#METABOLOMICS WORKBENCH jburgess_20160306_210457 STUDY_ID:ST000363 ANALYSIS_ID:AN000595 PROJECT_ID:PR000285 VERSION 1 CREATED_ON March 10, 2016, 9:48 pm #PROJECT PR:PROJECT_TITLE Metabolic Profiling of Anxiety Prone HSV-Latently Infected Obese Mice PR:PROJECT_TYPE Broad Spectrum LCMS PR:PROJECT_SUMMARY The biological factors that lead children from low socioeconomic backgrounds to PR:PROJECT_SUMMARY be at greater risk for the development of anxiety and learning problems are not PR:PROJECT_SUMMARY well understood. While it is clear that there are genetic components to the risk PR:PROJECT_SUMMARY of developing mental health disorders, a role for environmental factors in PR:PROJECT_SUMMARY inducing these problems has been suggested. Many of these factors that affect PR:PROJECT_SUMMARY the brain likely involve exposures that occur early in life. Two such factors PR:PROJECT_SUMMARY are the higher rates of herpes simplex virus (HSV)-1 seropositivity and PR:PROJECT_SUMMARY prevalence of obesity among these children. HSV-1 infection has been associated PR:PROJECT_SUMMARY with impaired cognition during childhood and mental health problems in PR:PROJECT_SUMMARY adulthood. Additionally, obese adults are shown to have higher HSV-1 titers. PR:PROJECT_SUMMARY Similarly, other studies have correlated a diet high in saturated fat and PR:PROJECT_SUMMARY obesity with increased risk of mood disorders and anxiety. A mouse model of PR:PROJECT_SUMMARY obese HSV-1 latent infection was developed. Broad spectrum metabolomics analysis PR:PROJECT_SUMMARY was performed to better understand the metabolomic profile of hippocampus and to PR:PROJECT_SUMMARY compare this metabolomics profile with that of the hypothalamus, microglia, and PR:PROJECT_SUMMARY peripheral blood mononuclear cells. Brain tissue samples for metabolomics PR:PROJECT_SUMMARY experiments were generated in the following manner: 3-week old mice were placed PR:PROJECT_SUMMARY on a 10% low fat (LF) diet and acclimated for one week prior to intranasal HSV-1 PR:PROJECT_SUMMARY infection or mock infection with PBS. Fourteen days post-infection mice were PR:PROJECT_SUMMARY randomized to either a 45% high fat diet (HF) or remained on the LF diet. Eight PR:PROJECT_SUMMARY weeks post-diet transition, mice were euthanized and brain tissue samples were PR:PROJECT_SUMMARY collected and processed for metabolomics. PR:INSTITUTE University of North Carolina , Chapel Hill PR:DEPARTMENT Gillings School of Public Health PR:LABORATORY Department of Nutrition PR:LAST_NAME Sheridan PR:FIRST_NAME Patricia PR:ADDRESS 2002 Hooker Research Center, CB#7461, Chapel Hill NC, 27599 PR:EMAIL patricia_sheridan@med.unc.edu PR:PHONE 919-843-6434 PR:FUNDING_SOURCE NIH, Common Fund, Pilot and Feasibility #STUDY ST:STUDY_TITLE Broad Spectrum MS analysis of mouse peripheral blood mononuclear cells (PBMC) ST:STUDY_TITLE from Anxiety Prone HSV-Latently Infected Obese Mice ST:STUDY_TYPE Broad Spectrum LCMS ST:STUDY_SUMMARY Mononuclear cell samples for metabolomics experiments were generated in the ST:STUDY_SUMMARY following manner: 3-week old mice were placed on a 10% low fat (LF) diet and ST:STUDY_SUMMARY acclimated for one week prior to intranasal HSV-1 infection or mock infection ST:STUDY_SUMMARY with PBS. Fourteen days post-infection mice were randomized to either a 45% high ST:STUDY_SUMMARY fat diet (HF) or remained on the LF diet. Eight weeks post-diet transition, mice ST:STUDY_SUMMARY were euthanized and mononuclear cell samples were collected and processed for ST:STUDY_SUMMARY metabolomics. ST:INSTITUTE RTI ST:DEPARTMENT Discovery Sciences ST:LABORATORY System and Translational Sciences ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 Cornwallis Rd, RTP,NC ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 30 ST:NUM_MALES 30 #SUBJECT SU:SUBJECT_TYPE mouse SU:SUBJECT_SPECIES Mus musculus SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 4.5 mo SU:WEIGHT_OR_WEIGHT_RANGE 29.1 g-49.2 g SU:GENDER male SU:ANIMAL_ANIMAL_SUPPLIER JAX SU:ANIMAL_HOUSING 4/cage SU:ANIMAL_FEED Purina chow #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 444 Blood444 HSV-1:No | Diet at Week 2:HF Weight (g) =38.1 SUBJECT_SAMPLE_FACTORS 430 Blood430 HSV-1:yes | Diet at Week 2:LF Weight (g) =25.2 SUBJECT_SAMPLE_FACTORS 431 Blood431 HSV-1:no | Diet at Week 2:LF Weight (g) =32.7 SUBJECT_SAMPLE_FACTORS 443 Blood443 HSV-1:No | Diet at Week 2:HF Weight (g) =37.8 SUBJECT_SAMPLE_FACTORS 441 Blood441 HSV-1:yes | Diet at Week 2:HF Weight (g) =38.6 SUBJECT_SAMPLE_FACTORS 428 Blood428 HSV-1:yes | Diet at Week 2:LF Weight (g) =28.9 SUBJECT_SAMPLE_FACTORS 427 Blood427 HSV-1:yes | Diet at Week 2:HF Weight (g) =31.4 SUBJECT_SAMPLE_FACTORS 445 Blood445 HSV-1:No | Diet at Week 2:HF Weight (g) =37.7 SUBJECT_SAMPLE_FACTORS 422 Blood422 HSV-1:no | Diet at Week 2:LF Weight (g) =30.3 SUBJECT_SAMPLE_FACTORS 436 Blood436 HSV-1:yes | Diet at Week 2:LF Weight (g) =29.8 SUBJECT_SAMPLE_FACTORS 442 Blood442 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.4 SUBJECT_SAMPLE_FACTORS 418 Blood418 HSV-1:no | Diet at Week 2:HF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 433 Blood433 HSV-1:no | Diet at Week 2:LF Weight (g) =26.4 SUBJECT_SAMPLE_FACTORS 439 Blood439 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.8 SUBJECT_SAMPLE_FACTORS 435 Blood435 HSV-1:yes | Diet at Week 2:LF Weight (g) =24.9 SUBJECT_SAMPLE_FACTORS 425 Blood425 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 434 Blood434 HSV-1:no | Diet at Week 2:LF Weight (g) =26.2 SUBJECT_SAMPLE_FACTORS 421 Blood421 HSV-1:no | Diet at Week 2:LF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 440 Blood440 HSV-1:yes | Diet at Week 2:HF Weight (g) =38.3 SUBJECT_SAMPLE_FACTORS 437 Blood437 HSV-1:yes | Diet at Week 2:LF Weight (g) =27.6 SUBJECT_SAMPLE_FACTORS 420 Blood420 HSV-1:no | Diet at Week 2:HF Weight (g) =33.8 SUBJECT_SAMPLE_FACTORS 423 Blood423 HSV-1:no | Diet at Week 2:LF Weight (g) =30.7 SUBJECT_SAMPLE_FACTORS 424 Blood424 HSV-1:no | Diet at Week 2:LF Weight (g) =29.8 SUBJECT_SAMPLE_FACTORS 429 Blood429 HSV-1:yes | Diet at Week 2:LF Weight (g) =28.6 SUBJECT_SAMPLE_FACTORS 419 Blood419 HSV-1:no | Diet at Week 2:HF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 432 Blood432 HSV-1:no | Diet at Week 2:LF Weight (g) =29.6 SUBJECT_SAMPLE_FACTORS 438 Blood438 HSV-1:yes | Diet at Week 2:LF Weight (g) =29.9 SUBJECT_SAMPLE_FACTORS 417 Blood417 HSV-1:no | Diet at Week 2:HF Weight (g) =32.6 SUBJECT_SAMPLE_FACTORS 426 Blood426 HSV-1:yes | Diet at Week 2:HF Weight (g) =30.9 SUBJECT_SAMPLE_FACTORS 446 Blood446 HSV-1:No | Diet at Week 2:HF Weight (g) =39 SUBJECT_SAMPLE_FACTORS - Total_Pool_01 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_02 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_03 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_04 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_05 HSV-1:- | Diet at Week 2:- Weight (g) =- #COLLECTION CO:COLLECTION_SUMMARY For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform CO:COLLECTION_SUMMARY (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser CO:COLLECTION_SUMMARY ceramic beads were added to the tubes, and a MagNA lyser was used to beat the CO:COLLECTION_SUMMARY samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for CO:COLLECTION_SUMMARY 5 min in between pulses. Sonication for 5 min was performed to the beaten cell CO:COLLECTION_SUMMARY samples followed by centrifugation at room temperature and at 16,000rcf for 4 CO:COLLECTION_SUMMARY min. A 350 µL aliquot of each experimental cell sample homogenized supernatant CO:COLLECTION_SUMMARY was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. #TREATMENT TR:TREATMENT_SUMMARY 3-week old mice were placed on a 10% low fat (LF) diet and acclimated for one TR:TREATMENT_SUMMARY week prior to intranasal HSV-1 infection or mock infection with PBS. Fourteen TR:TREATMENT_SUMMARY days post-infection mice were randomized to either a 45% high fat diet (HF) or TR:TREATMENT_SUMMARY remained on the LF diet. Eight weeks post-diet transition, mice were euthanized TR:TREATMENT_SUMMARY and brain tissue samples were collected and processed for metabolomics. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform SP:SAMPLEPREP_SUMMARY (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser SP:SAMPLEPREP_SUMMARY ceramic beads were added to the tubes, and a MagNA lyser was used to beat the SP:SAMPLEPREP_SUMMARY samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for SP:SAMPLEPREP_SUMMARY 5 min in between pulses. Sonication for 5 min was performed to the beaten cell SP:SAMPLEPREP_SUMMARY samples followed by centrifugation at room temperature and at 16,000rcf for 4 SP:SAMPLEPREP_SUMMARY min. A 350 µL aliquot of each experimental cell sample homogenized supernatant SP:SAMPLEPREP_SUMMARY was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. Analytical pooled SP:SAMPLEPREP_SUMMARY samples were created by combining a 125 µL aliquot from each experimental SP:SAMPLEPREP_SUMMARY sample into a labeled 20 mL glass vial. The QC pooled sample was vortexed for 30 SP:SAMPLEPREP_SUMMARY sec and 350 µL aliquots were transferred to 5 labeled 2.0 mL Lo-Bind eppendorf SP:SAMPLEPREP_SUMMARY tubes. Next, the supernatants of the study and QC pooled samples were dried SP:SAMPLEPREP_SUMMARY overnight using a lyophilizer. The residue was reconstituted in 50 µL of SP:SAMPLEPREP_SUMMARY reconstitution buffer (95:5 Water:Methanol, v/v) and mixed on an analog vortex SP:SAMPLEPREP_SUMMARY mixer for 1 min with speed set at 10. Then, the samples were centrifuged at room SP:SAMPLEPREP_SUMMARY temperature for 4 min at 16,000 rcf, and the supernatants were transferred to SP:SAMPLEPREP_SUMMARY autosampler vials for data acquisition. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x CH:CHROMATOGRAPHY_SUMMARY 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) CH:CHROMATOGRAPHY_SUMMARY were used as mobile phases and the metabolites were chromatographically CH:CHROMATOGRAPHY_SUMMARY separated using a gradient separation: CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Synapt G2 Si ESI-Q-TOF CH:COLUMN_NAME Acquity HSS T3 CH:METHODS_FILENAME RTI-RCMRC-RP #ANALYSIS AN:ANALYSIS_TYPE MS AN:ACQUISITION_DATE 7/21/15 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples Total_Pool_01 Total_Pool_02 Total_Pool_03 Total_Pool_04 Total_Pool_05 Blood417 Blood418 Blood419 Blood420 Blood443 Blood444 Blood445 Blood446 Blood425 Blood426 Blood427 Blood439 Blood440 Blood441 Blood442 Blood421 Blood422 Blood423 Blood424 Blood431 Blood432 Blood433 Blood434 Blood428 Blood429 Blood430 Blood435 Blood436 Blood437 Blood438 Factors HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet 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13.35_329.1535m/z 329.1534563 13.34903333 14.65_483.2451m/z 483.2450661 14.64505 15.07_781.9438m/z 781.9437851 15.0677 12.70_386.1824n 409.1716161 12.70113333 15.27_288.6338m/z 288.6338374 15.27246667 13.04_633.2803m/z 633.280345 13.04188333 15.07_773.9584m/z 773.9583861 15.0677 14.65_311.6279m/z 311.6278509 14.64505 15.17_715.9003n 716.9076121 15.17008333 15.07_991.6558m/z 991.6557531 15.0677 0.70_622.1278n 645.1230465 0.69565 15.21_421.2968m/z 421.2967961 15.21105 15.29_530.3171m/z 530.3171115 15.29295 14.14_452.3623m/z 452.3622726 14.1405 15.25_484.4264m/z 484.4264227 15.252 11.58_267.1517m/z 267.1517036 11.58226667 15.27_564.7914m/z 564.7914041 15.27246667 15.29_389.2958m/z 389.2957535 15.29295 4.39_205.0978m/z 205.0978105 4.386 15.56_448.3449n 487.2878943 15.55915 12.80_317.1706m/z 317.1706378 12.79615 5.26_339.0575m/z 339.0574684 5.259433333 10.59_251.1261m/z 251.1260531 10.58625 15.09_971.5846m/z 971.5846039 15.08818333 5.29_123.0421m/z 123.0420921 5.293 15.92_644.3867n 645.5245042 15.92036667 0.70_659.0765n 660.0837312 0.69565 6.06_139.0081m/z 139.0080773 6.063766667 8.97_137.0583m/z 137.0582944 8.9701 13.92_426.3543m/z 426.3543109 13.91525 11.44_253.1370m/z 253.1370195 11.43891667 5.74_123.0423m/z 123.0422519 5.7435 12.19_427.2474m/z 427.247442 12.1892 0.72_648.0916n 671.0768576 0.716133333 13.73_400.5074m/z 400.507376 13.73095 15.29_551.2950n 552.3023019 15.29295 15.07_820.9461n 804.9274117 15.0677 13.51_424.3360m/z 424.3360128 13.51285 15.07_816.9704n 799.9671002 15.0677 0.72_160.0610m/z 160.061002 0.716133333 13.21_398.3189m/z 398.318933 13.2057 15.27_104.1063m/z 104.1062701 15.27246667 15.25_280.6505m/z 280.6505423 15.252 15.37_259.6395m/z 259.6395367 15.37486667 11.26_283.1652m/z 283.1651606 11.262 10.25_162.0556m/z 162.0556083 10.24551667 13.35_279.1882m/z 279.188243 13.34903333 15.42_509.3405n 532.3343025 15.41581667 15.07_753.9196n 792.9299492 15.0677 15.56_371.1870m/z 371.1869675 15.55915 15.07_515.8078m/z 515.8078466 15.0677 3.83_219.1089n 242.1003003 3.83305 13.23_252.2654m/z 252.2654108 13.22616667 12.70_213.5720m/z 213.5719582 12.70113333 15.09_563.7743m/z 563.7742594 15.08818333 14.22_253.6274m/z 253.6273684 14.2224 10.91_163.0710m/z 163.0710054 10.91388333 5.26_371.0278m/z 371.0277953 5.259433333 15.23_407.3012m/z 407.3011845 15.23151667 0.70_292.0911n 315.080599 0.69565 13.59_296.2884m/z 296.2883808 13.58738333 0.70_315.2282m/z 315.2282401 0.69565 12.70_495.1167m/z 495.1166722 12.70113333 12.70_222.0757m/z 222.075726 12.70113333 14.73_816.8703m/z 816.8702874 14.72696667 13.89_293.2061m/z 293.2060603 13.89476667 4.54_226.1684n 249.1578158 4.542633333 15.52_431.2291m/z 431.2291272 15.5182 10.85_207.1291m/z 207.1291206 10.85245 15.72_979.1035n 963.0847634 15.72296667 3.37_145.0764m/z 145.0764392 3.36925 11.36_163.0746m/z 163.0745526 11.35701667 4.20_154.5766m/z 154.576624 4.2017 15.60_512.4584m/z 512.4583605 15.60011667 14.56_745.9192n 746.9264714 14.56315 5.74_210.0874n 193.084065 5.7435 14.91_418.7639m/z 418.763941 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