DOCSLIB.ORG

Evaluation and Optimization of the Translational Potential of Array

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Evaluation and Optimization of the Translational Potential of Array-Based Molecular Diagnostics by Dawn Nichole Kernagis Department of Pathology Duke University Date:_______________________ Approved: ___________________________ Michael Datto, Supervisor ___________________________ Richard Moon ___________________________ Thomas Ortel ___________________________ Herman Staats ___________________________ Richard Vann Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University 2012 ABSTRACT Evaluation and Optimization of the Translational Potential of Array-Based Molecular Diagnostics by Dawn Nichole Kernagis Department of Pathology Duke University Date:_______________________ Approved: ___________________________ Michael Datto, Supervisor ___________________________ Richard Moon ___________________________ Thomas Ortel ___________________________ Herman Staats ___________________________ Richard Vann An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University 2012 Copyright by Dawn Nichole Kernagis 2012 Abstract The translational potential of diagnostic and prognostic platforms developed using microarray technology is evident given the significant number of array-based studies describing clinically-relevant molecular subclasses of disease. However, the majority of array-based diagnostics have yet to make their way into the clinical laboratory. Current approaches tend to focus on development of multi-gene classifiers of disease subtypes, but very few studies assess the translational potential of these assays with the clinical laboratory in mind. Likewise, only a handful of studies focus on development of approaches to optimize array-based tests for the ultimate goal of clinical testing utility. Prior to translation into the clinical setting, molecular diagnostic platforms should demonstrate a number of characteristics to ensure optimal and efficient testing and patient care. Assays should be accurate and precise, technically and biologically robust, and should take into account normal sources of biological variance that could ultimately affect test results. The overarching goal of the research presented in this dissertation is to develop methods for evaluating and optimizing the translational potential of molecular diagnostics developed using expression microarray technology. The first research section of this dissertation is focused on our evaluation of the impact of intratumor heterogeneity on precision in microarray-based assays in breast cancer. We conducted genome-wide expression profiling on 50 needle core biopsies iv from 18 breast cancer patients. Global profiles of expression were characterized using unsupervised clustering methods and variance components models. Array-based measures of estrogen (ER) and progesterone receptor (PR) status were compared to immunohistochemistry. The precision of genomic predictors of ER pathway status, recurrence risk, and sensitivity to chemotherapeutics were evaluated by interclass correlation. Results demonstrated that intratumor variation was substantially less than the total variation observed across the patient population. Nevertheless, a fraction of genes exhibited significant intratumor heterogeneity in expression. A high degree of reproducibility was observed in single gene predictors of ER (intraclass correlation coefficient (ICC)=0.94) and PR expression (ICC=0.90), and in a multi-gene predictor of ER pathway activation (ICC=0.98) with high concordance with immunohistochemistry. Substantial agreement was also observed for multi-gene signatures of cancer recurrence (ICC=0.71), and chemotherapeutic sensitivity (ICC=0.72 and 0.64). Together, these results demonstrated that intratumor heterogeneity, although present at the level of individual gene expression, does not preclude precise micro-array based predictions of tumor behavior or clinical outcome in breast cancer patients. Leading into the second research section, we observed that in some cancer types, certain genes behave as molecular switches and have either an "on" or "off" expression state. Specifically, we observed these molecular switch genes exist in breast cancer as v robust diagnostic and prognostic markers, including ER, PR, and HER2, and define tumor subtypes associated with different treatment and patient survival. We hypothesized that clinically relevant molecular switch (bimodal) genes exist in epithelial ovarian cancer, a type of cancer with no established molecular subgroups. To test this hypothesis, we applied a bimodal discovery algorithm to a publically available ovarian cancer expression microarray dataset (GSE9891:285 tumors; 246 malignant serous (MS), 20 endometrioid (EM), 18 low malignant potential (LMP) ovarian carcinomas). Genes with robust bimodal expression were identified across all ovarian tumor types and within selected subtypes. Of these bimodal genes, 73 demonstrated differential expression between LMP vs. MS and EM, and 22 genes distinguished MS from EM. Fourteen bimodal genes had significant association with survival among MS tumors. When these genes were combined into a single survival score, the median survival for patients with a favorable versus unfavorable score was 65 versus 29 months (p<0.0001, HR=0.4221). Two independent datasets (n=53 high grade, advanced stage serous and n=119 advanced stage ovarian tumors) validated the survival score performance. Taken together, the results of this study revealed that genes with bimodal expression patterns not only define clinically relevant molecular subtypes of ovarian carcinoma, but also provide ideal targets for translation into the clinical laboratory. vi Finally, the third research section of this dissertation focuses on development of robust blood-based molecular markers of decompression stress (DS). DS is defined as the pathophysiological response to inert gas coming out of solution in the blood and tissues when a body experiences a reduction in ambient pressure. To date, there are no established molecular markers of DS. We hypothesized that comparing gene expression before and after human decompression exposures by genome-wide expression profiling would identify candidate molecular markers of DS. Peripheral blood was collected 1hr before and 2hr after 93 hyperoxic, mixed-gas experimental dives (n=54). Control arms included samples collected 1 hour before and 2 hours after high pressure oxygen breathing (n= 9) and surface exercise (n=9), and samples collected at 7am and 5pm for time of day (n=11). Pre and post-dive expression data collected from normoxic nitrox experimental dives were utilized for independent validation. Blood samples were collected into PaxGene RNA tubes. RNA was extracted and processed for globin reduction prior to cDNA synthesis and Affymetrix U133A GeneChip hybridization. Significance analysis revealed 746 genes differentially expressed following hyperoxic, mixed-gas decompression exposures (permutation adjusted p-value cutoff 1.0E-4). After filtering for significant genes in the hyperbaric oxygen, exercise, and diurnal control groups, 726 genes remained. Pathway analysis demonstrated a significant portion of genes were associated with innate immune response (p<0.0001). A 362 multi-gene vii signature of significant, covariant genes was then applied to the independent dataset and demonstrated differentiation between pre and post-dive samples (p=0.0058). Further investigation identified four individual, robust candidate gene markers of DS associated with immune and stress response. Our results showed that expression profiling of peripheral blood following decompression exposures, while controlling for experimental and normal sources of biological variance, identifies a reproducible multi- gene signature of differentially expressed genes, primarily comprising genes associated with innate immune response and independent of venous bubble grade or dive profile. Taken together, the research and results presented in this dissertation represent considerable advances in the development of approaches to guide microarray-based diagnostics towards the ultimate goal of clinical translation. viii Dedication This dissertation is dedicated to my parents, Frank and Patricia Kernagis, for their love, support, and encouragement to venture away from the well-worn path. ix Contents Abstract ......................................................................................................................................... iv Dedication ..................................................................................................................................... ix Contents ......................................................................................................................................... x List of Tables ................................................................................................................................ xv List of Figures ............................................................................................................................. xvi List of Abbreviations ................................................................................................................. xxi Acknowledgements ................................................................................................................ xxiii 1. Background
Recommended publications
  • Mutant IDH, (R)-2-Hydroxyglutarate, and Cancer

    Mutant IDH, (R)-2-Hydroxyglutarate, and Cancer

    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer Julie-Aurore Losman1 and William G. Kaelin Jr.1,2,3 1Department of Medical Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA; 2Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA Mutations in metabolic enzymes, including isocitrate whether altered cellular metabolism is a cause of cancer dehydrogenase 1 (IDH1) and IDH2, in cancer strongly or merely an adaptive response of cancer cells in the face implicate altered metabolism in tumorigenesis. IDH1 of accelerated cell proliferation is still a topic of some and IDH2 catalyze the interconversion of isocitrate and debate. 2-oxoglutarate (2OG). 2OG is a TCA cycle intermediate The recent identification of cancer-associated muta- and an essential cofactor for many enzymes, including tions in three metabolic enzymes suggests that altered JmjC domain-containing histone demethylases, TET cellular metabolism can indeed be a cause of some 5-methylcytosine hydroxylases, and EglN prolyl-4-hydrox- cancers (Pollard et al. 2003; King et al. 2006; Raimundo ylases. Cancer-associated IDH mutations alter the enzymes et al. 2011). Two of these enzymes, fumarate hydratase such that they reduce 2OG to the structurally similar (FH) and succinate dehydrogenase (SDH), are bone fide metabolite (R)-2-hydroxyglutarate [(R)-2HG]. Here we tumor suppressors, and loss-of-function mutations in FH review what is known about the molecular mechanisms and SDH have been identified in various cancers, in- of transformation by mutant IDH and discuss their im- cluding renal cell carcinomas and paragangliomas.
  • Gene PMID WBS Locus ABR 26603386 AASDH 26603386

    Gene PMID WBS Locus ABR 26603386 AASDH 26603386

    Supplementary material J Med Genet Gene PMID WBS Locus ABR 26603386 AASDH 26603386 ABCA1 21304579 ABCA13 26603386 ABCA3 25501393 ABCA7 25501393 ABCC1 25501393 ABCC3 25501393 ABCG1 25501393 ABHD10 21304579 ABHD11 25501393 yes ABHD2 25501393 ABHD5 21304579 ABLIM1 21304579;26603386 ACOT12 25501393 ACSF2,CHAD 26603386 ACSL4 21304579 ACSM3 26603386 ACTA2 25501393 ACTN1 26603386 ACTN3 26603386;25501393;25501393 ACTN4 21304579 ACTR1B 21304579 ACVR2A 21304579 ACY3 19897463 ACYP1 21304579 ADA 25501393 ADAM12 21304579 ADAM19 25501393 ADAM32 26603386 ADAMTS1 25501393 ADAMTS10 25501393 ADAMTS12 26603386 ADAMTS17 26603386 ADAMTS6 21304579 ADAMTS7 25501393 ADAMTSL1 21304579 ADAMTSL4 25501393 ADAMTSL5 25501393 ADCY3 25501393 ADK 21304579 ADRBK2 25501393 AEBP1 25501393 AES 25501393 AFAP1,LOC84740 26603386 AFAP1L2 26603386 AFG3L1 21304579 AGAP1 26603386 AGAP9 21304579 Codina-Sola M, et al. J Med Genet 2019; 56:801–808. doi: 10.1136/jmedgenet-2019-106080 Supplementary material J Med Genet AGBL5 21304579 AGPAT3 19897463;25501393 AGRN 25501393 AGXT2L2 25501393 AHCY 25501393 AHDC1 26603386 AHNAK 26603386 AHRR 26603386 AIDA 25501393 AIFM2 21304579 AIG1 21304579 AIP 21304579 AK5 21304579 AKAP1 25501393 AKAP6 21304579 AKNA 21304579 AKR1E2 26603386 AKR7A2 21304579 AKR7A3 26603386 AKR7L 26603386 AKT3 21304579 ALDH18A1 25501393;25501393 ALDH1A3 21304579 ALDH1B1 21304579 ALDH6A1 21304579 ALDOC 21304579 ALG10B 26603386 ALG13 21304579 ALKBH7 25501393 ALPK2 21304579 AMPH 21304579 ANG 21304579 ANGPTL2,RALGPS1 26603386 ANGPTL6 26603386 ANK2 21304579 ANKMY1 26603386 ANKMY2
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • 4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4).
  • Neutrophil Chemoattractant Receptors in Health and Disease: Double-Edged Swords

    Neutrophil Chemoattractant Receptors in Health and Disease: Double-Edged Swords

    Cellular & Molecular Immunology www.nature.com/cmi REVIEW ARTICLE Neutrophil chemoattractant receptors in health and disease: double-edged swords Mieke Metzemaekers1, Mieke Gouwy1 and Paul Proost 1 Neutrophils are frontline cells of the innate immune system. These effector leukocytes are equipped with intriguing antimicrobial machinery and consequently display high cytotoxic potential. Accurate neutrophil recruitment is essential to combat microbes and to restore homeostasis, for inflammation modulation and resolution, wound healing and tissue repair. After fulfilling the appropriate effector functions, however, dampening neutrophil activation and infiltration is crucial to prevent damage to the host. In humans, chemoattractant molecules can be categorized into four biochemical families, i.e., chemotactic lipids, formyl peptides, complement anaphylatoxins and chemokines. They are critically involved in the tight regulation of neutrophil bone marrow storage and egress and in spatial and temporal neutrophil trafficking between organs. Chemoattractants function by activating dedicated heptahelical G protein-coupled receptors (GPCRs). In addition, emerging evidence suggests an important role for atypical chemoattractant receptors (ACKRs) that do not couple to G proteins in fine-tuning neutrophil migratory and functional responses. The expression levels of chemoattractant receptors are dependent on the level of neutrophil maturation and state of activation, with a pivotal modulatory role for the (inflammatory) environment. Here, we provide an overview
  • The Role of EZH2 in the Induction and Maintenance of Acute Myeloid Leukaemia

    The Role of EZH2 in the Induction and Maintenance of Acute Myeloid Leukaemia

    The Role of EZH2 in the Induction and Maintenance of Acute Myeloid Leukaemia Faisal Basheer Downing College University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy September 2017 Declaration The work presented in this dissertation was carried out under the supervision of Professor Brian Huntly from November 2013 to November 2016 in the Department of Haematology, University of Cambridge. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit set by the Clinical Medicine and Clinical Veterinary Medicine Degree Committee. Faisal Basheer September 2017 i Acknowledgements First and foremost, I would like to thank my supervisor, Professor Brian Huntly, for providing me with the opportunity to work towards this thesis - for his help, encouragement and support over the last four years of my PhD. His dedicated high-level supervision, meticulous attention to detail and excellent direction throughout this time has undoubtedly helped me to maximise my potential in the lab throughout a highly interesting and rewarding project. I would also like to thank all of my colleagues and friends in the Huntly laboratory over the past four years - working together with all of you as a team has been an unforgettable and rewarding experience.
  • Supplementary Table 1: Adhesion Genes Data Set

    Supplementary Table 1: Adhesion Genes Data Set

    Supplementary Table 1: Adhesion genes data set PROBE Entrez Gene ID Celera Gene ID Gene_Symbol Gene_Name 160832 1 hCG201364.3 A1BG alpha-1-B glycoprotein 223658 1 hCG201364.3 A1BG alpha-1-B glycoprotein 212988 102 hCG40040.3 ADAM10 ADAM metallopeptidase domain 10 133411 4185 hCG28232.2 ADAM11 ADAM metallopeptidase domain 11 110695 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 195222 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 165344 8751 hCG20021.3 ADAM15 ADAM metallopeptidase domain 15 (metargidin) 189065 6868 null ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme) 108119 8728 hCG15398.4 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 117763 8748 hCG20675.3 ADAM20 ADAM metallopeptidase domain 20 126448 8747 hCG1785634.2 ADAM21 ADAM metallopeptidase domain 21 208981 8747 hCG1785634.2|hCG2042897 ADAM21 ADAM metallopeptidase domain 21 180903 53616 hCG17212.4 ADAM22 ADAM metallopeptidase domain 22 177272 8745 hCG1811623.1 ADAM23 ADAM metallopeptidase domain 23 102384 10863 hCG1818505.1 ADAM28 ADAM metallopeptidase domain 28 119968 11086 hCG1786734.2 ADAM29 ADAM metallopeptidase domain 29 205542 11085 hCG1997196.1 ADAM30 ADAM metallopeptidase domain 30 148417 80332 hCG39255.4 ADAM33 ADAM metallopeptidase domain 33 140492 8756 hCG1789002.2 ADAM7 ADAM metallopeptidase domain 7 122603 101 hCG1816947.1 ADAM8 ADAM metallopeptidase domain 8 183965 8754 hCG1996391 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 129974 27299 hCG15447.3 ADAMDEC1 ADAM-like,
  • Cellular Stress Pathways in Pediatric Bone Marrow Failure Syndromes: Many Roads Lead to Neutropenia

    Cellular Stress Pathways in Pediatric Bone Marrow Failure Syndromes: Many Roads Lead to Neutropenia

    nature publishing group Review Cellular stress pathways in pediatric bone marrow failure syndromes: many roads lead to neutropenia Taly Glaubach1–3, Alex C. Minella4 and Seth J. Corey1–3,5 The inherited bone marrow failure syndromes, like severe con- kidney, and gastrointestinal tract. Genetic lesions have been genital neutropenia (SCN) and Shwachman–Diamond syn- identified for all of these syndromes, although not all patients drome (SDS), provide unique insights into normal and impaired with these disorders have a known genetic cause. The inher- myelopoiesis. The inherited neutropenias are heterogeneous ited bone marrow failure syndromes offer genetically defined in both clinical presentation and genetic associations, and experiments of nature that provide unique opportunities for their causative mechanisms are not well established. SCN, for studying and understanding the regulatory networks involved example, is a genetically heterogeneous syndrome associated in hematopoiesis and how perturbations in blood cell function with mutations of ELANE, HAX1, GFI1, WAS, G6PC3, or CSF3R. The result in marrow production failure and peripheral cytopenias. genetic diversity in SCN, along with congenital neutropenias Here, we review the most recent developments in molecular associated with other genetically defined bone marrow failure basis of inherited bone marrow failure syndromes, particularly syndromes (e.g., SDS), suggests that various pathways may be SCN. One emerging theme is that different pathways involv- involved in their pathogenesis. Alternatively, all may lead to a ing cellular stress mechanisms drive apoptosis of blood cell final common pathway of enhanced apoptosis. The pursuit for precursors, resulting in cytopenia(s). These stress mechanisms a more complete understanding of the molecular mechanisms include endoplasmic reticulum (ER) stress and the unfold pro- that drive inherited neutropenias remains at the forefront­ tein response, defective ribosome assembly, and induction of of pediatric translational and basic science investigation.
  • Extraocular Muscle Characteristics Related to Myasthenia Gravis Susceptibility

    Extraocular Muscle Characteristics Related to Myasthenia Gravis Susceptibility

    Extraocular Muscle Characteristics Related to Myasthenia Gravis Susceptibility Rui Liu1., Hanpeng Xu2., Guiping Wang3, Jie Li4, Lin Gou2, Lihua Zhang1, Jianting Miao5*, Zhuyi Li5* 1 Department of Geratology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, P. R. China, 2 LONI, Department of Neurology, UCLA, Los Angeles, California, United States of America, 3 Department of Neurosurgery, 208th Hospital of PLA, Changchun, Jilin Province, P. R. China, 4 Department of Endocrinology, 451 Hospital of PLA,Xi’an, Shaanxi Province, P. R. China, 5 Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, P. R. China Abstract Background: The pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis. Methods: Female F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes. Results: In the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles.
  • FGF21 Regulates Hepatic Metabolic Pathways to Improve Steatosis and Inflammation

    FGF21 Regulates Hepatic Metabolic Pathways to Improve Steatosis and Inflammation

    ID: 20-0152 9 8 H Keinicke et al. Hepatic gene regulation by 9:8 755–768 FGF21 in DIO mice RESEARCH FGF21 regulates hepatic metabolic pathways to improve steatosis and inflammation Helle Keinicke1, Gao Sun2,*, Caroline M Junker Mentzel3, Merete Fredholm4, Linu Mary John5, Birgitte Andersen5, Kirsten Raun5 and Marina Kjaergaard5 1Insulin and Device Trial Operations, Novo Nordisk A/S, Søborg, Denmark 2Pharmacology and Histopathology, Novo Nordisk A/S, China 3Department of Experimental Animal Models, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark 4Department of Veterinary Clinical and Animal Science, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark 5Global Obesity and Liver Disease Research, Novo Nordisk A/S, Måløv, Denmark Correspondence should be addressed to M Kjaergaard: [email protected] *(G Sun is now at Pegbio Co., Ltd, Su Zhou, China) Abstract The prevalence of non-alcoholic fatty liver disease (NAFLD) has increased dramatically Key Words worldwide and, subsequently, also the risk of developing non-alcoholic steatohepatitis f fibroblast growth factor 21 (NASH), hepatic fibrosis, cirrhosis and cancer. Today, weight loss is the only available (FGF21) treatment, but administration of fibroblast growth factor 21 (FGF21) analogues have, f non-alcoholic fatty liver disease (NAFLD) in addition to weight loss, shown improvements on liver metabolic health but the f lipid metabolism mechanisms behind are not entirely clear. The aim of this study was to investigate the f glucose metabolism hepatic metabolic profile in response to FGF21 treatment. Diet-induced obese (DIO) f inflammation mice were treated with s.c. administration of FGF21 or subjected to caloric restriction by switching from high fat diet (HFD) to chow to induce 20% weight loss and changes were compared to vehicle dosed DIO mice.
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
  • Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

    Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

    Stem Cell Rev and Rep DOI 10.1007/s12015-016-9662-8 Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications Behnam Ahmadian Baghbaderani 1 & Adhikarla Syama2 & Renuka Sivapatham3 & Ying Pei4 & Odity Mukherjee2 & Thomas Fellner1 & Xianmin Zeng3,4 & Mahendra S. Rao5,6 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract We have recently described manufacturing of hu- help determine which set of tests will be most useful in mon- man induced pluripotent stem cells (iPSC) master cell banks itoring the cells and establishing criteria for discarding a line. (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Keywords Induced pluripotent stem cells . Embryonic stem Reports, 5(4), 647–659, 2015). In this manuscript, we de- cells . Manufacturing . cGMP . Consent . Markers scribe the detailed characterization of the two iPSC clones generated using this process, including whole genome se- quencing (WGS), microarray, and comparative genomic hy- Introduction bridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibra- Induced pluripotent stem cells (iPSCs) are akin to embryonic tion material and with a reporter subclone and lines made by a stem cells (ESC) [2] in their developmental potential, but dif- similar process from different donors. We believe that iPSCs fer from ESC in the starting cell used and the requirement of a are likely to be used to make multiple clinical products. We set of proteins to induce pluripotency [3]. Although function- further believe that the lines used as input material will be used ally identical, iPSCs may differ from ESC in subtle ways, at different sites and, given their immortal status, will be used including in their epigenetic profile, exposure to the environ- for many years or even decades.