#METABOLOMICS WORKBENCH epannkuk_20250521_092826 DATATRACK_ID:5933 STUDY_ID:ST004100 ANALYSIS_ID:AN006800 PROJECT_ID:PR002576
VERSION             	1
CREATED_ON             	August 8, 2025, 11:15 am
#PROJECT
PR:PROJECT_TITLE                 	Combined bacterial infection and radiation injury in the murine model:
PR:PROJECT_TITLE                 	Consequences on metabolomics based biodosimetry
PR:PROJECT_SUMMARY               	High-throughput biodosimetry assays are needed to assess ionizing radiation (IR)
PR:PROJECT_SUMMARY               	exposure in emergency situations that can predict the level of acute radiation
PR:PROJECT_SUMMARY               	syndrome (ARS) across a heterogenous segment of the population that may have
PR:PROJECT_SUMMARY               	secondary bacterial infections or combined injury. In this study, we investigate
PR:PROJECT_SUMMARY               	the impact of bacterial infection on metabolite-based biodosimetry using a
PR:PROJECT_SUMMARY               	well-established model: recombinant Listeria monocytogenes expressing ovalbumin
PR:PROJECT_SUMMARY               	(Lm-OVA) and C57BL/6 murine infection model. Male mice were infected with
PR:PROJECT_SUMMARY               	Lm-OVA, then subjected to 0, 2, or 6 Gy X-ray irradiation at 4 days
PR:PROJECT_SUMMARY               	post-infection (dpi). Biofluids were analyzed using untargeted metabolomics at 1
PR:PROJECT_SUMMARY               	day (d) post-irradiation (5 dpi).
PR:INSTITUTE                     	Georgetown University
PR:LAST_NAME                     	Pannkuk
PR:FIRST_NAME                    	Evan
PR:ADDRESS                       	3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia,
PR:ADDRESS                       	20057, USA
PR:EMAIL                         	elp44@georgetown.edu
PR:PHONE                         	2026875650
#STUDY
ST:STUDY_TITLE                   	Combined bacterial infection and radiation injury in the murine model:
ST:STUDY_TITLE                   	Consequences on metabolomics based biodosimetry (Serum analysis)
ST:STUDY_SUMMARY                 	High-throughput biodosimetry assays are needed to assess ionizing radiation (IR)
ST:STUDY_SUMMARY                 	exposure in emergency situations that can predict the level of acute radiation
ST:STUDY_SUMMARY                 	syndrome (ARS) across a heterogenous segment of the population that may have
ST:STUDY_SUMMARY                 	secondary bacterial infections or combined injury. In this study, we investigate
ST:STUDY_SUMMARY                 	the impact of bacterial infection on metabolite-based biodosimetry using a
ST:STUDY_SUMMARY                 	well-established model: recombinant Listeria monocytogenes expressing ovalbumin
ST:STUDY_SUMMARY                 	(Lm-OVA) and C57BL/6 murine infection model. Male mice were infected with
ST:STUDY_SUMMARY                 	Lm-OVA, then subjected to 0, 2, or 6 Gy X-ray irradiation at 4 days
ST:STUDY_SUMMARY                 	post-infection (dpi). Biofluids were analyzed using untargeted metabolomics at 1
ST:STUDY_SUMMARY                 	day (d) post-irradiation (5 dpi).
ST:INSTITUTE                     	Georgetown University
ST:LAST_NAME                     	Pannkuk
ST:FIRST_NAME                    	Evan
ST:ADDRESS                       	3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia,
ST:ADDRESS                       	20057, USA
ST:EMAIL                         	elp44@georgetown.edu
ST:PHONE                         	2026875650
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENDER                        	Male
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	669	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_042
SUBJECT_SAMPLE_FACTORS           	-	670	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_043
SUBJECT_SAMPLE_FACTORS           	-	671	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_055
SUBJECT_SAMPLE_FACTORS           	-	672	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_028
SUBJECT_SAMPLE_FACTORS           	-	673	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_036
SUBJECT_SAMPLE_FACTORS           	-	674	Sample source:Serum | Exposure:1_NF_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_051
SUBJECT_SAMPLE_FACTORS           	-	675	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_052
SUBJECT_SAMPLE_FACTORS           	-	676	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_016
SUBJECT_SAMPLE_FACTORS           	-	677	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_041
SUBJECT_SAMPLE_FACTORS           	-	678	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_011
SUBJECT_SAMPLE_FACTORS           	-	679	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_014
SUBJECT_SAMPLE_FACTORS           	-	680	Sample source:Serum | Exposure:2_NF_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_035
SUBJECT_SAMPLE_FACTORS           	-	681	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_026
SUBJECT_SAMPLE_FACTORS           	-	682	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_029
SUBJECT_SAMPLE_FACTORS           	-	683	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_030
SUBJECT_SAMPLE_FACTORS           	-	684	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_017
SUBJECT_SAMPLE_FACTORS           	-	685	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_027
SUBJECT_SAMPLE_FACTORS           	-	686	Sample source:Serum | Exposure:3_NF_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_039
SUBJECT_SAMPLE_FACTORS           	-	701	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_009
SUBJECT_SAMPLE_FACTORS           	-	702	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_024
SUBJECT_SAMPLE_FACTORS           	-	703	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_040
SUBJECT_SAMPLE_FACTORS           	-	704	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_012
SUBJECT_SAMPLE_FACTORS           	-	705	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_049
SUBJECT_SAMPLE_FACTORS           	-	706	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_050
SUBJECT_SAMPLE_FACTORS           	-	707	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_044
SUBJECT_SAMPLE_FACTORS           	-	708	Sample source:Serum | Exposure:6_Inf_6Gy | Irradiation:6Gy	RAW_FILE_NAME(Sample_ID)=POS_025
SUBJECT_SAMPLE_FACTORS           	-	709	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_037
SUBJECT_SAMPLE_FACTORS           	-	710	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_013
SUBJECT_SAMPLE_FACTORS           	-	711	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_038
SUBJECT_SAMPLE_FACTORS           	-	712	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_015
SUBJECT_SAMPLE_FACTORS           	-	713	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_022
SUBJECT_SAMPLE_FACTORS           	-	714	Sample source:Serum | Exposure:5_Inf_2Gy | Irradiation:2Gy	RAW_FILE_NAME(Sample_ID)=POS_031
SUBJECT_SAMPLE_FACTORS           	-	715	Sample source:Serum | Exposure:4_Inf_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_023
SUBJECT_SAMPLE_FACTORS           	-	716	Sample source:Serum | Exposure:4_Inf_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_054
SUBJECT_SAMPLE_FACTORS           	-	718	Sample source:Serum | Exposure:4_Inf_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_010
SUBJECT_SAMPLE_FACTORS           	-	719	Sample source:Serum | Exposure:4_Inf_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_048
SUBJECT_SAMPLE_FACTORS           	-	720	Sample source:Serum | Exposure:4_Inf_Sham | Irradiation:0Gy	RAW_FILE_NAME(Sample_ID)=POS_018
#COLLECTION
CO:COLLECTION_SUMMARY            	At 1 d post-irradiation, spot urines were collected, blood was collected via
CO:COLLECTION_SUMMARY            	cardiac puncture, and serum for metabolomics was separated using BD Microtainer
CO:COLLECTION_SUMMARY            	Tubes (REF 365967) with ~100 μL of whole blood added to each tube, kept at room
CO:COLLECTION_SUMMARY            	temperature for 30 min, then centrifuged (1300 x g, 4°C) for 10 min. Biofluids
CO:COLLECTION_SUMMARY            	were flash frozen and stored at -80°C until analysis. A separate aliquot of
CO:COLLECTION_SUMMARY            	blood was collected in a dipotassium EDTA Tube (BD Cat #365974) for collecting
CO:COLLECTION_SUMMARY            	complete blood count (CBC including levels of white blood cells [WBC],
CO:COLLECTION_SUMMARY            	lymphocyte [LYM], monocyte [MON], and neutrophil [NEU]) by VRL Diagnostics
CO:COLLECTION_SUMMARY            	(Gaithersburg, MD, http://www.vrlsat.com/).
CO:SAMPLE_TYPE                   	Blood (serum)
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	All animal experiments were approved by the Georgetown University Institutional
TR:TREATMENT_SUMMARY             	Animal Care and Use Committee (IACUC, protocol #2023-0012) and were conducted
TR:TREATMENT_SUMMARY             	under all relevant federal and state guidelines. Male C57BL/6 mice (10 weeks
TR:TREATMENT_SUMMARY             	old) were purchased from Charles River Laboratories (Frederick, MD) and provided
TR:TREATMENT_SUMMARY             	food (PicoLab Rodent Diet 20 #5053) and deionized water was provided ad libitum.
TR:TREATMENT_SUMMARY             	Mice were infected with a retro-orbital injection of Lm-OVA, which is a widely
TR:TREATMENT_SUMMARY             	used and reproducible infection method as the bacteria can bypass the gut stage
TR:TREATMENT_SUMMARY             	of infection. Lm-OVA stocks frozen at -80°C were grown overnight at 37°C while
TR:TREATMENT_SUMMARY             	shaking in BHI broth supplemented with 5 µg/mL erythromycin. Then, overnight
TR:TREATMENT_SUMMARY             	cultures were subcultured by diluting into fresh BHI broth supplemented with 5
TR:TREATMENT_SUMMARY             	µg/mL erythromycin and grown for 4 hours at 37°C while shaking. Bacteria CFU
TR:TREATMENT_SUMMARY             	was then quantified by measuring optical density at 600 nm. For primary
TR:TREATMENT_SUMMARY             	infections, bacterial culture was then diluted to 1x105 CFU/100 µL in sterile
TR:TREATMENT_SUMMARY             	1X PBS and 100 µL was injected per mouse. Both non-infected and infected mice
TR:TREATMENT_SUMMARY             	were randomly assigned to a zero-dose sham (0 Gy) or irradiated (2 or 6 Gy)
TR:TREATMENT_SUMMARY             	(Figure 1). We exposed mice to total body irradiation (TBI) in an acrylic,
TR:TREATMENT_SUMMARY             	12-slot mouse pie cage (MPC-1, Braintree Scientific, Braintree, MA) using a
TR:TREATMENT_SUMMARY             	specimen turntable (XD1905-0000, Precision X-Ray Inc, Branford, CT). The mice
TR:TREATMENT_SUMMARY             	were exposed to 0, 2, or 6 Gy X-ray IR (1.67 Gy/min; X-Rad 320, Precision X-Ray
TR:TREATMENT_SUMMARY             	Inc.; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For serum, a 5 μl aliquot was mixed with 195 μl of cold 66% acetonitrile
SP:SAMPLEPREP_SUMMARY            	containing internal standards (5 μM chlorpropamide [M+H]+ = 277.0414, [M-H]- =
SP:SAMPLEPREP_SUMMARY            	275.0257; deuterated amino acid mix). The deuterated amino acid mix contains 24
SP:SAMPLEPREP_SUMMARY            	different labeled metabolites, ranging in concentration from 222 μM for
SP:SAMPLEPREP_SUMMARY            	glutamine-d5 to 7 μM for 1-methylhistidine-d3. The cold 66% acetonitrile used
SP:SAMPLEPREP_SUMMARY            	for metabolite extraction contained the amino acid mixture adjusted to a
SP:SAMPLEPREP_SUMMARY            	concentration of 10 μM for glutamine-d5. Samples were then prepared as above.
SP:SAMPLEPREP_SUMMARY            	One μl aliquots of each biofluid sample were combined as a quality control (QC)
SP:SAMPLEPREP_SUMMARY            	sample. Additional QC samples included the creatinine in frozen human urine and
SP:SAMPLEPREP_SUMMARY            	metabolites in frozen human plasma NIST Standard Reference Materials. The QC
SP:SAMPLEPREP_SUMMARY            	samples were injected every 10 samples along with blanks.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	The LC and MS conditions for serum was as follows: LC solvent A (water/0.1%
CH:CHROMATOGRAPHY_SUMMARY        	formic acid [FA]), solvent B (acetonitrile/0.1% FA), and solvent C
CH:CHROMATOGRAPHY_SUMMARY        	(isopropanol/0.1% FA). Operating conditions for ESI were, capillary voltage 3
CH:CHROMATOGRAPHY_SUMMARY        	kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 600
CH:CHROMATOGRAPHY_SUMMARY        	L/Hr. The gradient for serum was: 4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min
CH:CHROMATOGRAPHY_SUMMARY        	2% A 98% B, 2 min 11.8% B 88.2% C, 0.5 min 50% A 50% B, and 1 min 98% A 2% B at
CH:CHROMATOGRAPHY_SUMMARY        	a flow rate of 0.5 ml/min, column temp 60 °C.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:SOLVENT_C                     	100% isopropanol; 0.1% formic acid
CH:FLOW_GRADIENT                 	4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min 2% A 98% B, 2 min 11.8% B 88.2% C,
CH:FLOW_GRADIENT                 	0.5 min 50% A 50% B, and 1 min 98% A 2% B
CH:FLOW_RATE                     	0.5 ml/min
CH:COLUMN_TEMPERATURE            	60
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Xevo-G2-S
MS:INSTRUMENT_TYPE               	QTRAP
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Operating conditions for ESI were, capillary voltage 3 kV, cone voltage 30 V,
MS:MS_COMMENTS                   	desolvation temperature 500°C, desolvation gas flow 600 L/Hr. The data are
MS:MS_COMMENTS                   	reported as normalized peak areas, normalized to all ions and internal std.
MS:MS_RESULTS_FILE               	ST004100_AN006800_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END