{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002445","ANALYSIS_ID":"AN003983","VERSION":"1","CREATED_ON":"January 17, 2023, 12:14 pm"},

"PROJECT":{"PROJECT_TITLE":"Mouse Neutrophil NTEM eicosanoids WT vs KI vs KO","PROJECT_SUMMARY":"Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, which are multi-phased physiological responses. Eicosanoids, a class of bioactive lipids, play a major regulatory role in these physiologies. In this study, the ablation of the ceramide-1-phosphate (C1P) interaction site in the eicosanoid biosynthetic enzyme, group IVA cytosolic phospholipase A2, in mice (cPLA2a-KI mice) resulted in enhanced and sustained neutrophil infiltration into both wounds and the peritoneum during the inflammatory phase of wound healing and sepsis. Enhanced neutrophil infiltration (i.e., neutrophilia) was associated with significant improvements in wound healing and the survival of mice to sepsis. As neutrophilia at the site of injury or infection normally associates with a poor outcome, our laboratory investigated this Neutrophil Conundrum by characterizing the cPLA2a-KI neutrophils, which showed enhanced N2-subtype markers, trans-endothelial migration, phagocytosis and VEGF with a concomitant decrease in TNFa, neutrophil extracellular trap production, N1-subtype markers, and endothelial cell damage versus wild-type neutrophils. This N2 polarization of cPLA2a-KI neutrophils was due to an induction of the 5-HETE/5-oxo-ETE biosynthetic pathway and activation of the OXER1 receptor. Unbiased proteomics identified perturbations in the pentose phosphate pathway (PPP) specific to OXER1 signaling in the cPLA2a-KI neutrophils, and modulation of the PPP recapitulated specific aspects of the N2 phenotype. Thus, C1P via cPLA2a negatively regulates 5-oxo-ETE biosynthesis, which controls neutrophil polarization via the PPP","INSTITUTE":"University of South Florida","DEPARTMENT":"CAS","LABORATORY":"Chalfant","LAST_NAME":"Maus","FIRST_NAME":"Kenneth","ADDRESS":"4202 E Fowler Ave, Tampa, FL, 33620, USA","EMAIL":"kmaus@usf.edu","PHONE":"8139283137"},

"STUDY":{"STUDY_TITLE":"The interaction of ceramide-1-phosphate with group IVA cytosolic phospholipase A2 modulates neutrophil polarization during inflammatory responses","STUDY_SUMMARY":"Bone marrow-derived mouse neutrophils from wildtype, cPLA2alpha-knockin (interaction site on cPLA2alpha for its substrate C1P was ablated), and cPLA2alpha-knockout (cPLA2alpha gene mutated) mice were exposed to 4 hours of trans-endothelial migration and resulting eicosanoids were analyzed (eg, PGE2, PGD2, 5-HETE, and 5-oxo-ETE).","INSTITUTE":"University of South Florida","LAST_NAME":"Maus","FIRST_NAME":"Kenneth","ADDRESS":"4202 E Fowler Ave, CMMB - NES 107 - Chalfant Lab","EMAIL":"kmaus@usf.edu","PHONE":"8139283137"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"balbc, blk6 background","AGE_OR_AGE_RANGE":"10-14 weeks","GENDER":"Male and female","ANIMAL_HOUSING":"USF College of MEdicine","ANIMAL_LIGHT_CYCLE":"Normal","ANIMAL_FEED":"Ad libdum","ANIMAL_WATER":"Ad libdum"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Kenny 1",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 2",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 3",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 4",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 5",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 6",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 7",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 8",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 9",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 10",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 11",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 12",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 13",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny 14",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny and Anika Eicosanoids_reinject.WIFF"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 1",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 2",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 3",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 4",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 5",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 6",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 7",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 8",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 9",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 10",
"Factors":{"Genotype":"Wild-type","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 11",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 12",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 13",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 14",
"Factors":{"Genotype":"cPLA2a-KI","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 15",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 16",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 17",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Kenny Eicosanoids 18",
"Factors":{"Genotype":"cPLA2a-KO","Treatment":"NTEM"},
"Additional sample data":{"RAW_FILE_NAME":"Kenny Eicosanoids.wiff"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"HUVECs were seeded on the upper wells Corning® Transwell® polycarbonate membrane inserts with 3.0 µm pores (Sigma-Aldrich CLS3415) inserted into a 24-well tissue culture plate at a density of 1.6 x 106 cells per ml in 250 µl volume. Inflammatory response was induced by adding TNFα (Cayman #32020, 20 ng/ml) 16-18 hours before addition of PNs. PNs were then isolated from mouse bone marrow and added to the upper well while HUVEC media containing lipopolysaccharides (LPS) from E. coli (Millipore Sigma #L3023, 1 µg/ml) was added to the bottom well as a chemoattractant. After time indicated (e.g., 4 hours), media and cells from the upper and lower wells were extracted, counted, combined, centrifuged, and washed in preparation for UHPLC-MS/MS.","SAMPLE_TYPE":"Bone marrow"},

"TREATMENT":{"TREATMENT_SUMMARY":"Eicosanoids and/or inhibitors (Cayman Chemical) were applied at the following concentrations: 1.0 nM 5-HETE (#34210), 1.0 nM 5-oxo-ETE (#34250), 7.5 nM MK886 (#21753), 25 µM Gue1654 (#29686), 100 nM physcion (Santa Cruz Biotech SC-205805). Cells were allowed to equilibrate with inhibitors for 30 minutes before experimentation via NTEM and subsequent collection and processing for UHPLC-MS/MS."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Media collection: Add proper volumes to achieve final concentrations of 10% MeOH and 0.5% glacial acetic acid. Then add 20 μL eicosanoid internal standard. Example: For 4 mL media add 400 μL of 100% MeOH and 20 μL of glacial acetic acid. • Example, for 24 samples, prep for 26: o 2.6 mL 100% MeOH (100 μL x 26) o 130 μL glacial acetic acid (5 μL x 26) o 520 μL eicosanoid IS (20 μL x 26) Columns: Set the manometer on the vacuum chamber to 5 mmHg. Precondition columns: Elute 2 mL MeOH, stop, then elute 2 mL H2O. Load sample and tighten the caps. Wash: Add 2 mL of 5% MeOH to the sample vial. Vortex and apply to the column under vacuum. Allow to run dry for 30 seconds. Stop vacuum, remove manifold & lay on side. Change out glass vials and elute: Apply 2 mL Isopropyl alcohol to column and equilibrate for 1 minute. Elute under vacuum and run dry for 30 seconds. Concentration: The solvent is removed by speed vac for !5 hours. Eicosanoids are re-dissolved in 100 μL of 50% EtOH in HPLC water. Centrifuge @ 4000 RPM for 10 minutes to pellet. Transfer supernatant into glass 0.1 mL mini conicals and load into white slots in HPLC tray."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Shimadzu Nexara X2 LC-30AD","COLUMN_NAME":"MilliporeSigma Supelco Ascentis Express C18 (150 x 4.6 mm, 2.7 um)","SOLVENT_A":"20% acetonitrile/80% water; 0.02% formic acid","SOLVENT_B":"20% acetonitrile/80% isopropanol; 0.02% formic acid","FLOW_GRADIENT":"The column was equilibrated with 100% Solvent A for 5 min and then 10 µl of sample was injected. 100% Solvent A was used for the first two minutes of elution. Solvent B was increased in a linear gradient to 25% Solvent B at 3 min, to 30% at 6 min., to 55% at 6.1 min, to 70% at 10 min, and to 100% at 10.10 min. 100% Solvent B was held constant until 13.0 min, where it was decreased to 0% Solvent B and 100% Solvent A from 13.0 min to 13.1 min. From 13.1 min to 14.0 min. Solvent A was held constant at 100%","FLOW_RATE":"0.5 ml/min","COLUMN_TEMPERATURE":"40"},

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

"MS":{"INSTRUMENT_NAME":"ABI Sciex 5500 QTrap","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Eicosanoids were analyzed via mass spec using an AB Sciex Triple Quad 5500 Mass Spectrometer as previously described by Mitra et al., 2007 (FEBS Lett 581, 581–793). Q1 and Q3 were set to detect distinctive precursor and product ion pairs. Ions were fragmented in Q2 using N2 gas for collisionally induced dissociation. Analysis used multiple-reaction monitoring in negative-ion mode. Eicosanoids were monitored using precursor → product MRM pairs. The mass spectrometer parameters as previously described by Chalfant et al., 2001 and Wijesinghe et al., 2013 were: Curtain Gas: 20; CAD: Medium; Ion Spray Voltage: -4500V; Temperature: 300°C; Gas 1: 40; Gas 2: 60; Declustering Potential, Collision Energy, and Cell Exit Potential vary per transition"},

"MS_METABOLITE_DATA":{
"Units":"pmol/mL",

"Data":[{"Metabolite":"PGE2","Kenny 1":"7.58E+03","Kenny 2":"5.27E+03","Kenny 3":"4.67E+03","Kenny 4":"5.29E+03","Kenny 5":"6.18E+03","Kenny 6":"4.18E+03","Kenny 7":"4.70E+05","Kenny 8":"4.60E+05","Kenny 9":"4.50E+05","Kenny 10":"6.78E+05","Kenny 11":"4.96E+05","Kenny 12":"3.98E+05","Kenny 13":"4.65E+05","Kenny 14":"3.77E+05","Kenny Eicosanoids 1":"4.03E+05","Kenny Eicosanoids 2":"1.60E+05","Kenny Eicosanoids 3":"1.37E+05","Kenny Eicosanoids 4":"2.17E+05","Kenny Eicosanoids 5":"4.40E+05","Kenny Eicosanoids 6":"3.87E+05","Kenny Eicosanoids 7":"1.02E+05","Kenny Eicosanoids 8":"2.49E+05","Kenny Eicosanoids 9":"6.01E+05","Kenny Eicosanoids 10":"7.00E+05","Kenny Eicosanoids 11":"5.22E+05","Kenny Eicosanoids 12":"8.84E+03","Kenny Eicosanoids 13":"1.40E+05","Kenny Eicosanoids 14":"1.14E+05","Kenny Eicosanoids 15":"1.14E+06","Kenny Eicosanoids 16":"1.12E+06","Kenny Eicosanoids 17":"1.19E+06","Kenny Eicosanoids 18":"1.22E+06"},{"Metabolite":"PGD2","Kenny 1":"0.00E+00","Kenny 2":"0.00E+00","Kenny 3":"0.00E+00","Kenny 4":"0.00E+00","Kenny 5":"0.00E+00","Kenny 6":"0.00E+00","Kenny 7":"1.38E+05","Kenny 8":"1.19E+05","Kenny 9":"1.58E+05","Kenny 10":"2.15E+05","Kenny 11":"1.47E+05","Kenny 12":"1.23E+05","Kenny 13":"1.24E+05","Kenny 14":"1.25E+05","Kenny Eicosanoids 1":"2.83E+04","Kenny Eicosanoids 2":"9.96E+03","Kenny Eicosanoids 3":"5.12E+03","Kenny Eicosanoids 4":"7.80E+03","Kenny Eicosanoids 5":"2.85E+04","Kenny Eicosanoids 6":"1.69E+04","Kenny Eicosanoids 7":"8.09E+03","Kenny Eicosanoids 8":"1.77E+04","Kenny Eicosanoids 9":"4.45E+04","Kenny Eicosanoids 10":"3.72E+04","Kenny Eicosanoids 11":"2.36E+04","Kenny Eicosanoids 12":"8.53E+02","Kenny Eicosanoids 13":"1.03E+04","Kenny Eicosanoids 14":"5.76E+03","Kenny Eicosanoids 15":"6.22E+04","Kenny Eicosanoids 16":"5.77E+04","Kenny Eicosanoids 17":"6.58E+04","Kenny Eicosanoids 18":"8.15E+04"},{"Metabolite":"5 HETE","Kenny 1":"1.40E+05","Kenny 2":"8.07E+04","Kenny 3":"1.25E+05","Kenny 4":"9.24E+04","Kenny 5":"1.01E+05","Kenny 6":"1.27E+05","Kenny 7":"4.79E+04","Kenny 8":"3.89E+04","Kenny 9":"4.36E+04","Kenny 10":"5.87E+04","Kenny 11":"4.59E+04","Kenny 12":"4.86E+04","Kenny 13":"6.80E+04","Kenny 14":"6.50E+04","Kenny Eicosanoids 1":"9.14E+04","Kenny Eicosanoids 2":"4.69E+04","Kenny Eicosanoids 3":"3.49E+04","Kenny Eicosanoids 4":"6.45E+04","Kenny Eicosanoids 5":"1.01E+05","Kenny Eicosanoids 6":"7.77E+04","Kenny Eicosanoids 7":"5.13E+03","Kenny Eicosanoids 8":"1.61E+04","Kenny Eicosanoids 9":"3.90E+04","Kenny Eicosanoids 10":"5.31E+04","Kenny Eicosanoids 11":"2.63E+04","Kenny Eicosanoids 12":"6.40E+02","Kenny Eicosanoids 13":"5.94E+03","Kenny Eicosanoids 14":"4.87E+03","Kenny Eicosanoids 15":"4.37E+04","Kenny Eicosanoids 16":"3.53E+04","Kenny Eicosanoids 17":"3.51E+04","Kenny Eicosanoids 18":"4.01E+04"},{"Metabolite":"5-OxoETE","Kenny 1":"8.29E+03","Kenny 2":"5.62E+03","Kenny 3":"7.66E+03","Kenny 4":"5.19E+03","Kenny 5":"5.51E+03","Kenny 6":"1.51E+04","Kenny 7":"9.68E+03","Kenny 8":"1.62E+04","Kenny 9":"7.14E+03","Kenny 10":"1.05E+04","Kenny 11":"7.12E+03","Kenny 12":"8.10E+03","Kenny 13":"1.54E+04","Kenny 14":"1.17E+04","Kenny Eicosanoids 1":"8.29E+03","Kenny Eicosanoids 2":"3.13E+03","Kenny Eicosanoids 3":"3.06E+03","Kenny Eicosanoids 4":"3.73E+03","Kenny Eicosanoids 5":"7.31E+03","Kenny Eicosanoids 6":"5.47E+03","Kenny Eicosanoids 7":"0.00E+00","Kenny Eicosanoids 8":"1.30E+03","Kenny Eicosanoids 9":"2.47E+03","Kenny Eicosanoids 10":"2.69E+03","Kenny Eicosanoids 11":"1.92E+03","Kenny Eicosanoids 12":"1.95E+02","Kenny Eicosanoids 13":"6.28E+02","Kenny Eicosanoids 14":"3.75E+02","Kenny Eicosanoids 15":"4.60E+03","Kenny Eicosanoids 16":"3.65E+03","Kenny Eicosanoids 17":"2.11E+03","Kenny Eicosanoids 18":"1.93E+03"}],

"Metabolites":[{"Metabolite":"PGE2","PubChem ID":"5280360","KEGG ID":"C00584"},{"Metabolite":"PGD2","PubChem ID":"448457","KEGG ID":"C00696"},{"Metabolite":"5 HETE","PubChem ID":"5280733","KEGG ID":"C04805"},{"Metabolite":"5-OxoETE","PubChem ID":"5283159","KEGG ID":"C14732"}]
}

}