{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001252","ANALYSIS_ID":"AN002078","VERSION":"1","CREATED_ON":"September 24, 2019, 3:24 pm"},

"PROJECT":{"PROJECT_TITLE":"Eicosanoid Expression in Dermal Fibroblasts","PROJECT_SUMMARY":"Quantitative lipidomics studies examining eicosanoid levels in primary dermal fibroblasts","INSTITUTE":"University of South Florida, Tampa","LAST_NAME":"Chalfant","FIRST_NAME":"Charles","ADDRESS":"4202 E Fowler Ave, Tampa, FL 33620","EMAIL":"cechalfant@usf.edu","PHONE":"8139747103"},

"STUDY":{"STUDY_TITLE":"Eicosanoid profiles of dermal fibroblasts","STUDY_SUMMARY":"The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2 with an ablated C1P interaction site (KI) to examine the cPLA2/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (PGE2) and increased levels of specific HETE species (5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2 was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key biological mechanisms by a defined protein:lipid interaction in vivo and provide new insights into cPLA2 function.","INSTITUTE":"University of South Florida, Tampa","LAST_NAME":"Chalfant","FIRST_NAME":"Charles","ADDRESS":"4202 E Fowler Ave, Tampa, FL 33620","EMAIL":"cechalfant@usf.edu","PHONE":"8139747103"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Patrick Media 1",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 2",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 3",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 4",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 5",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 6",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 7",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 8",
"Factors":{"Genotype":"Wild Type","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 9",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 10",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 11",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 12",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 13",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 14",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 15",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 16",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 17",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 18",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 19",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 20",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 21",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 22",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 23",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 24",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Unscratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 25",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 26",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 27",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 28",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 29",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 30",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 31",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 32",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 33",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 34",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 35",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 36",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 37",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 38",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 39",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 40",
"Factors":{"Genotype":"cPLA2α KI","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 41",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 42",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 43",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 44",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 45",
"Factors":{"Genotype":"Wild Type","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 46",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 47",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
},
{
"Subject ID":"-",
"Sample ID":"Patrick Media 48",
"Factors":{"Genotype":"cPLA2α KO","Treatment":"Scratched"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"pDFs obtained from WT, KI, and KO mice were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 ul pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h.","SAMPLE_TYPE":"Fibroblasts"},

"TREATMENT":{"TREATMENT_SUMMARY":"Primary Dermal Fibroblasts were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 l pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were stored at − 80 °C until the time of analysis. Lipids were extracted for eicosanoids. Eicosanoid Preparation Eicosanoids were extracted using a modified extraction process from previously described. (1, 2). 4 mL of media was combined with an IS mixture comprised of comprised of 10% methanol (400 μl), glacial acetic acid (20 μl), and internal standard (20 μl) containing the following deuterated eicosanoids (1.5 pmol/μl, 30 pmol total) (All standards purchased from Cayman Chemicals): (d4) 6keto-Prostaglandin F1α, (d4) Prostaglandin F2α, (d4) Prostaglandin E2, (d4) Prostaglandin D2, (d8) 5-Hydroxyeicosa-tetranoic acid (5-HETE), (d8) 12-Hydroxyeicosa-tetranoic acid (12-HETE) (d8) 15-Hydroxyeicosa-tetranoic acid (15-HETE), (d6) 20-Hydroxyeicosa-tetranoic acid (20-HETE), (d11) 8,9 Epoxyeicosa-trienoic acid, (d8) 14,15 Epoxyeicosa-trienoic acid, (d8) Arachidonic acid, (d5) Eicosapentaenoic acid, (d5) Docosahexaenoic acid, (d4) Prostaglandin A2, (d4) Leukotriene B4, (d4) Leukotriene C4, (d4) Leukotriene D4, (d4) Leukotriene E4, (d5) 5(S),6(R)-Lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso Prostaglandin F2α, (d11) (±)14,15-DHET, (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) Prostaglandin E1, (d4) Thromboxane B2, (d6) dihmo gamma linoleic acid, (d5) Resolvin D2, (d5) Resolvin D1, (d5) Maresin 2, and (d5) Resolvin D3. Samples and vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH2O (50:50;100 μl) prior to UPLC ESI-MS/MS analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Shimadzu Nexera X2","COLUMN_NAME":"Acentis Express C18 column"},

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

"MS":{"INSTRUMENT_NAME":"ABI Sciex 5500 QTrap","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"MALDI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Sciex Analyst software was used for analysis"},

"MS_METABOLITE_DATA":{
"Units":"pmol of lipid",

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