#METABOLOMICS WORKBENCH jburgess_20160304_9108141_mwtab.txt DATATRACK_ID:535 jburgess_20160304_101433 DATATRACK_ID:662 STUDY_ID:ST000358 ANALYSIS_ID:AN001447 PROJECT_ID:PR000285 VERSION 1 CREATED_ON November 3, 2017, 11:25 am #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 hippocampus from Anxiety Prone HSV-Latently ST:STUDY_TITLE Infected Obese Mice ST:STUDY_TYPE Broad Spectrum LCMS ST:STUDY_SUMMARY Brain tissue 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 brain tissue 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 31 ST:NUM_MALES 31 #SUBJECT SU:SUBJECT_TYPE mouse SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 5.5 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 409 Hipp409 HSV-1:yes | Diet at Week 2:HF Weight (g) =37.1 SUBJECT_SAMPLE_FACTORS 394 Hipp394 HSV-1:yes | Diet at Week 2:HF Weight (g) =48.2 SUBJECT_SAMPLE_FACTORS 391 Hipp391 HSV-1:no | Diet at Week 2:LF Weight (g) =31.2 SUBJECT_SAMPLE_FACTORS 414 Hipp414 HSV-1:yes | Diet at Week 2:LF Weight (g) =36.6 SUBJECT_SAMPLE_FACTORS 398 Hipp398 HSV-1:yes | Diet at Week 2:LF Weight (g) =35.2 SUBJECT_SAMPLE_FACTORS 392 Hipp392 HSV-1:no | Diet at Week 2:LF Weight (g) =34 SUBJECT_SAMPLE_FACTORS 386 Hipp386 HSV-1:no | Diet at Week 2:HF Weight (g) =34.8 SUBJECT_SAMPLE_FACTORS 396 Hipp396 HSV-1:yes | Diet at Week 2:HF Weight (g) =48.4 SUBJECT_SAMPLE_FACTORS 388 Hipp388 HSV-1:no | Diet at Week 2:HF Weight (g) =44.1 SUBJECT_SAMPLE_FACTORS 412 Hipp412 HSV-1:yes | Diet at Week 2:HF Weight (g) =34.7 SUBJECT_SAMPLE_FACTORS 413 Hipp413 HSV-1:yes | Diet at Week 2:LF Weight (g) =36 SUBJECT_SAMPLE_FACTORS 389 Hipp389 HSV-1:no | Diet at Week 2:LF Weight (g) =34 SUBJECT_SAMPLE_FACTORS 411 Hipp411 HSV-1:yes | Diet at Week 2:HF Weight (g) =40.2 SUBJECT_SAMPLE_FACTORS 408 Hipp408 HSV-1:no | Diet at Week 2:LF Weight (g) =29.1 SUBJECT_SAMPLE_FACTORS 403 Hipp403 HSV-1:no | Diet at Week 2:HF Weight (g) =48.6 SUBJECT_SAMPLE_FACTORS 415 Hipp415 HSV-1:yes | Diet at Week 2:LF Weight (g) =34.8 SUBJECT_SAMPLE_FACTORS 401 Hipp401 HSV-1:no | Diet at Week 2:HF Weight (g) =49.2 SUBJECT_SAMPLE_FACTORS 405 Hipp405 HSV-1:no | Diet at Week 2:LF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 395 Hipp395 HSV-1:yes | Diet at Week 2:HF Weight (g) =40.9 SUBJECT_SAMPLE_FACTORS 402 Hipp402 HSV-1:no | Diet at Week 2:HF Weight (g) =38.8 SUBJECT_SAMPLE_FACTORS 390 Hipp390 HSV-1:no | Diet at Week 2:LF Weight (g) =34.5 SUBJECT_SAMPLE_FACTORS 410 Hipp410 HSV-1:yes | Diet at Week 2:HF Weight (g) =37.8 SUBJECT_SAMPLE_FACTORS 385 Hipp385 HSV-1:no | Diet at Week 2:HF Weight (g) =41.6 SUBJECT_SAMPLE_FACTORS 397 Hipp397 HSV-1:yes | Diet at Week 2:LF Weight (g) =33.1 SUBJECT_SAMPLE_FACTORS 399 Hipp399 HSV-1:yes | Diet at Week 2:LF Weight (g) =33 SUBJECT_SAMPLE_FACTORS 393 Hipp393 HSV-1:yes | Diet at Week 2:HF Weight (g) =44.1 SUBJECT_SAMPLE_FACTORS 407 Hipp407 HSV-1:no | Diet at Week 2:LF Weight (g) =34.7 SUBJECT_SAMPLE_FACTORS 406 Hipp406 HSV-1:no | Diet at Week 2:LF Weight (g) =31.4 SUBJECT_SAMPLE_FACTORS 387 Hipp387 HSV-1:no | Diet at Week 2:HF Weight (g) =31.9 SUBJECT_SAMPLE_FACTORS 400 Hipp400 HSV-1:yes | Diet at Week 2:LF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 416 Hipp416 HSV-1:yes | Diet at Week 2:LF Weight (g) =35.2 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 Brain tissue samples were homogenized in 50:50 Acetonitrile:Water (10 µL to CO:COLLECTION_SUMMARY every 1 mg of tissue) with washed ceramic beads on a MagNA Lyser, using two 30 CO:COLLECTION_SUMMARY sec pulses at 2,000 rpm with a 1 min chilling step in between pulses. Samples CO:COLLECTION_SUMMARY were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm and centrifuged at CO:COLLECTION_SUMMARY room temperature for 4 min at 16,000 rcf. A 50 µL aliquot of each experimental CO:COLLECTION_SUMMARY brain tissue homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind CO:COLLECTION_SUMMARY eppendorf tube. Analytical pooled samples were created by combining 15 µL CO:COLLECTION_SUMMARY aliquot from each experimental sample in a 2.0 mL Lo-Bind eppendorf tube. The QC CO:COLLECTION_SUMMARY pooled sample was vortexed for 30 sec and 50 µL aliquots were transferred to 5 CO:COLLECTION_SUMMARY labeled 2.0 mL Lo-Bind eppendorf tubes. Next, 350 µL of acetonitrile was added CO:COLLECTION_SUMMARY to study sample and QC pooled sample tubes. The samples were vortexed on a CO:COLLECTION_SUMMARY multi-tube vortexer for 2 min at 5,000 rpm, and centrifuged at room temperature CO:COLLECTION_SUMMARY for 4 min at 16,000 rcf. The supernatants were transferred to new labeled tubes CO:COLLECTION_SUMMARY and dried on a lyophilizer overnight. Dried samples were reconstituted in 50 µL CO:COLLECTION_SUMMARY of 95:5 Water:Methanol, mixed on an analog vortex mixer for 1 min with speed set CO:COLLECTION_SUMMARY at 10. Then, the samples were centrifuged at room temperature for 4 min at CO:COLLECTION_SUMMARY 16,000 rcf, and the supernatants were transferred to autosampler vials for data CO:COLLECTION_SUMMARY acquisition. UPLC-MS Methods: UPLC-MS spectra were collected for all samples. CO:COLLECTION_SUMMARY UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x CO:COLLECTION_SUMMARY 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% CO:COLLECTION_SUMMARY formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) CO:COLLECTION_SUMMARY were used as mobile phases and the metabolites were chromatographically CO:COLLECTION_SUMMARY separated using a gradient separation: (see the 3. CO:COLLECTION_SUMMARY Sheridan-Mice-Hypothalamus_RP-Metadata and Analytical Metadata.xlsx file for the CO:COLLECTION_SUMMARY flow gradient). Mass spectroscopy analysis was performed using a Synapt G2 Si CO:COLLECTION_SUMMARY ESI-Q-TOF using a 10 µL injection volume. UPLC-MS data were collected over CO:COLLECTION_SUMMARY 70-1000 m/z in both positive and negative modes. CO:SAMPLE_TYPE brain homogenate #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 Brain tissue samples were homogenized in 50:50 Acetonitrile:Water (10 µL to SP:SAMPLEPREP_SUMMARY every 1 mg of tissue) with washed ceramic beads on a MagNA Lyser, using two 30 SP:SAMPLEPREP_SUMMARY sec pulses at 2,000 rpm with a 1 min chilling step in between pulses. Samples SP:SAMPLEPREP_SUMMARY were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm and centrifuged at SP:SAMPLEPREP_SUMMARY room temperature for 4 min at 16,000 rcf. A 50 µL aliquot of each experimental SP:SAMPLEPREP_SUMMARY brain tissue homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind SP:SAMPLEPREP_SUMMARY eppendorf tube. Analytical pooled samples were created by combining 15 µL SP:SAMPLEPREP_SUMMARY aliquot from each experimental sample in a 2.0 mL Lo-Bind eppendorf tube. The QC SP:SAMPLEPREP_SUMMARY pooled sample was vortexed for 30 sec and 50 µL aliquots were transferred to 5 SP:SAMPLEPREP_SUMMARY labeled 2.0 mL Lo-Bind eppendorf tubes. Next, 350 µL of acetonitrile was added SP:SAMPLEPREP_SUMMARY to study sample and QC pooled sample tubes. The samples were vortexed on a SP:SAMPLEPREP_SUMMARY multi-tube vortexer for 2 min at 5,000 rpm, and centrifuged at room temperature SP:SAMPLEPREP_SUMMARY for 4 min at 16,000 rcf. The supernatants were transferred to new labeled tubes SP:SAMPLEPREP_SUMMARY and dried on a lyophilizer overnight. Dried samples were reconstituted in 50 µL SP:SAMPLEPREP_SUMMARY of 95:5 Water:Methanol, mixed on an analog vortex mixer for 1 min with speed set SP:SAMPLEPREP_SUMMARY at 10. Then, the samples were centrifuged at room temperature for 4 min at SP:SAMPLEPREP_SUMMARY 16,000 rcf, and the supernatants were transferred to autosampler vials for data SP:SAMPLEPREP_SUMMARY 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 Waters Acquity CH:COLUMN_NAME Acquity HSS T3 CH:METHODS_FILENAME RTI-RCMRC-RP #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST000358_AN001447_Results.txt UNITS:intensity #END