge)","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"STUDY":{"STUDY_TITLE":"Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem Cell-Derived Model of Multiple Sclerosis - cellular lipidomics","STUDY_SUMMARY":"Lipidomics analysis was performed on directly induced neural stem/progenitor cell (iNSC) lines derived from fibroblasts of patients with progressive multiple sclerosis (PMS) versus age matched controls (AMC) treated or untreated with cholesterol synthesis inhibitor simvastatin.","INSTITUTE":"University of Colorado Denver","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","AGE_OR_AGE_RANGE":"25-63","GENDER":"Male and female"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"A",
"Sample ID":"AMC A P1",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-001"}
},
{
"Subject ID":"A",
"Sample ID":"AMC A P2",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-002"}
},
{
"Subject ID":"A",
"Sample ID":"AMC A P3",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-003"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B P1",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-004"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B P2",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-005"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B P3",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-006"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C P1",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-007"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C P2",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-008"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C P3",
"Factors":{"Disease status":"AMC","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-009"}
},
{
"Subject ID":"A",
"Sample ID":"AMC A SV P1",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-010"}
},
{
"Subject ID":"A",
"Sample ID":"AMC A SV P2",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-011"}
},
{
"Subject ID":"A",
"Sample ID":"AMC A SV P3",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-012"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B SV P1",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-013"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B SV P2",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-014"}
},
{
"Subject ID":"B",
"Sample ID":"AMC B SV P3",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-015"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C SV P1",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-016"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C SV P2",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-017"}
},
{
"Subject ID":"C",
"Sample ID":"AMC C SV P3",
"Factors":{"Disease status":"AMC","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-018"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A P1",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-019"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A P2",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-020"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A P3",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-021"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B P1",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-022"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B P2",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-023"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B P3",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-024"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C P1",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-025"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C P2",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-026"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C P3",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-027"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D P1",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-028"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D P2",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-029"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D P3",
"Factors":{"Disease status":"PMS","Treatment":"untreated","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-030"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A SV P1",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-031"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A SV P2",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-032"}
},
{
"Subject ID":"A",
"Sample ID":"PMA A SV P3",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-033"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B SV P1",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-034"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B SV P2",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-035"}
},
{
"Subject ID":"B",
"Sample ID":"PMA B SV P3",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-036"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C SV P1",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-037"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C SV P2",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-038"}
},
{
"Subject ID":"C",
"Sample ID":"PMS C SV P3",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-039"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D SV P1",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-040"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D SV P2",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-041"}
},
{
"Subject ID":"D",
"Sample ID":"PMS D SV P3",
"Factors":{"Disease status":"PMS","Treatment":"SV","Sample source":"induced neural stem cell"},
"Additional sample data":{"RAW_FILE_NAME":"DS1-93-042"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"3-5 mm skin biopsies were collected in Biopsy Collection Medium (RPMI 1460 [Thermo Fisher] with 1X Antibiotic-Antimycotic [Thermo Fisher]), cut into smaller pieces (<1 mm) and plated onto a TC-treated 35 mm dish in Biopsy Plating Medium, composed by Knockout DMEM (Thermo Fisher), 2 mM GlutaMax (Thermo Fisher), 0.1 mM non-essential amino acids (Thermo Fisher), 0.1 mM β-mercaptoethanol (Thermo Fisher), 10% Fetal Bovine Serum (FBS) (Thermo Fisher), 1X penicillin-streptomycin (P/S) (Thermo Fisher) and 1% nucleosides (Millipore). Once the first fibroblasts migrated out of the biopsies, the cultures were maintained in growth medium (DMEM Glutamax I [Thermo Fisher] supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1 mM sodium pyruvate (Thermo Fisher) at 37°C with 5 % CO2 and fed every 3-4 days. After reaching 90% confluency the fibroblasts were detached with trypsin-EDTA 0.05% for 5 min followed by neutralization in DMEM and spun down at 300xg for 5 min. They were split 1:4 into growth media. Fibroblasts at passage 3-5 were reprogrammed to iPSCs using the integration free technology based on non-modified RNA plus microRNA (kit from REPROCELL, formerly Stemgent), following manufacturer’s instructions. About 25x10^3 fibroblasts/well were plated onto Matrigel-coated 12-well plates in culture medium for 24 hours and then in NuFF-conditioned Pluriton reprogramming medium with B18R. Cells were transfected for 11 consecutive days using Stemfect as following: day 0 microRNA only, days 1-3 RNA only, day 4 microRNA plus RNA, days 5-11 RNA only. From day 11, TRA-1-60+ colonies (live stained) were manually picked and re-plated on mouse embryonic fibroblasts in HUESM medium (Knockout-DMEM, 20% knock-out serum, glutamax 2 mM, NEAA 0.1 mM, 1X P/S and β-mercaptoethanol 0.1 mM [Thermo Fisher]). Pluripotent colonies were passaged and adapted to feeder-free conditions with hESC matrigel (Corning) and mTeSR1 (STEMCELL Technologies) medium. The WIBJ iPSC line, obtained from Cambridge BioResource as a gift from Alessandra Granada, was reprogrammed from fibroblasts using the CytoTune 1 non-integrating kit (Thermo Fisher). iPSC media was changed every day. When confluent, cells were lifted using accutase (Thermo Fisher), spun at 300xg for 5 min, and split 1:10-1:20 onto hESC-matrigel coated plates with Y-27632 (10 uM) (Thermo Fisher) in mTeSR1 media. To generate directly induced neural stem cells (iNSCs) from fibroblasts we used a nonintegrating Sendai virus-based direct conversion strategy, as previously described.(https://doi.org/10.3791/52831) Briefly, fibroblasts were seeded at 75,000/well in fibroblast media in a non-coated 12-well. On the next day the fibroblasts were transduced using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher) with hKOS (MOI: 3), hc-Myc (MOI: 3), hKlf4 (MOI: 3). The day following transfection, the medium was switched to neural induction medium (NIM) [DMEM:F12 and Neurobasal (1:1), supplemented with N2 supplement (1x, ThermoFisher), 1% glutamax, B27 supplement (1x, ThermoFisher), CHIR99021 (3 µM, Cell Guidance Systems), SB-431542 (2 µM, Cayman Chemicals) and hLIF (10 ng/ml, Cell Signaling Technology)], and cells were moved to 39°C with 5% CO2 to achieve viral clearance over 14 days. A few samples were collected here to generate positive controls for quality control assays. Medium changes were performed every other day. Following 25 days of transfection, iNSC colonies were manually selected, seeded onto Growth Factor Reduced (GFR) Matrigel Matrix (1:20 in DMEM/F12) coated plates for expansion, and subjected to quality control assays. iNSCs were maintained in NIM media until 70% confluent, then lifted using accutase (ThermoFisher), spun at 300xg for 3 mins, and plated onto GFR-Matrigel coated plates with Y-27632 (10 µM, Miltenyi Biotec) between 1:3-1:5 in NIM media. Media was changed every second day as needed. Experiments were performed on cells from passages 20-40.","SAMPLE_TYPE":"Stem cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"iNSCs were seeded in NIM with Y-27632 (10 µM) at a density of 100,000 cells/cm2 GFR-matrigel coated wells, in technical triplicates per line. Simvastatin-treated cells were exposed to 10 uM simvastatin for 48 hours. Sample collection was performed on ice. Conditioned culture medium was removed then cells were lifted using accutase and spun at 300 x g for 5 mins. Cell pellets were resuspended in PBS for counting and spun once more at 300 x g for 5 mins. Supernatants were aspirated and cell pellets were stored at -80 degrees C until sample processing."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Lipids from frozen cell pellets were extracted with cold methanol at a concentration of 2 million cells/mL. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.","PROCESSING_STORAGE_CONDITIONS":"4℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Negative C18","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)","SOLVENT_A":"75% water/25% acetonitrile; 5 mM ammonium acetate","SOLVENT_B":"90% isopropanol/10% acetonitrile; 5 mM ammonium acetate","FLOW_GRADIENT":"0-3 min 10-95% B at 0.3 mL/min, 3-4.2 min hold at 95% B at 0.3 mL/min, 4.2-4.3 min drop to 10% B at 0.45 mL/min, 4.3-4.9 min hold at 10%B while lowering flow rate to 0.4 mL/min, 4.9-5 min hold at 10%B while lowering flow rate to 0.3 mL/min.","FLOW_RATE":"0.3 mL/min - 0.45 mL/min","COLUMN_TEMPERATURE":"45","SAMPLE_INJECTION":"10 uL"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

"Data":[{"Metabolite":"Cer(d18:1_16:0)","AMC A P1":"22235.42","AMC A P2":"793.99","AMC A P3":"13464.16","AMC B P1":"34633.09","AMC B P2":"58833.82","AMC B P3":"36699.34","AMC C P1":"47205.47","AMC C P2":"62109.32","AMC C P3":"73676.11","AMC A SV P1":"9039.71","AMC A SV P2":"7800.35","AMC A SV P3":"4389.32","AMC B SV P1":"15368.12","AMC B SV P2":"25433.56","AMC B SV P3":"24338.35","AMC C SV P1":"39489.91","AMC C SV P2":"43178.03","AMC C SV P3":"36364.69","PMA A P1":"50486.74","PMA A P2":"60686.98","PMA A P3":"51689.62","PMA B P1":"52074.44","PMA B P2":"44574.18","PMA B P3":"40911.24","PMS C P1":"43122.38","PMS C P2":"80221.13","PMS C P3":"83332.94","PMS D P1":"106614.08","PMS D P2":"52421.88","PMS D P3":"75072.90","PMA A SV P1":"25518.33","PMA A SV P2":"44567.18","PMA A SV P3":"67147.39","PMA B SV P1":"48243.85","PMA B SV P2":"50524.74","PMA B SV P3":"47209.64","PMS C SV P1":"52081.53","PMS C SV P2":"38495.13","PMS C SV P3":"62567.63","PMS D SV P1":"48890.28","PMS D SV P2":"36520.15","PMS D SV P3":"32818.10"},{"Metabolite":"Cer(d18:1_18:0)","AMC A P1":"2145.51","AMC A P2":"83.42","AMC A P3":"1553.33","AMC B P1":"4624.20","AMC B P2":"5720.74","AMC B P3":"4627.40","AMC C P1":"8348.58","AMC C P2":"13326.07","AMC C P3":"10939.28","AMC A SV P1":"1980.08","AMC A SV P2":"1028.44","AMC A SV P3":"615.97","AMC B SV P1":"2787.66","AMC B SV P2":"4245.58","AMC B SV P3":"6609.51","AMC C SV P1":"8905.42","AMC C SV P2":"9760.60","AMC C SV P3":"7138.53","PMA A P1":"8039.41","PMA A P2":"11165.39","PMA A P3":"9554.83","PMA B P1":"8457.41","PMA B P2":"6791.28","PMA B P3":"4634.97","PMS C P1":"9564.45","PMS C P2":"16746.69","PMS C P3":"16279.26","PMS D P1":"22091.51","PMS D P2":"9384.68","PMS D P3":"11574.87","PMA A SV P1":"7867.45","PMA A SV P2":"13658.29","PMA A SV P3":"16153.70","PMA B SV P1":"11293.71","PMA B SV P2":"14414.41","PMA B SV P3":"11967.88","PMS C SV P1":"16256.09","PMS C SV P2":"12623.26","PMS C SV P3":"16247.26","PMS D SV P1":"17498.29","PMS D SV P2":"14689.29","PMS D SV P3":"8417.66"},{"Metabolite":"Cer(d18:1_24:1)","AMC A P1":"4099.87","AMC A P2":"149.13","AMC A P3":"2407.23","AMC B P1":"6019.82","AMC B P2":"8279.25","AMC B P3":"4920.60","AMC C P1":"7593.53","AMC C P2":"11683.51","AMC C P3":"12900.51","AMC A SV P1":"1436.37","AMC A SV P2":"1125.93","AMC A SV P3":"609.06","AMC B SV P1":"2250.75","AMC B SV P2":"3034.58","AMC B SV P3":"3694.47","AMC C SV P1":"5273.96","AMC C SV P2":"5671.74","AMC C SV P3":"5071.63","PMA A P1":"13701.17","PMA A P2":"17629.67","PMA A P3":"14565.97","PMA B P1":"14133.42","PMA B P2":"9855.42","PMA B P3":"7935.71","PMS C P1":"10965.09","PMS C P2":"25860.88","PMS C P3":"22440.36","PMS D P1":"21339.26","PMS D P2":"14039.85","PMS D P3":"15362.42","PMA A SV P1":"4737.05","PMA A SV P2":"8681.69","PMA A SV P3":"13722.04","PMA B SV P1":"10220.81","PMA B SV P2":"10557.25","PMA B SV P3":"12604.72","PMS C SV P1":"11902.53","PMS C SV P2":"10818.08","PMS C SV P3":"13855.79","PMS D SV P1":"9749.51","PMS D SV 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