#METABOLOMICS WORKBENCH epannkuk_20210723_090352_mwtab.txt DATATRACK_ID:2769 STUDY_ID:ST001892 ANALYSIS_ID:AN003073 PROJECT_ID:000000 VERSION 1 CREATED_ON July 29, 2021, 6:37 pm #PROJECT PR:PROJECT_TITLE Small molecule signatures of mice lacking T-cell p38 alternate activation, a PR:PROJECT_TITLE model for immunosuppression conditions, after exposure to total body radiation PR:PROJECT_SUMMARY Introduction Novel biodosimetry assays are needed in the event of PR:PROJECT_SUMMARY radiological/nuclear emergencies for both immediate triage and identifying PR:PROJECT_SUMMARY delayed effects of acute radiation exposure. Genetically engineered mouse models PR:PROJECT_SUMMARY are used to assess how genotypic variation in the general population may affect PR:PROJECT_SUMMARY post-irradiation classification performance. Here, we used a mouse model that PR:PROJECT_SUMMARY lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double PR:PROJECT_SUMMARY knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory PR:PROJECT_SUMMARY responses may affect dose reconstruction. Objectives To determine if deficient PR:PROJECT_SUMMARY alternative p38 activation differentially affects biofluid metabolic signatures PR:PROJECT_SUMMARY post-irradiation compared to wild-type (WT). Methods Untargeted global PR:PROJECT_SUMMARY metabolomics was used to assess biofluid signatures between WT and DKI mice (8 PR:PROJECT_SUMMARY – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine PR:PROJECT_SUMMARY was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral PR:PROJECT_SUMMARY features of interest were identified using the machine learning algorithm Random PR:PROJECT_SUMMARY Forests and MetaboLyzer. Validated metabolite panels were constructed and PR:PROJECT_SUMMARY classification performance was assessed by determining the area under the PR:PROJECT_SUMMARY receiver operating characteristic curve (AUROC). Results A multidimensional PR:PROJECT_SUMMARY scaling plot showed excellent separation of IR exposed groups in WT with PR:PROJECT_SUMMARY slightly dampened responses in DKI mice. For both urine and serum, excellent PR:PROJECT_SUMMARY sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups PR:PROJECT_SUMMARY irrespective of genotype using identical metabolite panels. Similarly, excellent PR:PROJECT_SUMMARY to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy PR:PROJECT_SUMMARY post-irradiation mice for both genotypes, however, model performance declined PR:PROJECT_SUMMARY (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these PR:PROJECT_SUMMARY results suggest less influence of the alternative p38 activation pathway for PR:PROJECT_SUMMARY dose reconstruction compared to other radiosensitive genotypes. PR:INSTITUTE Georgetown University PR:LAST_NAME Pannkuk PR:FIRST_NAME Evan PR:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 PR:EMAIL elp44@georgetown.edu PR:PHONE 2026875650 #STUDY ST:STUDY_TITLE Small molecule signatures of mice lacking T-cell p38 alternate activation, a ST:STUDY_TITLE model for immunosuppression conditions, after exposure to total body radiation ST:STUDY_TITLE (part II) ST:STUDY_SUMMARY Introduction Novel biodosimetry assays are needed in the event of ST:STUDY_SUMMARY radiological/nuclear emergencies for both immediate triage and identifying ST:STUDY_SUMMARY delayed effects of acute radiation exposure. Genetically engineered mouse models ST:STUDY_SUMMARY are used to assess how genotypic variation in the general population may affect ST:STUDY_SUMMARY post-irradiation classification performance. Here, we used a mouse model that ST:STUDY_SUMMARY lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double ST:STUDY_SUMMARY knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory ST:STUDY_SUMMARY responses may affect dose reconstruction. Objectives To determine if deficient ST:STUDY_SUMMARY alternative p38 activation differentially affects biofluid metabolic signatures ST:STUDY_SUMMARY post-irradiation compared to wild-type (WT). Methods Untargeted global ST:STUDY_SUMMARY metabolomics was used to assess biofluid signatures between WT and DKI mice (8 ST:STUDY_SUMMARY – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine ST:STUDY_SUMMARY was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral ST:STUDY_SUMMARY features of interest were identified using the machine learning algorithm Random ST:STUDY_SUMMARY Forests and MetaboLyzer. Validated metabolite panels were constructed and ST:STUDY_SUMMARY classification performance was assessed by determining the area under the ST:STUDY_SUMMARY receiver operating characteristic curve (AUROC). Results A multidimensional ST:STUDY_SUMMARY scaling plot showed excellent separation of IR exposed groups in WT with ST:STUDY_SUMMARY slightly dampened responses in DKI mice. For both urine and serum, excellent ST:STUDY_SUMMARY sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups ST:STUDY_SUMMARY irrespective of genotype using identical metabolite panels. Similarly, excellent ST:STUDY_SUMMARY to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy ST:STUDY_SUMMARY post-irradiation mice for both genotypes, however, model performance declined ST:STUDY_SUMMARY (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these ST:STUDY_SUMMARY results suggest less influence of the alternative p38 activation pathway for ST:STUDY_SUMMARY dose reconstruction compared to other radiosensitive genotypes. ST:INSTITUTE Georgetown University ST:LAST_NAME Pannkuk ST:FIRST_NAME Evan ST:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 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 - 1 Genotype_irradiation:WT_sham Post-irradiation=1d; RAW_FILE_NAME=POS_26 SUBJECT_SAMPLE_FACTORS - 2 Genotype_irradiation:WT_sham Post-irradiation=1d; RAW_FILE_NAME=POS_38 SUBJECT_SAMPLE_FACTORS - 3 Genotype_irradiation:WT_sham Post-irradiation=1d; RAW_FILE_NAME=POS_48 SUBJECT_SAMPLE_FACTORS - 4 Genotype_irradiation:WT_sham Post-irradiation=1d; RAW_FILE_NAME=POS_55 SUBJECT_SAMPLE_FACTORS - 5 Genotype_irradiation:WT_sham Post-irradiation=1d; RAW_FILE_NAME=POS_56 SUBJECT_SAMPLE_FACTORS - 6 Genotype_irradiation:WT_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_08 SUBJECT_SAMPLE_FACTORS - 7 Genotype_irradiation:WT_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_09 SUBJECT_SAMPLE_FACTORS - 8 Genotype_irradiation:WT_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_13 SUBJECT_SAMPLE_FACTORS - 9 Genotype_irradiation:WT_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_27 SUBJECT_SAMPLE_FACTORS - 10 Genotype_irradiation:WT_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_28 SUBJECT_SAMPLE_FACTORS - 11 Genotype_irradiation:WT_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_10 SUBJECT_SAMPLE_FACTORS - 12 Genotype_irradiation:WT_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_12 SUBJECT_SAMPLE_FACTORS - 13 Genotype_irradiation:WT_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_33 SUBJECT_SAMPLE_FACTORS - 14 Genotype_irradiation:WT_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_39 SUBJECT_SAMPLE_FACTORS - 15 Genotype_irradiation:WT_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_49 SUBJECT_SAMPLE_FACTORS - 16 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_14 SUBJECT_SAMPLE_FACTORS - 17 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_16 SUBJECT_SAMPLE_FACTORS - 18 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_34 SUBJECT_SAMPLE_FACTORS - 19 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_41 SUBJECT_SAMPLE_FACTORS - 20 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_42 SUBJECT_SAMPLE_FACTORS - 21 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_57 SUBJECT_SAMPLE_FACTORS - 22 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_58 SUBJECT_SAMPLE_FACTORS - 23 Genotype_irradiation:dki_sham Post-irradiation=1d; RAW_FILE_NAME=POS_59 SUBJECT_SAMPLE_FACTORS - 24 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_20 SUBJECT_SAMPLE_FACTORS - 25 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_21 SUBJECT_SAMPLE_FACTORS - 26 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_22 SUBJECT_SAMPLE_FACTORS - 27 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_23 SUBJECT_SAMPLE_FACTORS - 28 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_24 SUBJECT_SAMPLE_FACTORS - 29 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_35 SUBJECT_SAMPLE_FACTORS - 30 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_36 SUBJECT_SAMPLE_FACTORS - 31 Genotype_irradiation:dki_2Gy Post-irradiation=1d; RAW_FILE_NAME=POS_52 SUBJECT_SAMPLE_FACTORS - 32 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_11 SUBJECT_SAMPLE_FACTORS - 33 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_07 SUBJECT_SAMPLE_FACTORS - 34 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_15 SUBJECT_SAMPLE_FACTORS - 35 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_25 SUBJECT_SAMPLE_FACTORS - 36 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_29 SUBJECT_SAMPLE_FACTORS - 37 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_37 SUBJECT_SAMPLE_FACTORS - 38 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_40 SUBJECT_SAMPLE_FACTORS - 39 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_46 SUBJECT_SAMPLE_FACTORS - 40 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_47 SUBJECT_SAMPLE_FACTORS - 41 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_50 SUBJECT_SAMPLE_FACTORS - 42 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_51 SUBJECT_SAMPLE_FACTORS - 43 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_53 SUBJECT_SAMPLE_FACTORS - 44 Genotype_irradiation:dki_7Gy Post-irradiation=1d; RAW_FILE_NAME=POS_54 #COLLECTION CO:COLLECTION_SUMMARY Serum was collected after irradiation CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY WT C57Bl/6 mice (C57BL/6NCrl strain code #027) were obtained from Charles River TR:TREATMENT_SUMMARY Laboratories (Frederick, MD) and DKI mice were kindly provided by the Laboratory TR:TREATMENT_SUMMARY of Immune Cell Biology, National Cancer Institute (P.I. Jonathan D. Ashwell, TR:TREATMENT_SUMMARY M.D.) (Jirmanova et al. 2011). Animals were bred/irradiated (12 h light / 12 h TR:TREATMENT_SUMMARY dark cycle conditions) at Georgetown University and water and food (PicoLab TR:TREATMENT_SUMMARY Rodent Diet 20 #5053) were provided ad libitum according to Georgetown TR:TREATMENT_SUMMARY University Institutional Animal Care and Use Committee (GUACUC) protocols TR:TREATMENT_SUMMARY (2016-1152). Before irradiation and biofluid collection the mice were acclimated TR:TREATMENT_SUMMARY to metabolic cages for 24 h. Male mice that were 8 – 10 weeks old were exposed TR:TREATMENT_SUMMARY to a total body ionization (TBI) x-ray dose (~1.67 Gy/min; X-Rad 320, Precision TR:TREATMENT_SUMMARY X-Ray Inc, Branford, CT; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum) of TR:TREATMENT_SUMMARY 0, 2, or 7 Gy. All urine samples were collected over a 24 h period in a TR:TREATMENT_SUMMARY metabolic cage pre-irradiation and at days 1, 3, and 7 d post-irradiation TR:TREATMENT_SUMMARY (Figure S1). Blood for metabolomics was collected at 1 d via cheek bleed from TR:TREATMENT_SUMMARY the submandibular vein and serum was separated in a BD microtainer serum TR:TREATMENT_SUMMARY separator tube and centrifuged for 10 min (10,000 x g, 4°C). Serum samples from TR:TREATMENT_SUMMARY sham-irradiated mice were used as a control (Figure S1). All biofluids were TR:TREATMENT_SUMMARY flash frozen and stored at -80°C until further use. Seven days TR:TREATMENT_SUMMARY post-irradiation, blood was collected in a dipotassium EDTA Tube (BD Cat TR:TREATMENT_SUMMARY #365974) via the facial vein from each animal and subjected to a complete blood TR:TREATMENT_SUMMARY count by VRL Diagnostics (Gaithersburg, MD, http://www.vrlsat.com/) (Figure S2). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Biofluids were prepared as previously described (Pannkuk et al. 2018;2020). SP:SAMPLEPREP_SUMMARY Urine (20 μl) was deproteinated with 50% acetonitrile (80 μl) containing SP:SAMPLEPREP_SUMMARY internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), SP:SAMPLEPREP_SUMMARY incubated on ice for 10 min, vortexed for 30 seconds, and centrifuged for 10 min SP:SAMPLEPREP_SUMMARY (10,000 x g, 4°C). Serum (5 μl) was prepared as above but was deproteinated SP:SAMPLEPREP_SUMMARY with 66% acetonitrile (195 μl). A quality control (QC) sample was prepared by SP:SAMPLEPREP_SUMMARY mixing 1 μl from each sample and prepared as above. SP:PROCESSING_STORAGE_CONDITIONS -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phases consisted of the following: solvent A (water/0.1% formic acid CH:CHROMATOGRAPHY_SUMMARY [FA]), solvent B (ACN/0.1% FA), solvent C (isopropanol [IPA]/ACN (90:10)/0.1% CH:CHROMATOGRAPHY_SUMMARY FA). The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, CH:CHROMATOGRAPHY_SUMMARY 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for CH:CHROMATOGRAPHY_SUMMARY serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min CH:CHROMATOGRAPHY_SUMMARY 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow CH:CHROMATOGRAPHY_SUMMARY rate of 0.5 ml/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Negative and positive electrospray ionization (ESI) data-independent modes were MS:MS_COMMENTS used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = MS:MS_COMMENTS 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage MS:MS_COMMENTS 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow MS:MS_COMMENTS 1000 L/Hr. MS:MS_RESULTS_FILE ST001892_AN003073_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END