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Sticht Center on Aging January 26, 2016 Promote the health and independence of older adults by fostering multi-disciplinary collaboration in basic and clinical research, research training, professional education and community outreach. The Sticht Center on . . . Affiliated Programs Affiliated Faculty Pepper Older Americans Independence Center (NIA - P30) >100 Faculty from 22 Departments Alzheimer’s Center / Kulynych Center for Memory and Cognition Research John A. Hartford COE Donald W. Reynolds GEP Services / Equipment Available Metabolic Assessments (Medgraphics): • V02 Max • Resting Metabolic Rate (RMR) DXA Imaging Assessments (Hologic Horizon) CT, MRI imaging for body composition, blood flow, and perfusion. Tools and Protocols to help ‘gerontologize’ clinical research. Tissue collection (fat, muscle, blood, spinal fluid) Wake Forest School of Medicine Services Available Recruitment of Older Adults • Targeted direct mail, VITAL newsletter (11,650 older adults in our database) Transportation • Trained drivers and vans to provide transportation for study participants unable to drive or find a ride, a common barrier to recruitment /adherence particularly for functionally impaired and minority participants. Wake Forest School of Medicine Equipment / Training Available Physical Function Assessments: • Leg strength and power (Biodex & Leg Press) • Gait speed (GaitRite Mat) • Postural Sway (Force Plate) • Short Physical Performance Battery (SPPB) • Grip Strength Cognitive Function Assessments: • Variety including MCI / Dementia diagnosis Wake Forest School of Medicine National Leading in Behavioral intervention trials in older adults across multiple morbidities. Multicenter study coordination in older adults Impact of obesity on older adults Interventions to manipulate brain metabolism to affect cognitive function Application of functional assessment to inform clinical care. Wake Forest School of Medicine In the Offing . .. • ENRGISE – a 300 persons RCT to reduce IL-6 with omega-3 FA and/or losartan. • U13 Conference Grant – applying physical and cognitive functional assessment to guide clinical care • NCI funded effort to understand the vascular aging effects of chemotherapy • Trial of the Epigenomic / Transcriptomic Changes with Weight Loss • Alzheimer’s Disease Center (P30) Application • Trial of the interventions to reduce sedentary time. Wake Forest School of Medicine Promoting Translational Research • Team (People) • Models / Approaches • Facilitating Resources Wake Forest School of Medicine TRIAL OF VITAMIN D SUPPLEMENTATION AND NEUROMUSCULAR FUNCTIOIN IN OLDER ADULTS • Denise Houston & Osvaldo Delbono • What level of Vitamin D stores is associated with optimal muscle function? • DH – Nutrition, Epidemiology • OD – Physiology of Aging Muscle (primarily rodent) Wake Forest School of Medicine EPIGENETIC ROLES IN REGULATION OF CHOLESTEROL METABOLISM AND CVD RISK • Yongmei Liu • Developed methodology to quantify transcriptome / methylome on large scale. • Working with a large team – identified gene modules involved cellular cholesterol transport to be associated with coronary calcification and diabetes • This R01 explores microRNA regulation of these gene modules, and works in cell culture to elucidate mechanism. Wake Forest School of Medicine GoMET (Jingzhong Ding, Liu, Kritchevsky, Nicklas, Applegate, McClain, Justice) • Based on this work . . . • Aging Center funded a project to show that transcripomic modules are sensitive to change. • Pilot study to evaluate metformin for its possible anti-aging effects based on changes in aging-related modules. • Preliminary data for a large trial (TAME) being planned with a national group. Wake Forest School of Medicine People • Should have something concrete to contribute and in depth. • Must be willing to try to understand the problem from the perspectives of persons from other disciplines. • Must be willing to devote time and energy to finding the overlaps that permit translation to occur. • Should not be asked to do anything that is more than a small departure from the direction that their work is heading. Wake Forest School of Medicine Models / Approaches What is handed off across translational divides? • Biologic Specimens • Imaging • Phenotyping that can be done in both humans and model systems. • Information about pathways and translational relevance. Wake Forest School of Medicine Facilitating Resources • Pilot funds • Methods development funds • Project management expertise • Specimen and Image Repositories • Cores • Access to persons with relevant phenotypes & controls. Wake Forest School of Medicine My job as a champion . . . • Be aware of what’s going on institutionally / nationally that might be applied to problems in aging. • Actively engage investigators to work on agingrelated problems. • Provide $ to promote the collection of new data in support of translation. • Mobilize other Aging Center resources to build out the skills needed for a successful translational project. Wake Forest School of Medicine Roundtable: New Research Equipment and Services Proteomics & Metabolomics Shared Resource (PMSR) Mass spectrometry – the driver of Multi-Omics Mass Spectrometry Proteogenomics Wake Forest Baptist Medical Center Functional Genomics 17 Q-Exactive HF for proteomics research Nanospray Flex™ Ion Source Thermo Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer w/ Dionex Ultimate-3000 nano-UPLC system o High-resolution mass spectrometry with high mass accuracy and fast scan speed provides in depth analysis of complex biological samples. o Various applications cover the identification and quantification of proteins, peptides and small molecules. Wake Forest Baptist Medical Center 18 Typical Workflows in Proteomics Cells – Tissue – Biological Fluids (Saliva, Blood, CSF, Urine) Organelle Fractionation, Laser Capture Microdissection (LCM) Depletion of Abundant Proteins Proteome Fraction of Interest/Enrichment in particular PTMs In-solution/in-gel proteolysis with trypsin SRM/MRM mode (single or multiple reaction monitoring) Targeted Method Wake Forest Baptist Medical Center nanoLC-MS and MS/MS analysis Data Dependent or Independent mode Non-Targeted Method (Discovery) 19 Common Proteomics Studies Static Studies: Organellar Proteomics – “spatial proteomics” Protein-drug interactions Chemical Profiling PTM (post-translational modifications) Proteomics Pipeline ID a single band/spot on a PAGE gel Dynamic Studies: Determine changes in protein(s) expression, PTMs or localization in response to perturbations (e.g. oncogenic mutations, drug/radiation treatment) or as function of time, pH, oxidation status of the microenvironment. Walther & Mann J Cell Biol. 2010, 190, 491-500. Common Proteomics Studies crosstalk of various signaling/metabolic networks drug effects on signaling/metabolism time course studies changes in mE & effects on signaling/metabolism tissue comparison e.g. normal vs diseased, host vs regenerated Clinical Proteomics Studies – Biomarker Discovery and Validation Genomic analysis Disease vs. Normal mRNA level Proteomic analysis (MS, Quantitation: labelfree, TMT, Super SILAC) Discovery Candidate genes (tissue, biological fluids) One Success Story: IVDMIA/OVA1 in vitro diagnostic multivariate index assay a qualitative serum test that combines the results of five immunoassays using a proprietary algorithm to yield a single score indicating the likelihood that a woman's pelvic mass is malignant. Verification in situ hybridization/Immunohistochemistry Mass spectrometry (SISCAPA/MRM) Large # samples (blood, population) Small # candidates Clinical Validation (blood, population) Diagnostic Prognostic Wake Forest Baptist Medical Center Therapeutic 22 Main applications Bottom-up Proteomics Data-dependent acquisition (DDA) Targeted Proteomics Parallel reaction monitoring (PRM) Qualitative & Quantitative Proteomics Data-independent acquisition (DIA) Wake Forest Baptist Medical Center 23 Bottom-up proteomics Data-dependent acquisition (DDA) - Giuseppe Orlando/WFIRM Protein sample in lysis buffer Native human tissue (e.g. total lysate, SILAC proteins) Protein separation & profiling Decellularization (e.g. SDS-PAGE) Modified protein/peptide enrichment Extracellular matrix (ECM) Enzymatic digestion (e.g. Trypsin) Fractionation & solubilization High-pH reversed phase peptide fractionation DDA analysis LC-MS/MS (DDA) Proteome profile of ECM structure Quantifier labeling Data processing (e.g. DB search, compiling) Wake Forest Baptist Medical Center 24 Targeted proteomics Parallel reaction monitoring (PRM) Brittany Bondic Henderson, Division of Endocrinology, Diabetes, and Metabolism Protein sample in lysis buffer (e.g. total lysate, SILAC proteins) Enzymatic digestion (e.g. Trypsin) Data processing Normal human thyroid (e.g. DB search, compiling) Target peptide candidates Tumorized (e.g. BLAST/alignment) A A set of signature molecules with altered expression C B LC-MS/MS LC-MS/MS (DDA) (PRM) Data processing Data processing (e.g. DB search, compiling) (e.g. MS/MS quantification) PRM analysis Selective & sensitive quantitation Wake Forest Baptist Medical Center 25 All ions fragmentation Data-independent acquisition (DIA) Target identification Protein sample in lysis buffer (e.g. RIPA) Enzymatic digestion (e.g. Trypsin) High-pH reversed phase peptide fractionation Mass calibration LC-MS/MS (DIA) Wake Forest Baptist Medical Center Data independent acquisition (All ions fragmentarion) Identification + Quantification Molecular targets DB search & compilation Complimentary SRM for target quantification Data processing (e.g. Reporter ion quantification) Post-SRM (e.g. Skyline) 26 Shimadzu LCMS-8050 for Metabolomics Research Shimadzu LCMS-8050 Mass Spectrometer w/ Shimadzu Prominence UPLC system o High sensitivity, rapid scan rate, and fast polarity switching allows for quantitative MRM analysis of hundreds of metabolites simultaneously, in both the positive and negative modes. Wake Forest Baptist Medical Center 27 Research Relevance of New Equipment • The PMSR has partnered with Shimazdu to implement new methods for targeted metabolomics studies within the core. • The methods to be implemented will allow for the rapid quantitative analysis of over 300 metabolites. • Main new applications will include pharmacokinetic studies, measurements of eicosanoids, amino acids, nucleotides, and biomarkers of oxidative stress. Wake Forest Baptist Medical Center 28 Metabolite List for a Single 2 Column Method 2,3-Bisphosphoglyceric acid 3-Phosphoglyceric acid Dihydroxyacetone phosphate Fructose 1,6-bisphophate Glucose 1-phosphate Glucose 6-phosphate Glucose 3-phosphate Phosphoenolpyruvic acid Lactic acid Pyruvic acid Fructose 6-phosphate Glyceraldehyde 3-phosphate 6-Phosphogluconic acid Erythrose 4-phosphate Ribose 5-phosphate Ribulose 5-phosphate Sedoheptulose 7-phosphate Acetyl coenzyme A Succinyl coenzyme A 2-Ketoglutaric acid Aconitic acid Citric acid Fumaric acid Isocitric acid Malic acid Succinic acid Cystathionine Cysteine Homocysteine Methionine 5-Glutamylcysteine Glutathione Oxidized Glutathione S-Adenosylhomocysteine S-Adenosylmethionine Alanine Arginine Asparagine Aspartic Acid Asymmetric dimethylarginine Citrulline Cysteine Cystine Homocystine Glutamic acid Glutamine Glycine Dimethylglycine Histidine Isoleucine Leucine Lysine Methionine Methionine sulfoxide Ornitine Phenylalanine Proline 4-Hydroxyproline Serine Symmetric dimethylarginine Threonine Tryptophan Tyrosine Valine NAD NADH NADP NADPH FAD FMN Adenine Cytosine Guanine Thymine Uracil Xanthine Adenosine Cytidine Guanosine Inosine Thymidine Uridine Adenosine 3',5'-cyclic monophosphate Adenosine diphosphate Adenosine monophosphate Adenosine triphosphate Cytidine 3',5'-cyclic monophosphate Cytidine diphosphate Cytidine monophosphate Cytidine triphosphate Guanosine 3',5'-cyclic monophosphate Guanosine diphosphate Guanosine monophosphate Guanosine triphosphate Thymidine diphosphate Thymidine monophosphate Thymidine triphosphate Uridine diphosphate Uridine monophosphate Uridine triphosphate 4-Aminobutyric acid Adenylsuccinic acid Argininosuccinic acid Cholic acid Creatine Nicotinic acid Ophthalmic acid Orotic acid Pantothenic acid Taurocholic acid Uric acid 2-Aminobutyric acid Acetylcarnitine Acetylcholine Allantoin Carnitine Carnosine Choline Citocholine Creatinine Cysteamine Dopa Dopamine Epinephrine Histamine Hypoxanthine Kynurenine Niacinamide Serotonin Glycolytic system Pentose-phosphate pathway TCA Cycle Methylation and Transsulfuration Cycle Amino Acids Co-enzymes Organic Acids Other Metabolites Contact Information for Service Requests Contact Hanes Building, Room 2029, 1 Medical Center Blvd. Proteomics services and equipment Contact: Jingyun Lee, Ph.D ([email protected]) Hanes Building, Room 2027, 1 Medical Center Blvd. Metabolomics services and equipment Contact: Brian Fulp ([email protected]) Room-Suite 250-2W003, 575 Patterson Ave. MALDI/HPLC services and equipment Contact: Mark Morris ([email protected]) Leadership Cristina Furdui, Ph.D., Co-Director [email protected], tel (336) 716-2697 Mark Lively, Ph.D., Co-Director [email protected] Wake Forest Baptist Medical Center 30 Roundtable: New Research Equipment and Services Cancer Genomics Core Lab Co-directors: Lance Miller and Greg Hawkins Illumina NextSeq 500 Up to 400 million reads (high throughput) Up to 130 million read (mid throughput) Data format: base-space fastq, fasta, BAM, VCF 1 base pair sequencing Run times (11-29 hours) Manual library construction Read lengths: single 1 X 50 to 1 X 150 bp paired up to 2 X 150 bp Capacity: High throughput 1 genome (20-30 X coverage) 8 RNAseq (up to 50 SE million reads) >9 exomes (>30X coverage) Capacity: Mid-throughput 3 exomes (>30X coverage) 3 RNAseq (up to 40 million reads) Data analysis BaseSpace (individual accounts) Off-line: GATK, BWA, Bowtie, Cufflinks Tophat, (lot’s of others) Wake Forest Baptist Medical Center 32 Illumina MiSeq DX 1-25 million reads (high throughput) Data format: base-space fastq, fasta, BAM, VCF 1 base pair sequencing Run times (5-24 hours) Manual library construction Read lengths: single 1 X 50 to 1 X 150 bp paired up to 2 X 150 bp Capacity: High throughput 8-28 samples per run (clinical) >96 samples research Type of Samples ChIP-seq Low throughput RNAseq Sequencing panels CLIA Approved Cystic Fibrosis Custom assay kit Wake Forest Baptist Medical Center Data analysis BaseSpace (individual accounts) Off-line: GATK, BWA, Bowtie, Cufflinks Tophat, (lot’s of others) 33 Current Applications for NGS RNAseq whole transcriptome, targeted gene panels: mRNA/microRNA/lncRNA, splice variants, gene fusions, allele specific expression, genetic variants ChIPseq Microbiome 16s ribosomal RNA sequencing whole bacterial genome Exome Sequencing identification of coding variations human, mouse, primate (vervet) Targeted Sequencing custom single gene or panel of genes Methylation Sequencing whole genome (bisulfite) or methylation capture (Methyl binding protein) o Whole Genome Sequencing human, mouse, primate (vervet) o Targeted Panels (MiSeq) predesigned (example: Cancer) custom designed Wake Forest Baptist Medical Center 34 Contact Information • Co-Directors: Lance Miller, Greg Hawkins • Staff: Lou Craddock, Wei Cui, Jamie Haywood • Analyst: Dr. Jeff Chou • Location: Hanes 4026/4028, NRC 311/312 • Contact email: [email protected] [email protected] [email protected] • Lab Phone Number: 713-5103 Wake Forest Baptist Medical Center 35