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Disease and Disease Technology Subgroup

Disease and Disease Technology Subgroup. Jared, Marta, Sutee, Denise, Ryan, Inyoul, Burak, Zhiyuan, Qin. Liver Toxicity MRM Pulmonary Fibrosis Frontotemporal Dementia Huntington’s Disease Glioblastoma Multiforme. Rodent and/or human primary liver cells. Different mouse strains.

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Disease and Disease Technology Subgroup

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  1. Disease and Disease Technology Subgroup Jared, Marta, Sutee, Denise, Ryan, Inyoul, Burak, Zhiyuan, Qin

  2. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  3. Rodent and/or human primary liver cells Different mouse strains New biomarker discovery Blood handling Molecular measurements Liver toxicity biomarkers Treat with selected drugs/chemicals (Acetaminophen) Serum proteomePathology confirmation Secretome Using computational tools and MPSS database to analyze transcriptome Pre-selected liver-specific proteins/target Using SPR, MS, and other targeted proteomics methods to monitor the changes in sera Using SPR, DEAL, MS, ELISA, and immuno-blotting to validate the changes in human sera • Liver-specific biomarkers in blood • reflecting liver status for • chemical exposure monitoring • treatment strategy • prognosis, etc.

  4. three mice inbred strains B6 >> AJ >> SJL Suspectibility of three mice inbred strains to Acetaminophen ALT (IU/L)

  5. THE TEAM Caltech Jim Heath Lab Plexera China/USA Institute for Systems Biology Seattle, Washington Hood Lab Zhiyuan Hu Chris Lausted Shizhen Qin Xiaowei Yan Gustavo Glusman Lee Rowen Hyuntae Yoo Bingyun Sun Amy Brightman Kai Wang Shile Zhang Wei Yan ASB San Diego, CA ABMAX Beijing, China Nanostring Seattle, WA

  6. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  7. GNF RNA expression database H GeneCards Select tryptic peptides as standards Prepare peptide standards Protein list Generate a target list ISB MPSS data set depleted serum Full scan LC/MS/MS Spike-in heavy peptide standards digestion Raw data L Perform ProteinProphet search Data conversion, visualization, analysis and interpretation cleaning Run MRM sample ready to run Prepare serum samples Spike in standards Build MRM method Select MRM transitions from MS spectrum

  8. Fold change in in serum from Acetaminophen treated mouse Liver proteins Novel biomarkers for hepatotoxicity (human data, GlaxoSmithKline) Acetaminophen induced liver injury - markers identified through gene expression data from a panel of inbred mouse strains (other groups) Liver specific MRM Team: Shizhen Qin, Li Gray

  9. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  10. Pulmonary Fibrosis • Pulmonary Fibrosis is a chronic disease that causes inflammation (swelling and irritation) and fibrosis (scarring) of tissue in the lungs. • Approximately 200,000 Americans have it, with an estimated 50,000 new cases each year. • There is no effective treatment for pulmonary fibrosis. Many people live only about 3 to 5 years after diagnosis.

  11. Model Systems • Cell culture • tracheal epithelial cells, bronchial epithelial cells, alveolar epithelial cells (type I and II), pulmonary fibroblasts, microvascular endothelial cells • treatment with TGFß or NECA (5’-N-Ethylcarboxamido adenosine) • Mouse models • bleomycin treated mice • adenosine deaminase (ADA) deficient mice

  12. Methods Currently in Use • Microarrays • mRNA • miRNA • qPCR • SYBR • TaqMan • Mass Spectrometry • Shotgun • iTRAQ

  13. Acknowledgements • Galas group • Kai, Richard, Jessica, Shile, David, Ji-Hoon and Nikita • Ohio State • Univ. of Texas • Battelle, DTRA

  14. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  15. FrontotemporalDementia (FTD) degenerative brain disease that primarily affects the frontal and and temporal lobes of the brain. characterized by an accumulation of abnormal forms of tau proteins in the brain the most common cause of early-onset dementia in people under age 60. affects approximately 250,000 Americans a year, or about 6.7 people per 100,000 among people ages 45 to 64. Due to the symptoms, FTD can be mistaken for Alzheimer's disease, Parkinson's disease or a primarily psychiatric disorder like depression or schizophrenia. There is no treatment or cure yet that can reverse the damage, but medications can help relieve the symptoms.

  16. FTD and AD: Similarities & Differences

  17. Research Plan Four transgenic mouse groups for experiments: 1) rTg(tauP301L) : express mutant tau 2) rTg(tauwt) : express wild type tau 3) rTg(PrP)_RML : express prion protein and develop prion disease 4) rTg(PrP)_Clean : express prion protein and stay normal Brains and plasmawill be harvested : monthly up to 9 months for the FTD mouse groups at 10 day intervals for the prion disease mouse groups Multiple Comparisonswill be made to identify shared and unique molecules (miRNAs, Genes, Proteins) and pathways between FTD and prion disease

  18. People Institute for Systems Biology Seattle, Washington Leroy Hood David Galas Hyuntae Yoo David Baxter Inyoul Lee Array core facility Proteomics core facility McLaughlin Research Institute Great Falls, Montana George Carlson Miranda Orr Rose Pitstick Mouse core facility Axonal Microtubule Assembly through GSK signaling pathway

  19. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  20. Huntington Disease • HD occurs in 1 out of 10,000 individuals • Mood swings,  cognitive functions, chorea • Symptoms are correlated with  spiny neurons • Basal ganglia, caudate nucleus and cortex • “Cause”: mutant Htt gene • (Expanded triplet repeat)

  21. Research at ISB (contemplated) • Computational • Model construction • “Synthetic lethal” networks • Symptom networks • Protein-protein interaction networks • IPS cells • Perturbable • Replicate samples • Model of hard-to-query tissues • (neurons)

  22. Genome Analysis • We are designing a complete genome sequencing project to detect genes that modify and influence the HD phenotype, and that may interact with Htt. • Biomarker • Metabolomics • Serum proteomics, microRNAs • Would like to predict HD onset, progression, use as surrogate endpoints for therapeutic trials • Proteomics • Does Htt (specifically) bind DNA • ChIP seq • We would like to determine proteins that interact with Htt • Mutant and wild type • Cytoplasmic and nuclear locations • Soluble / aggregate • Several cell types

  23. People • Jared Roach • project leader • Nat Goodman • advisory • More to come… A number of researchers have jumped on the “BDNF pathway” bandwagon. A more systematic approach might advance the field.

  24. Liver Toxicity • MRM • Pulmonary Fibrosis • Frontotemporal Dementia • Huntington’s Disease • Glioblastoma Multiforme

  25. Glioblastoma Multiforme (GBM) Age <18: 0.41% Ages 18-35: 1.65% Age >70: 21% Ages 35-50: 16% Ages 50-70: 59.9% • The most common primary brain tumor of adults – 52% of all primary brain tumors • Common symptoms: Headaches, memory loss, seizures and behavioral changes • primarily due to increased pressure of the brain caused by rapid growth of the tumor • WHO classification of Brain Tumors • Grade II – Low grade diffuse Astrocytoma • Grade III – Anaplastic Astrocytoma • Grade IV - GBM • Primary GBMs vs Secondary GBMs • Age Distribution

  26. GBM Treatment • Median Survival Time from the time of diagnosis •  Without treatment is 3 months •  With treatment  12 months •  Most patients die within 24 months • Standard care of radiation-plus-temozolomide (TMZ) •  2 month improvement vs radiation alone • Economic Burden to the health care system •  $49,242 per patient > 20 x compared to control cancer-free subjects * • * Kutikova et al J. of Neuro-Oncology 2007

  27. Pten Pten Pten Rbf Rbf Research Plan Inducible Astrocytoma Model Assessment Normal K-Ras Normal Diffuse grade II-III astrocytoma Diffuse grade II-III astrocytoma accelerated no Brain Tumor Ras sk. tu. Rbf, Ras AA angiogenesis invasion Rbf, Ras GBM Pten+/- angiogenesis invasion necrosis Rbf, Ras GBM +sk. tu. Pten 2 mo 4 mo 1 yr time post 4OHT:

  28. Systems Biology with Strategy for GBM with Genetically Engineered Mouse Models • mRNA • microRNA • protein Brain Tissue • Data Analysis • C-Monkey - Inferelator • Network analysis and • exploration protein microRNA Blood informed biomarker candidate selection • Brain Specific Proteins • Disease Classifiers

  29. The Team • ISB • Lee Hood • David Galas • Qiang Tian • Leslie Chen • Burak Kutlu • NCI- CAPR-SAIC • Terry Van Dyke • Sergeui Kozlov • Yurong Song • Zoe Weaver • Simone Difilippantonio • UNC • Ryan Miller • Ryan Bash

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