DOCSLIB.ORG

Genomics-Based Characterization Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

GENOMICS-BASED CHARACTERIZATION OF TUMOR SUPPRESSOR GENES IN THE CARDIOVASCULAR SYSTEM: A ROLE FOR ADENOMATOSIS POLYPOSIS COLI GENE IN HUMAN CARDIOVASCULAR DEVELOPMENT AND DISEASE Mojgan Rezvani A thesis submitted in conforrnity with the requirements for the degree of Doctor of Philosophy Graduate lnstitute of Medical Sciences University of Toronto 8 Copyright by Mojgan Rezvani (2001) 1 National Library Bibliotheque nationale du Canada Acquisitions and Acquisitions et Bibtiogtaphic Services services bibtiographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 OttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. antorisation. GENOMICS-BASED CHARACTERIZATION OF TUMOR SUPPRESSOR GENES IN THE CARDIOVASCULAR SYSTEM: A ROLE FOR ADENOMATOSIS P9LYPOSIS COLI GENE IN HUMAN CARDIOVASCULAR DEVELOPMENT AND DISEASE Mojgan Rezvani, Ph.D. (2001) Graduate lnstitute of Medical Sciences University of Toronto CANADA ABST RACT Advances in sequence-based genome research using expressed sequence tag (EST) technology offers the opportunity for large-scale gene discovery. Complernenting the EST technology, the rapidly developing area of bioinformatics devoted to the collection, organization and analysis of DNA and protein sequences has advanced significantly our understanding of gene regulation during development and in disease states. The EST data can be analyzed in silico (cornputer based) to establish expression prof iles of cDNA libraries derived f rom specific tissues or organs. Expression profiles of different cDNA libraries can be compared by in silico Northern analysis as a tool to identify differentially expressed genes and to predict their functional roles in regulation of biological processes. In the current study, in silico Northem analysis of cardiac-derived EST's was employed to identify differentially expressed genes during cardiac development and in disease. Large scale expression profiling of EST's from cDNA libraries created from human fetal, normal adult and adult hypertrophie hearts revealed developrnentalfy and disease-dependent differentiat expression of severat genes involved in regulation of cell number and growth, including tumor suppressor and apoptosis-related genes, leading to the hypothesis that temporal changes in expression of these growth regulating genes may underlie developmental- and disease-specific alterations in cardiac growth phenotype. The tumor suppressor adenomatosis polyposis coli (APC) and its interacting protein P-catenin were found to be differentially expressed during cardiac development and in hypertrophic disease, resulting in higher abundance of APC in normal adult heart relative to hypertrophied and fetal heatts, and in reciprocal changes in p-catenin protein levels. Loss of function studies using antisense inhibition of APC translation in murine CzCi2 myoblasts inhibited cell proliferation and myotube formation and led to an increase in cell death, in parallel with an increase in basal p-catenin protein levels. Three novel APC protein isofoms were found to be expressed in the heart in a developmental- and disease-specific manner, and cornmensurate with this, expression levels of altematively spliced brain-specific (BS)- and exon-l containing APC gene isoforms were also found to Vary in developmental- and disease-specific manner. We conclude that APC plays a direct role in cardiac myocyte growth and differentiation and that differential and switching of altematively spliced and/or post-translationally modified APC isofoms may underlie, at least in part, some of the developmental- and disease- dependent alterations in cardiac growth phenotype. iii ACKNOWLEDGEMENTS 1 would like to thank my melitor, Dr. CC. Liew, for outstanding supervision through out the course of this degree, and Dr. M. Rabinovitch and Dr. Peter Liu for their excellent guidance and encouragement as members of my supervisory cornmittee. Thanks to IMS for their generous support of University of Toronto Open scholarship for the first year of my Ph.D. program as well as the yearly Merit Awards to date. Thanks to Heart and Stroke Foundation of Canada for awarding a research traineeship for the past four years. I would also like to thank my colleagues, David Hwang, Adam Dempsey, David Barrans, Ken Shaw, Christopher Ton and Dimitri Stamatiou and express my special gratitude to Eva Cukennan and Jack Liew for their helpful insights. Also. special thanks to Dr. V. Dzau and Dr. R. Pratts group, at the Brigham and women's hospital, Harvard Medical School in Boston for the opportunity of experiencing further scientific interaction as a exchange visiting scientist in the last year of my Ph.D. degree. I would like to specially thank Dr. L. G. Mello for his helpful comments and for sharing his extensive experience to prepare this thesis. I would like to specially thank my parents, Ahmad Rezvani and Fateme Haji and rny sisters Nooshin and Rashin for their unconditional love and support. Finally, I would like to express my eternal gratitude to the love of my life, rny husband, Günay Mete without whom none of this would have been possible. His undying support, inspiration, motivation and love have been the force to get me through it all. This Ph.D. degree belongs to hirn just as much as it belongs to me. TABLE Of CONTENTS Title Page i Abstract ii Acknowledgement iv Table of Contents v List of Abreviations viii List of Figures and tables ix CHAPTER 1: Introduction Preamble Hurnan Genome Project Expressed Sequence Tag (EST) Technology In Silico Northern Analysis EST Technology and Cardiovascular Genomics Genes lnvoived in Regulation of Growth in the Heart Tumor Suppressors, Cell Cycle and Cardiac Growth Suppressor Genes in Myocardium Tumor Suppressor APC in Cardiac Development and Disease A. Biochemistry and molecular biology of the APC gene B. Physiology and Pathophysiology of the APC gene Wingless 1 wnt Signaling Pathway p-Catenin Rationale, Hypothesis and Overview of Current Study A. Rationale B. Specific Hypotheses C. Experimental approach D. Significance CHAPTER 2: Apoptosis-related Genes Expressed in Cardiovascular ûevetoptnent and Disease: An EST Approach Abst ract lntroduction Genes lnvolved in Apoptosis - A Cardiovascular Perspective Interfeukin-converting enzyme (ICE) family: Caspases Bcl-2 Family Apoptosis-related Genes ldentified in the Cardiovascular System a) Effectors MA-3 Nip Family Stannin b) Suppressors DAD-1 Apoptosis Inhibifory Protein 47 c) Intemidiate Regulators 47 Tumor Necrosis Factor (TNF) and Fas Receptor Systems47 p38 Family Member p38-2G4 49 Apoptotic Genes Studied in Our Laboratory 49 Zinc Finger Proteins 49 ~53 50 APC 51 Summary and Future Directions 51 Acknowledgment 53 Refe rences 54 CHAPTER 3: Role of Adenornatous Polyposis Coli in Human Cardiac Development and Disease Abstract 63 Introduction 64 Experimental Procedures Total RNA extraction frorn Tissue and Cell Culture 67 cDNA Library Construction and LargeScale Sequencing 67 of cDNA Libraries Sequence and Digital Northern Analysis 67 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 68 Quantification of RT-PCR Results 69 lmmunoblotting Analysis 69 Ceil Culture 70 Antisense and Uptake Study 70 Cellular Proliferation Assay 71 Cellular Differentiation Assay 71 Statistics 72 Results Sequence and Computer-based Digital Northern Analysis 73 In Vitro Gene Expression Analysis (RT-PCR) 73 Protein Expression Level (Western Blot) 78 Cellular Growth and Differentiation Assay 78 Discussion 87 Acknowledgment 92 References 93 CHAPTER 4: Characterization of APC lsoforms in Caidiovascular System During Development and Disease Overview 97 Methods 1. Clones from human cDNA libraries 101 1 .l Isolation of cDNA clones cDNA clones 1 O1 1.2. Full Length AB1 Sequencing 1 O1 2. 5' Rapid Arnpllication of cDNA Ends (RACE) 101 2.t . RNA tsotation 2.2. Gene Specific Primer Design 2.3. PCR Amplification 2.4. Sequencing and Sequence Analysis of PCR Fragments 3. Sequencing of RT-PCR Products Arnplified with Prirnen Encompassing Exons 1 to 15 and Exon BS to 15 3.1 . Total RNA Extraction frorn Tissue 3.2. Primer Design 3.3. Reverse Transcription Polymerase Chain Reaction 3.4. Quantification of RT-PCR Results 3.5. Sequencing 3.6. Sequence Alignment and Analysis Results 1. Clones from human cDNA libraries 2. 5' RACE 2.1. PCR and Re-PCR 2.2. Sequencing of 5' RACE Products 2.3. Sequence Analysis 3. RT-PCR products amplified with primers spanning exons 1 to 15 and exons BS to 15 3.1. Differential Expression and Isofom Switching of APC in Myocardial Development and Disease 3.2. Sequence and BLAST Results Discussion CHAPTER 5: General Discussion, Conclusion and Future Directions 1. Synthesis of major findings 2. Surnmary 3. Conclusions 4. Overall significance of the findings 5. Future Directions REFERENCES
Recommended publications
  • Defining Functional Interactions During Biogenesis of Epithelial Junctions

    Defining Functional Interactions During Biogenesis of Epithelial Junctions

    ARTICLE Received 11 Dec 2015 | Accepted 13 Oct 2016 | Published 6 Dec 2016 | Updated 5 Jan 2017 DOI: 10.1038/ncomms13542 OPEN Defining functional interactions during biogenesis of epithelial junctions J.C. Erasmus1,*, S. Bruche1,*,w, L. Pizarro1,2,*, N. Maimari1,3,*, T. Poggioli1,w, C. Tomlinson4,J.Lees5, I. Zalivina1,w, A. Wheeler1,w, A. Alberts6, A. Russo2 & V.M.M. Braga1 In spite of extensive recent progress, a comprehensive understanding of how actin cytoskeleton remodelling supports stable junctions remains to be established. Here we design a platform that integrates actin functions with optimized phenotypic clustering and identify new cytoskeletal proteins, their functional hierarchy and pathways that modulate E-cadherin adhesion. Depletion of EEF1A, an actin bundling protein, increases E-cadherin levels at junctions without a corresponding reinforcement of cell–cell contacts. This unexpected result reflects a more dynamic and mobile junctional actin in EEF1A-depleted cells. A partner for EEF1A in cadherin contact maintenance is the formin DIAPH2, which interacts with EEF1A. In contrast, depletion of either the endocytic regulator TRIP10 or the Rho GTPase activator VAV2 reduces E-cadherin levels at junctions. TRIP10 binds to and requires VAV2 function for its junctional localization. Overall, we present new conceptual insights on junction stabilization, which integrate known and novel pathways with impact for epithelial morphogenesis, homeostasis and diseases. 1 National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK. 2 Computing Department, Imperial College London, London SW7 2AZ, UK. 3 Bioengineering Department, Faculty of Engineering, Imperial College London, London SW7 2AZ, UK. 4 Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK.
  • NICU Gene List Generator.Xlsx

    NICU Gene List Generator.Xlsx

    Neonatal Crisis Sequencing Panel Gene List Genes: A2ML1 - B3GLCT A2ML1 ADAMTS9 ALG1 ARHGEF15 AAAS ADAMTSL2 ALG11 ARHGEF9 AARS1 ADAR ALG12 ARID1A AARS2 ADARB1 ALG13 ARID1B ABAT ADCY6 ALG14 ARID2 ABCA12 ADD3 ALG2 ARL13B ABCA3 ADGRG1 ALG3 ARL6 ABCA4 ADGRV1 ALG6 ARMC9 ABCB11 ADK ALG8 ARPC1B ABCB4 ADNP ALG9 ARSA ABCC6 ADPRS ALK ARSL ABCC8 ADSL ALMS1 ARX ABCC9 AEBP1 ALOX12B ASAH1 ABCD1 AFF3 ALOXE3 ASCC1 ABCD3 AFF4 ALPK3 ASH1L ABCD4 AFG3L2 ALPL ASL ABHD5 AGA ALS2 ASNS ACAD8 AGK ALX3 ASPA ACAD9 AGL ALX4 ASPM ACADM AGPS AMELX ASS1 ACADS AGRN AMER1 ASXL1 ACADSB AGT AMH ASXL3 ACADVL AGTPBP1 AMHR2 ATAD1 ACAN AGTR1 AMN ATL1 ACAT1 AGXT AMPD2 ATM ACE AHCY AMT ATP1A1 ACO2 AHDC1 ANK1 ATP1A2 ACOX1 AHI1 ANK2 ATP1A3 ACP5 AIFM1 ANKH ATP2A1 ACSF3 AIMP1 ANKLE2 ATP5F1A ACTA1 AIMP2 ANKRD11 ATP5F1D ACTA2 AIRE ANKRD26 ATP5F1E ACTB AKAP9 ANTXR2 ATP6V0A2 ACTC1 AKR1D1 AP1S2 ATP6V1B1 ACTG1 AKT2 AP2S1 ATP7A ACTG2 AKT3 AP3B1 ATP8A2 ACTL6B ALAS2 AP3B2 ATP8B1 ACTN1 ALB AP4B1 ATPAF2 ACTN2 ALDH18A1 AP4M1 ATR ACTN4 ALDH1A3 AP4S1 ATRX ACVR1 ALDH3A2 APC AUH ACVRL1 ALDH4A1 APTX AVPR2 ACY1 ALDH5A1 AR B3GALNT2 ADA ALDH6A1 ARFGEF2 B3GALT6 ADAMTS13 ALDH7A1 ARG1 B3GAT3 ADAMTS2 ALDOB ARHGAP31 B3GLCT Updated: 03/15/2021; v.3.6 1 Neonatal Crisis Sequencing Panel Gene List Genes: B4GALT1 - COL11A2 B4GALT1 C1QBP CD3G CHKB B4GALT7 C3 CD40LG CHMP1A B4GAT1 CA2 CD59 CHRNA1 B9D1 CA5A CD70 CHRNB1 B9D2 CACNA1A CD96 CHRND BAAT CACNA1C CDAN1 CHRNE BBIP1 CACNA1D CDC42 CHRNG BBS1 CACNA1E CDH1 CHST14 BBS10 CACNA1F CDH2 CHST3 BBS12 CACNA1G CDK10 CHUK BBS2 CACNA2D2 CDK13 CILK1 BBS4 CACNB2 CDK5RAP2
  • Cldn19 Clic2 Clmp Cln3

    Cldn19 Clic2 Clmp Cln3

    NewbornDx™ Advanced Sequencing Evaluation When time to diagnosis matters, the NewbornDx™ Advanced Sequencing Evaluation from Athena Diagnostics delivers rapid, 5- to 7-day results on a targeted 1,722-genes. A2ML1 ALAD ATM CAV1 CLDN19 CTNS DOCK7 ETFB FOXC2 GLUL HOXC13 JAK3 AAAS ALAS2 ATP1A2 CBL CLIC2 CTRC DOCK8 ETFDH FOXE1 GLYCTK HOXD13 JUP AARS2 ALDH18A1 ATP1A3 CBS CLMP CTSA DOK7 ETHE1 FOXE3 GM2A HPD KANK1 AASS ALDH1A2 ATP2B3 CC2D2A CLN3 CTSD DOLK EVC FOXF1 GMPPA HPGD K ANSL1 ABAT ALDH3A2 ATP5A1 CCDC103 CLN5 CTSK DPAGT1 EVC2 FOXG1 GMPPB HPRT1 KAT6B ABCA12 ALDH4A1 ATP5E CCDC114 CLN6 CUBN DPM1 EXOC4 FOXH1 GNA11 HPSE2 KCNA2 ABCA3 ALDH5A1 ATP6AP2 CCDC151 CLN8 CUL4B DPM2 EXOSC3 FOXI1 GNAI3 HRAS KCNB1 ABCA4 ALDH7A1 ATP6V0A2 CCDC22 CLP1 CUL7 DPM3 EXPH5 FOXL2 GNAO1 HSD17B10 KCND2 ABCB11 ALDOA ATP6V1B1 CCDC39 CLPB CXCR4 DPP6 EYA1 FOXP1 GNAS HSD17B4 KCNE1 ABCB4 ALDOB ATP7A CCDC40 CLPP CYB5R3 DPYD EZH2 FOXP2 GNE HSD3B2 KCNE2 ABCB6 ALG1 ATP8A2 CCDC65 CNNM2 CYC1 DPYS F10 FOXP3 GNMT HSD3B7 KCNH2 ABCB7 ALG11 ATP8B1 CCDC78 CNTN1 CYP11B1 DRC1 F11 FOXRED1 GNPAT HSPD1 KCNH5 ABCC2 ALG12 ATPAF2 CCDC8 CNTNAP1 CYP11B2 DSC2 F13A1 FRAS1 GNPTAB HSPG2 KCNJ10 ABCC8 ALG13 ATR CCDC88C CNTNAP2 CYP17A1 DSG1 F13B FREM1 GNPTG HUWE1 KCNJ11 ABCC9 ALG14 ATRX CCND2 COA5 CYP1B1 DSP F2 FREM2 GNS HYDIN KCNJ13 ABCD3 ALG2 AUH CCNO COG1 CYP24A1 DST F5 FRMD7 GORAB HYLS1 KCNJ2 ABCD4 ALG3 B3GALNT2 CCS COG4 CYP26C1 DSTYK F7 FTCD GP1BA IBA57 KCNJ5 ABHD5 ALG6 B3GAT3 CCT5 COG5 CYP27A1 DTNA F8 FTO GP1BB ICK KCNJ8 ACAD8 ALG8 B3GLCT CD151 COG6 CYP27B1 DUOX2 F9 FUCA1 GP6 ICOS KCNK3 ACAD9 ALG9
  • Tumor Suppressor and DNA Damage Response Panel 2

    Tumor Suppressor and DNA Damage Response Panel 2

    Tumor suppressor and DNA damage response panel 2 (55 analytes) Gene Symbol Target protein name UniProt ID (& link) Modification* *blanks mean the assay detects the ACT Actin; ACTA2 ACTA1 ACTB ACTG1 ACTC1 ACTG2 Q562L2 non‐modified peptide sequence CASC5 cancer susceptibility candidate 5 Q8NG31 pS767 CASC5 cancer susceptibility candidate 5 Q8NG31 CASP3 caspase 3, apoptosis‐related cysteine peptidase P42574 pS26 CDC25B cell division cycle 25 homolog B (S. pombe) P30305 pS16 CDC25B cell division cycle 25 homolog B (S. pombe) P30305 pS323 CDC25B cell division cycle 25 homolog B (S. pombe) P30305 CDC25C cell division cycle 25 homolog B (S. pombe) P30307 pS216 CDC25C cell division cycle 25 homolog B (S. pombe) P30307 CDCA8 cell division cycle associated 8 Q53HL2 pT16 CDK1 cyclin‐dependent kinase 1 P06493 pT161 CDK1 cyclin‐dependent kinase 1 P06493 CDK7 cyclin‐dependent kinase 7 P50613 pT17 CHEK1 CHK1 checkpoint homolog (S. pombe) O14757 pS286 CHEK2 CHK2 checkpoint homolog (S. pombe) O96017 pS379 CHEK2 CHK2 checkpoint homolog (S. pombe) O96017 pT387 CHEK2 CHK2 checkpoint homolog (S. pombe) O96017 GAPDH Glyceraldehyde‐3‐phosphate dehydrogenase P04406 LAT linker for activation of T cells O43561 pS224 LMNB1 lamin B1 P20700 pT2; pS23 LMNB1 lamin B1 P20700 pT2 LMNB1 lamin B1 P20700 pS23 MCM6 minichromosome maintenance complex component 6 Q14566 pS762 MCM6 minichromosome maintenance complex component 6 Q14566 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) Q00987 pS166 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) Q00987 MKI67 antigen identified by monoclonal antibody Ki‐67 P46013 pT181 MKI67 antigen identified by monoclonal antibody Ki‐67 P46013 MKI67 antigen identified by monoclonal antibody Ki‐67 P46013 pT246 MRE11A MRE11 meiotic recombination 11 homolog A (S.
  • Proteomic Expression Profile in Human Temporomandibular Joint

    Proteomic Expression Profile in Human Temporomandibular Joint

    diagnostics Article Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction Andrea Duarte Doetzer 1,*, Roberto Hirochi Herai 1 , Marília Afonso Rabelo Buzalaf 2 and Paula Cristina Trevilatto 1 1 Graduate Program in Health Sciences, School of Medicine, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba 80215-901, Brazil; [email protected] (R.H.H.); [email protected] (P.C.T.) 2 Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru 17012-901, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-41-991-864-747 Abstract: Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human’s health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (n = 3), CH (n = 4) and MD (n = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three Citation: Doetzer, A.D.; Herai, R.H.; TMD conditions showed different protein expression profiles, and novel proteins were identified Buzalaf, M.A.R.; Trevilatto, P.C.
  • Illuminating the Divergent Role of Filamin C Mutations in Human Cardiomyopathy

    Illuminating the Divergent Role of Filamin C Mutations in Human Cardiomyopathy

    Journal of Clinical Medicine Review Cardiac Filaminopathies: Illuminating the Divergent Role of Filamin C Mutations in Human Cardiomyopathy Matthias Eden 1,2 and Norbert Frey 1,2,* 1 Department of Internal Medicine III, University of Heidelberg, 69120 Heidelberg, Germany; [email protected] 2 German Centre for Cardiovascular Research, Partner Site Heidelberg, 69120 Heidelberg, Germany * Correspondence: [email protected] Abstract: Over the past decades, there has been tremendous progress in understanding genetic alterations that can result in different phenotypes of human cardiomyopathies. More than a thousand mutations in various genes have been identified, indicating that distinct genetic alterations, or combi- nations of genetic alterations, can cause either hypertrophic (HCM), dilated (DCM), restrictive (RCM), or arrhythmogenic cardiomyopathies (ARVC). Translation of these results from “bench to bedside” can potentially group affected patients according to their molecular etiology and identify subclinical individuals at high risk for developing cardiomyopathy or patients with overt phenotypes at high risk for cardiac deterioration or sudden cardiac death. These advances provide not only mechanistic insights into the earliest manifestations of cardiomyopathy, but such efforts also hold the promise that mutation-specific pathophysiology might result in novel “personalized” therapeutic possibilities. Recently, the FLNC gene encoding the sarcomeric protein filamin C has gained special interest since FLNC mutations were found in several distinct and possibly overlapping cardiomyopathy phenotypes. Specifically, mutations in FLNC were initially only linked to myofibrillar myopathy (MFM), but are now increasingly found in various forms of human cardiomyopathy. FLNC thereby Citation: Eden, M.; Frey, N. Cardiac represents another example for the complex genetic and phenotypic continuum of these diseases.
  • Gene Therapy Rescues Cardiac Dysfunction in Duchenne Muscular

    Gene Therapy Rescues Cardiac Dysfunction in Duchenne Muscular

    JACC: BASIC TO TRANSLATIONAL SCIENCE VOL.4,NO.7,2019 ª 2019 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/). PRECLINICAL RESEARCH Gene Therapy Rescues Cardiac DysfunctioninDuchenneMuscular Dystrophy Mice by Elevating Cardiomyocyte Deoxy-Adenosine Triphosphate a b c d,e Stephen C. Kolwicz, JR,PHD, John K. Hall, PHD, Farid Moussavi-Harami, MD, Xiolan Chen, PHD, d,e b,d,e e,f,g, b,e,g, Stephen D. Hauschka, PHD, Jeffrey S. Chamberlain, PHD, Michael Regnier, PHD, * Guy L. Odom, PHD * VISUAL ABSTRACT Kolwicz, S.C. Jr. et al. J Am Coll Cardiol Basic Trans Science. 2019;4(7):778–91. HIGHLIGHTS rAAV vectors increase cardiac-specific expression of RNR and elevate cardiomyocyte 2-dATP levels. Elevated myocardial RNR and subsequent increase in 2-dATP rescues the performance of failing myocardium, an effect that persists long term. ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2019.06.006 JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 4, NO. 7, 2019 Kolwicz, Jr., et al. 779 NOVEMBER 2019:778– 91 Nucleotide-Based Cardiac Gene Therapy Restores Function in dmd Mice We show the ability to increase both cardiac baseline function and high workload contractile performance in ABBREVIATIONS aged (22- to 24-month old) mdx4cv mice, by high-level muscle-specific expression of either microdystrophin AND ACRONYMS or RNR. mDys = microdystrophin Five months post-treatment, mice systemically injected with rAAV6 vector carrying a striated muscle-specific CK8 regulatory cassette driving expression of microdystrophin in both skeletal and cardiac muscle, exhibited the = miniaturized murine creatine kinase regulatory greatest effect on systolic function.
  • Supplementary Data

    Supplementary Data

    Supplementary Fig. 1 A B Responder_Xenograft_ Responder_Xenograft_ NON- NON- Lu7336, Vehicle vs Lu7466, Vehicle vs Responder_Xenograft_ Responder_Xenograft_ Sagopilone, Welch- Sagopilone, Welch- Lu7187, Vehicle vs Lu7406, Vehicle vs Test: 638 Test: 600 Sagopilone, Welch- Sagopilone, Welch- Test: 468 Test: 482 Responder_Xenograft_ NON- Lu7860, Vehicle vs Responder_Xenograft_ Sagopilone, Welch - Lu7558, Vehicle vs Test: 605 Sagopilone, Welch- Test: 333 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Figure S1. Venn diagrams comparing probe sets regulated by Sagopilone treatment (10mg/kg for 24h) between individual models (Welsh Test ellipse p-value<0.001 or 5-fold change). A Sagopilone responder models, B Sagopilone non-responder models. Supplementary Figure S2. Pathway analysis of genes regulated by Sagopilone treatment in responder xenograft models 24h after Sagopilone treatment by GeneGo Metacore; the most significant pathway map representing cell cycle/spindle assembly and chromosome separation is shown, genes upregulated by Sagopilone treatment are marked with red thermometers. Supplementary Figure S3. GeneGo Metacore pathway analysis of genes differentially expressed between Sagopilone Responder and Non-Responder models displaying –log(p-Values) of most significant pathway maps. Supplementary Tables Supplementary Table 1. Response and activity in 22 non-small-cell lung cancer (NSCLC) xenograft models after treatment with Sagopilone and other cytotoxic agents commonly used in the management of NSCLC Tumor Model Response type
  • Supplementary Table 2

    Supplementary Table 2

    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
  • Bioinformatic Analysis Reveals the Importance of Epithelial-Mesenchymal Transition in the Development of Endometriosis

    Bioinformatic Analysis Reveals the Importance of Epithelial-Mesenchymal Transition in the Development of Endometriosis

    www.nature.com/scientificreports OPEN Bioinformatic analysis reveals the importance of epithelial- mesenchymal transition in the development of endometriosis Meihong Chen1,6, Yilu Zhou2,3,6, Hong Xu4, Charlotte Hill2, Rob M. Ewing2,3, Deming He1, Xiaoling Zhang1 ✉ & Yihua Wang2,3,5 ✉ Background: Endometriosis is a frequently occurring disease in women, which seriously afects their quality of life. However, its etiology and pathogenesis are still unclear. Methods: To identify key genes/ pathways involved in the pathogenesis of endometriosis, we recruited 3 raw microarray datasets (GSE11691, GSE7305, and GSE12768) from Gene Expression Omnibus database (GEO), which contain endometriosis tissues and normal endometrial tissues. We then performed in-depth bioinformatic analysis to determine diferentially expressed genes (DEGs), followed by gene ontology (GO), Hallmark pathway enrichment and protein-protein interaction (PPI) network analysis. The fndings were further validated by immunohistochemistry (IHC) staining in endometrial tissues from endometriosis or control patients. Results: We identifed 186 DEGs, of which 118 were up-regulated and 68 were down-regulated. The most enriched DEGs in GO functional analysis were mainly associated with cell adhesion, infammatory response, and extracellular exosome. We found that epithelial-mesenchymal transition (EMT) ranked frst in the Hallmark pathway enrichment. EMT may potentially be induced by infammatory cytokines such as CXCL12. IHC confrmed the down-regulation of E-cadherin (CDH1) and up-regulation of CXCL12 in endometriosis tissues. Conclusions: Utilizing bioinformatics and patient samples, we provide evidence of EMT in endometriosis. Elucidating the role of EMT will improve the understanding of the molecular mechanisms involved in the development of endometriosis. Endometriosis is a frequently occurring gynaecological disease characterised by chronic pelvic pain, dysmenor- rhea and infertility1.
  • WNT16 Is a New Marker of Senescence

    WNT16 Is a New Marker of Senescence

    Table S1. A. Complete list of 177 genes overexpressed in replicative senescence Value Gene Description UniGene RefSeq 2.440 WNT16 wingless-type MMTV integration site family, member 16 (WNT16), transcript variant 2, mRNA. Hs.272375 NM_016087 2.355 MMP10 matrix metallopeptidase 10 (stromelysin 2) (MMP10), mRNA. Hs.2258 NM_002425 2.344 MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), mRNA. Hs.375129 NM_002422 2.300 HIST1H2AC Histone cluster 1, H2ac Hs.484950 2.134 CLDN1 claudin 1 (CLDN1), mRNA. Hs.439060 NM_021101 2.119 TSPAN13 tetraspanin 13 (TSPAN13), mRNA. Hs.364544 NM_014399 2.112 HIST2H2BE histone cluster 2, H2be (HIST2H2BE), mRNA. Hs.2178 NM_003528 2.070 HIST2H2BE histone cluster 2, H2be (HIST2H2BE), mRNA. Hs.2178 NM_003528 2.026 DCBLD2 discoidin, CUB and LCCL domain containing 2 (DCBLD2), mRNA. Hs.203691 NM_080927 2.007 SERPINB2 serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2), mRNA. Hs.594481 NM_002575 2.004 HIST2H2BE histone cluster 2, H2be (HIST2H2BE), mRNA. Hs.2178 NM_003528 1.989 OBFC2A Oligonucleotide/oligosaccharide-binding fold containing 2A Hs.591610 1.962 HIST2H2BE histone cluster 2, H2be (HIST2H2BE), mRNA. Hs.2178 NM_003528 1.947 PLCB4 phospholipase C, beta 4 (PLCB4), transcript variant 2, mRNA. Hs.472101 NM_182797 1.934 PLCB4 phospholipase C, beta 4 (PLCB4), transcript variant 1, mRNA. Hs.472101 NM_000933 1.933 KRTAP1-5 keratin associated protein 1-5 (KRTAP1-5), mRNA. Hs.534499 NM_031957 1.894 HIST2H2BE histone cluster 2, H2be (HIST2H2BE), mRNA. Hs.2178 NM_003528 1.884 CYTL1 cytokine-like 1 (CYTL1), mRNA. Hs.13872 NM_018659 tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain (TNFRSF10D), 1.848 TNFRSF10D Hs.213467 NM_003840 mRNA.
  • Variants in Exons 5 and 6 of ACTB Cause Syndromic Thrombocytopenia

    Variants in Exons 5 and 6 of ACTB Cause Syndromic Thrombocytopenia

    ARTICLE DOI: 10.1038/s41467-018-06713-0 OPEN Variants in exons 5 and 6 of ACTB cause syndromic thrombocytopenia Sharissa L. Latham 1, Nadja Ehmke2,3, Patrick Y.A. Reinke1,4, Manuel H. Taft 1, Dorothee Eicke5, Theresia Reindl1, Werner Stenzel6, Michael J. Lyons7, Michael J. Friez7, Jennifer A. Lee7, Ramona Hecker8, Michael C. Frühwald9, Kerstin Becker10, Teresa M. Neuhann10, Denise Horn2, Evelin Schrock11, Indra Niehaus11, Katharina Sarnow11, Konrad Grützmann12, Luzie Gawehn11, Barbara Klink 11, Andreas Rump11, Christine Chaponnier13, Constanca Figueiredo5, Ralf Knöfler14, Dietmar J. Manstein1,4 & Nataliya Di Donato 11 1234567890():,; Germline mutations in the ubiquitously expressed ACTB, which encodes β-cytoplasmic actin (CYA), are almost exclusively associated with Baraitser-Winter Cerebrofrontofacial syn- drome (BWCFF). Here, we report six patients with previously undescribed heterozygous variants clustered in the 3′-coding region of ACTB. Patients present with clinical features distinct from BWCFF, including mild developmental disability, microcephaly, and thrombo- cytopenia with platelet anisotropy. Using patient-derived fibroblasts, we demonstrate cohort specific changes to β-CYA filament populations, which include the enhanced recruitment of thrombocytopenia-associated actin binding proteins (ABPs). These perturbed interactions are supported by in silico modeling and are validated in disease-relevant thrombocytes. Co- examination of actin and microtubule cytoskeleton constituents in patient-derived mega- karyocytes and thrombocytes indicates that these β-CYA mutations inhibit the final stages of platelet maturation by compromising microtubule organization. Our results define an ACTB- associated clinical syndrome with a distinct genotype-phenotype correlation and delineate molecular mechanisms underlying thrombocytopenia in this patient cohort. 1 Institute for Biophysical Chemistry, Hannover Medical School, Hannover 30625, Germany.