{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002304","ANALYSIS_ID":"AN003765","VERSION":"1","CREATED_ON":"October 6, 2022, 12:21 pm"},

"PROJECT":{"PROJECT_TITLE":"White-nose syndrome disrupts the splenic lipidome of little brown bats (Myotis lucifugus) at early disease stages","PROJECT_SUMMARY":"The fungal disease of bats, white-nose syndrome (WNS), is caused by the pathogen Pseudogymnoascus destructans (Pd). WNS-positive little brown bats (Myotis lucifugus) can exhibit an immune response during infection that include increases in cytokine and pro-inflammatory mediator gene levels. While bioactive lipid mediators (oxylipins) formed by enzymatic oxidation of polyunsaturated fatty acids (PUFAs) can contribute to this type of immune response, their role in WNS pathophysiology have not been investigated. Nonenzymatic conversion of PUFAs can also occur due to reactive oxygen species (ROS), however, these enantiomeric isomers will lack the same signaling properties. In this study, we performed a series of targeted lipidomic approaches on laboratory Pd-inoculated bats to assess changes in their splenic lipidome, including the formation of lipid mediators at early stages of WNS. Hepatic lipids previously identified were also resolved to a higher structural detail. We compared WNS-susceptible M. lucifugus to a WNS-resistant species, the big brown bat (Eptesicus fuscus). Altered splenic lipid levels were only observed in M. lucifugus, with lower total levels of glycerophospholipids (GPs) and free fatty acids (FFAs) in the Pd-inoculated group compared to the sham-inoculated group. Lower concentrations of splenic GPs were observed in lipid compounds containing 18:2 or saturated acyl chains. Differences in splenic FFAs included both omega-3 (including docosahexaenoic acid [DHA]) and omega-6 compounds. Increased levels of an enantiomeric monohydroxy DHA (4-hydroxydocosahexaenoic [HDoHE], 10-HDoHE, and 13-HDoHE) mixture, suggesting nonenzymatic formation, along with 6-keto-PGF1a were found. Changes in previously identified hepatic lipids were confined to omega-3 constituents. Together, these results suggest that increased oxidative stress, but not an inflammatory response, is occurring in bats at early stages of WNS that proceeds fat depletion.","INSTITUTE":"Georgetown University","LAST_NAME":"Pannkuk","FIRST_NAME":"Evan","ADDRESS":"3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, 20057, USA","EMAIL":"elp44@georgetown.edu","PHONE":"2026875650"},

"STUDY":{"STUDY_TITLE":"White-nose syndrome disrupts the splenic lipidome of little brown bats (Myotis lucifugus) at early disease stages","STUDY_SUMMARY":"The fungal disease of bats, white-nose syndrome (WNS), is caused by the pathogen Pseudogymnoascus destructans (Pd). WNS-positive little brown bats (Myotis lucifugus) can exhibit an immune response during infection that include increases in cytokine and pro-inflammatory mediator gene levels. While bioactive lipid mediators (oxylipins) formed by enzymatic oxidation of polyunsaturated fatty acids (PUFAs) can contribute to this type of immune response, their role in WNS pathophysiology have not been investigated. Nonenzymatic conversion of PUFAs can also occur due to reactive oxygen species (ROS), however, these enantiomeric isomers will lack the same signaling properties. In this study, we performed a series of targeted lipidomic approaches on laboratory Pd-inoculated bats to assess changes in their splenic lipidome, including the formation of lipid mediators at early stages of WNS. Hepatic lipids previously identified were also resolved to a higher structural detail. We compared WNS-susceptible M. lucifugus to a WNS-resistant species, the big brown bat (Eptesicus fuscus). Altered splenic lipid levels were only observed in M. lucifugus, with lower total levels of glycerophospholipids (GPs) and free fatty acids (FFAs) in the Pd-inoculated group compared to the sham-inoculated group. Lower concentrations of splenic GPs were observed in lipid compounds containing 18:2 or saturated acyl chains. Differences in splenic FFAs included both omega-3 (including docosahexaenoic acid [DHA]) and omega-6 compounds. Increased levels of an enantiomeric monohydroxy DHA (4-hydroxydocosahexaenoic [HDoHE], 10-HDoHE, and 13-HDoHE) mixture, suggesting nonenzymatic formation, along with 6-keto-PGF1a were found. Changes in previously identified hepatic lipids were confined to omega-3 constituents. Together, these results suggest that increased oxidative stress, but not an inflammatory response, is occurring in bats at early stages of WNS that proceeds fat depletion.","INSTITUTE":"Georgetown University","LAST_NAME":"Pannkuk","FIRST_NAME":"Evan","ADDRESS":"3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, 20057, USA","EMAIL":"elp44@georgetown.edu","PHONE":"2026875650"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Myotis lucifugus"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Mylu",
"Sample ID":"20",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"25",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"27",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"46",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"47",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"21",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"23",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"24",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"26",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"28",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"29",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"70",
"Factors":{"Treatment":"PBST"}
},
{
"Subject ID":"Mylu",
"Sample ID":"31",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"34",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"36",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"44",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"32",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"35",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"38",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"39",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"40",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"41",
"Factors":{"Treatment":"Pd"}
},
{
"Subject ID":"Mylu",
"Sample ID":"43",
"Factors":{"Treatment":"Pd"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Bats were housed within mesh cages (Exo-terra Flexarium©; 22 cm x 35 cm x 43 cm) that were placed within temperature/humidity-controlled incubators (Caron© Environmental Chamber model 6041; 90.1 cm x 84.5 cm x 228.9 cm set to 8°C and 98% humidity) to encourage hibernation. Four cages were used to house the bats, separated by species and inoculation group; M. lucifugus (one sham-treatment: 16 individuals/cage, 10 male and 6 female; one Pd-inoculated; E. fuscus (8 individuals/cage, 4 male and 4 female). Bats were infected with a sham inoculum (20 μl, Phosphate Buffered Saline with Tween®20 (PBS-T)) or a Pd inoculum (20 μl, PBST containing 5x105 conidia) on wing and tail membranes. We monitored arousal frequency and sickness behavior using motion-activated infrared video. During the 71-day period bats found unresponsive or extremely sluggish were humanely euthanized (CO2 inhalation after isoflurane anesthesia and cervical dislocation), and were not included in this experiment. Several M. lucifugus showed signs of morbidity around day 55. We therefore, terminated the experiment 71 – 77 days post-inoculation (mid-March) which coincided with early stages of WNS progression. Disease severity was assessed by fungal load by qPCR33, UV fluorescence indicative of hyphae invasion, survival rate, and behavioral changes 32. E. fuscus was chosen as a WNS-resistant control and we found changes in their splenic lipidome to be minor. Tissue samples were collected, flash frozen, and shipped to Georgetown University Medical Center on dry ice and stored at -80°C until analysis.","SAMPLE_TYPE":"Spleen"},

"TREATMENT":{"TREATMENT_SUMMARY":"Bats were housed within mesh cages (Exo-terra Flexarium©; 22 cm x 35 cm x 43 cm) that were placed within temperature/humidity-controlled incubators (Caron© Environmental Chamber model 6041; 90.1 cm x 84.5 cm x 228.9 cm set to 8°C and 98% humidity) to encourage hibernation. Four cages were used to house the bats, separated by species and inoculation group; M. lucifugus (one sham-treatment: 16 individuals/cage, 10 male and 6 female; one Pd-inoculated; E. fuscus (8 individuals/cage, 4 male and 4 female). Bats were infected with a sham inoculum (20 μl, Phosphate Buffered Saline with Tween®20 (PBS-T)) or a Pd inoculum (20 μl, PBST containing 5x105 conidia) on wing and tail membranes. We monitored arousal frequency and sickness behavior using motion-activated infrared video. During the 71-day period bats found unresponsive or extremely sluggish were humanely euthanized (CO2 inhalation after isoflurane anesthesia and cervical dislocation), and were not included in this experiment. Several M. lucifugus showed signs of morbidity around day 55. We therefore, terminated the experiment 71 – 77 days post-inoculation (mid-March) which coincided with early stages of WNS progression. Disease severity was assessed by fungal load by qPCR33, UV fluorescence indicative of hyphae invasion, survival rate, and behavioral changes 32. E. fuscus was chosen as a WNS-resistant control and we found changes in their splenic lipidome to be minor. Tissue samples were collected, flash frozen, and shipped to Georgetown University Medical Center on dry ice and stored at -80°C until analysis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Tissue samples were homogenized in 300 μL of extraction buffer (isopropanol [IPA]) containing internal standards for the above lipid classes per the manufacturer’s instructions. A 10 μL aliquot of the tissue/extraction buffer mixture was collected for determining protein concentration. The samples were vortexed for 30 seconds, incubated on ice for 30 min, and then centrifuged for 10 min (10,000 x g, 4°C). The supernatant was transferred to a MS vial for LC-MS analysis. A pooled sample of all aliquots was prepared as a quality control and run every 10 samples in addition to the NIST SRM 1950 plasma mix. The NIST SRM 1950 plasma was prepared by aliquoting 20 µL in 100 μL of chilled IPA containing internal standards. Samples were vortexed for 1 min, incubated on ice for 30 min then incubated at -20°C for 2 hours for complete protein precipitation, then centrifuged for 20 min (10,000 x g, 4°C)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Shimadzu","COLUMN_NAME":"Xbridge amide 3.5 µm, 4.6 X 100 mm column"},

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

"MS":{"INSTRUMENT_NAME":"ABI Sciex 5500 QTrap","INSTRUMENT_TYPE":"QTRAP","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"the qlm file was imported into MultiQuant v 2.0"},

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

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}

}