Transcript The Sticht Center on - Wake Forest Clinical and Translational

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.
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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.
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Promoting Translational Research
• Team (People)
• Models / Approaches
• Facilitating Resources
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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)
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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
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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.
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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
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nanoLC-MS and
MS/MS analysis
Data Dependent or
Independent mode
Non-Targeted Method
(Discovery)
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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
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Therapeutic
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Main applications
Bottom-up
Proteomics
Data-dependent acquisition (DDA)
Targeted
Proteomics
Parallel reaction monitoring (PRM)
Qualitative & Quantitative
Proteomics
Data-independent acquisition (DIA)
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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)
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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
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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)
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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
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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
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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
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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
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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)
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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
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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
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