Summary of Study ST002304

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001477. The data can be accessed directly via it's Project DOI: 10.21228/M8T40Q This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST002304
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
Submit Date	2022-09-22
Analysis Type Detail	LC-MS
Release Date	2023-09-22
Release Version	1

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Project:

Project ID:	PR001477
Project DOI:	doi: 10.21228/M8T40Q
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

Subject:

Subject ID:	SU002390
Subject Type:	Mammal
Subject Species:	Myotis lucifugus

Factors:

Subject type: Mammal; Subject species: Myotis lucifugus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA226669	26	PBST
SA226670	28	PBST
SA226671	20	PBST
SA226672	24	PBST
SA226673	29	PBST
SA226674	70	PBST
SA226675	27	PBST
SA226676	23	PBST
SA226677	46	PBST
SA226678	25	PBST
SA226679	21	PBST
SA226680	47	PBST
SA226681	39	Pd
SA226682	40	Pd
SA226683	41	Pd
SA226684	38	Pd
SA226685	43	Pd
SA226686	44	Pd
SA226687	31	Pd
SA226688	34	Pd
SA226689	36	Pd
SA226690	32	Pd
SA226691	35	Pd

Showing results 1 to 23 of 23

Showing results 1 to 23 of 23

Collection:

Collection ID:	CO002383
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 ID:	TR002402
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.

Treatment ID:

TR002402

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.

Sample Preparation:

Sampleprep ID:	SP002396
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).

Sampleprep ID:

SP002396

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).

Combined analysis:

Analysis ID	AN003765
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	UNSPECIFIED
Units	peak area

Chromatography:

Chromatography ID:	CH002784
Instrument Name:	Shimadzu
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003508
Analysis ID:	AN003765
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	the qlm file was imported into MultiQuant v 2.0
Ion Mode:	UNSPECIFIED