DOCSLIB.ORG

Human Disease Variants in the Ryanodine Receptor Have Effects in Vivo for Caenorhabditis Elegans Neuromuscular Function

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Human Disease Variants in the Ryanodine Receptor Have Effects in Vivo for Caenorhabditis Elegans Neuromuscular Function Human disease variants in the ryanodine receptor have effects in vivo for Caenorhabditis elegans neuromuscular function Brittany Laura Graham Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biology February 2020 ii The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Brittany Laura Graham to be identified as Author of this work has been asserted by Brittany Laura Graham in accordance with the Copyright, Designs and Patents Act 1988. iii Acknowledgements I would like to express my appreciation to my supervisor Ian Hope; for providing the opportunity, for all the scientific advice, for his feedback during my write-up and for his overall support through this process. Thank you for training me to think like a scientist. I would also like to thank my co-supervisor Marie-Anne Shaw. Her encouragement, comments and different point of view have helped widen my research and made me a better scientist. I must also acknowledge the White Rose DTP, the BBSRC and the FBS graduate office at the University of Leeds without whom this project would not have been possible. My special thanks are extended to NemaMetrix for hosting me on my internship and their help in generating the variant strains used in this thesis. Additional thanks go to David Pertab and Alex Jubb, for generating two further variant strains used in this research. I also appreciate the advice and many discussions had with Elpi Kalogeropoulou, Célia Ferreira and Ros Clifford in the Hope lab. I would like to recognise the help provided by the Cohen group at the University of Leeds, particularly Omer Yuval and Rob Holbrook. The multi-worm tracker and guidance on coding and software were vital to the crawling analysis in this research. My friends in the postgrad office: Zatul, Ellie, Francis, Dayah, Rosie, Nok, Victoria, Jens, Jake and Brad, provided an enjoyable environment to work for which I will always be thankful. To all my friends, wherever in the world you are, thank you for being there for me. A special mention made to Elpi and Rosie whose friendships have meant the world to me. Tanner, I am forever grateful for your endless reassurance, positivity and belief in me. Finally, to my family, I would not be where I am without your unconditional love and encouragement. I am thankful to have your support in anything I decide to do. I could not end without thanking my mum for the cups of tea that kept me going, and for everything else. Having a strong and proud woman as a role model made me believe I could do and be anything. iv I would like to dedicate this thesis to women in science. “I was taught that the way of progress was neither swift nor easy.” – Marie Curie The women before me have made my way easier, and I hope to do the same for the future women in science. v Abstract The ryanodine receptor is a key intracellular calcium ion channel in nerve and muscle cells. A number of links have been made between the ryanodine receptor and age- related muscular and neurodegeneration. Variants of the RYR1 gene are associated with malignant hyperthermia, which is a hypermetabolic reaction to inhalational anaesthetics. I used eight genome-edited Caenorhabditis elegans strains expressing ryanodine receptors with modifications equivalent to known RYR1 disease variants to characterise the consequences of these variants on neuromuscular function in vivo. Animals expressing variant ryanodine receptors exhibited increased sensitivity to halothane when both homozygous and heterozygous for the variants. This mirrors the situation in humans where malignant hyperthermia is inherited in an autosomal dominant pattern. Novel, subtle, locomotion defects were found in animals expressing variant ryanodine receptors even in the absence of halothane. In-depth analysis of crawling suggested that these variant channels release excessive calcium, resulting in increased muscle contraction. Exaggerated age-related degeneration of locomotion was observed in variant strains, which may be a result of excessive calcium release via variant ryanodine receptors throughout the lifespan resulting in cellular damage. Cholinergic pharmacological agents were used to characterise if the consequences of the variants were pre and/ or post-synaptic. A range of complex phenotypes were found, reflecting the complexity of regulatory inputs to the ryanodine receptor. The altered properties of variant ryanodine receptors and effects of long-term calcium mishandling found here are anticipated to have consequences for human carriers, which may be in the nerve cells. Assessment of these phenotypes in a whole organism was important to fully appreciate the significant effects of variant ryanodine receptors. vi Table of Contents Acknowledgements ................................................................................................ iii Abstract ................................................................................................................... v Table of Contents ...................................................................................................... vi List of Tables ............................................................................................................ x List of Figures .......................................................................................................... xi List of Abbreviations .............................................................................................. xiii Chapter 1 General introduction ................................................................................ 1 1.1. The ryanodine receptor ............................................................................................ 1 1.1.1. Ryanodine receptor isoforms and evolution ............................................................................. 2 1.1.2. Structure of the ryanodine receptor.......................................................................................... 5 1.1.3. Control of ryanodine receptor activation .................................................................................. 9 1.1.4. Ryanodine receptor related diseases ...................................................................................... 12 1.1.5. Malignant hyperthermia and its triggering agents .................................................................. 15 1.2. Caenorhabditis elegans as a model ......................................................................... 18 1.2.1. C. elegans as a model for malignant hyperthermia ................................................................. 23 1.3. Focus of this research ............................................................................................. 26 1.3.1. Modelling RyR1-related disease in C. elegans ......................................................................... 26 1.3.2. Pre- and postsynaptic effects of RYR1 gene variants .............................................................. 26 1.4. Thesis outline ......................................................................................................... 27 Chapter 2 General Methods ................................................................................... 28 2.1. C. elegans maintenance and strains ........................................................................ 28 2.1.1. C. elegans maintenance ........................................................................................................... 28 2.1.2. C. elegans strains ..................................................................................................................... 28 2.2. Generating strains through CRISPR-Cas9 genome editing ......................................... 29 2.2.1. Genotyping ryanodine receptor variant strains ...................................................................... 33 2.3. Synchronisation and ageing .................................................................................... 34 2.3.1. Generating an age-synchronised population of C. elegans ..................................................... 34 2.3.2. Ageing C. elegans ..................................................................................................................... 35 2.3.3. Statistical and graphical analysis ............................................................................................. 35 Chapter 3 Confirming a malignant hyperthermia related phenotype in genome- edited RYR1 variant C. elegans: modelling malignant hyperthermia in the worm .... 38 3.1. Introduction ........................................................................................................... 38 3.1.1. Aim of this chapter ................................................................................................................... 38 3.1.2. A malignant hyperthermia related phenotype in C. elegans .................................................. 38 vii 3.2. Methods................................................................................................................. 39 3.2.1. Halothane and thrashing assays .............................................................................................. 39 3.2.2. L1 thrashing assay ...................................................................................................................
Load more

Recommended publications
  • Autoantibodies in Neurological Diseases
    Autoantibodies in neurological diseases Hu, Ri, Yo, Tr CV2 Amphiphysin Amphiphysin GM1 Ma/Ta CV2 Amphiphysin Cerebellum Intestine Hippocampus Control transfection CV2 GM2 SOX1 PNMAP 2 (MMa2/Ta) Zic4 PNMP A2 GM3G ITPR1 (MMa2/Ta) RiR CARP YoY RiR GD1G a GAD Hippocampus HEp-2 cells Cerebellum NMDAR (transf. cells) Recoverin HuH Titin Anti-Hu positive Anti-NMDA-receptor positive YoY GD1G b Recoverin Gangliosides MAG HuH GT1b SOS X1 Myelin Aquaporin-4 Titin GQ1G b MOG Zic4 VGKC (LGI1 + CASPR2) Cerebellum Intestine Cerebellum Control transfection NMDA receptors GAD65G AMPA receptors Tr (DNER) GABAB receptors DPPX Control CoC ntrol CoC ntrol IgLON5 Hippocampus HEp-2 cells Optic nerve AQP-4 (transf. cells) Glycine receptors Anti-Yo positive Anti-aquaporin-4 positive AChR Indirect immunofl uorescence EUROLINE Examples of relevant target antigens EUROIMMUN AG · Seekamp 31 · 23560 Lübeck (Germany) · Tel +49 451/5855-0 · Fax 5855-591 · [email protected] · www.euroimmun.com 2 Autoantibodies IIFT pattern Test systems Anti-Hu (ANNA-1*) IIFT: Granular fl uorescence of almost all neuronal nuclei on the substrates cerebellum and hippocampus. The Autoantibodies against basic, RNA- cell nuclei of the plexus myentericus (intestinal tissue) binding proteins of the neuronal cell are also positive. nuclei of the central and peripheral nervous system EUROLINE: Positive reaction of the recombinant Hu antigen (HuD). Associated diseases: encephalomyelitis, subacute sensory neuronopathy (Denny-Brown syndrome), autonomous neuropathy Associated tumours: small-cell lung carcinoma, Cerebellum Intestine neuroblastoma Anti-Ri (ANNA-2*) IIFT: Granular fl uorescence of almost all neuronal nuclei on the substrates cerebellum and hippocampus. The Autoantibodies against neuronal cell substrate intestine (plexus myentericus) shows no reac- nuclei of the central nervous system tion.
    [Show full text]
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model
    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
    [Show full text]
  • 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.
    [Show full text]
  • Transcriptomic Analysis of Native Versus Cultured Human and Mouse Dorsal Root Ganglia Focused on Pharmacological Targets Short
    bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Transcriptomic analysis of native versus cultured human and mouse dorsal root ganglia focused on pharmacological targets Short title: Comparative transcriptomics of acutely dissected versus cultured DRGs Andi Wangzhou1, Lisa A. McIlvried2, Candler Paige1, Paulino Barragan-Iglesias1, Carolyn A. Guzman1, Gregory Dussor1, Pradipta R. Ray1,#, Robert W. Gereau IV2, # and Theodore J. Price1, # 1The University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, 800 W Campbell Rd. Richardson, TX, 75080, USA 2Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine # corresponding authors [email protected], [email protected] and [email protected] Funding: NIH grants T32DA007261 (LM); NS065926 and NS102161 (TJP); NS106953 and NS042595 (RWG). The authors declare no conflicts of interest Author Contributions Conceived of the Project: PRR, RWG IV and TJP Performed Experiments: AW, LAM, CP, PB-I Supervised Experiments: GD, RWG IV, TJP Analyzed Data: AW, LAM, CP, CAG, PRR Supervised Bioinformatics Analysis: PRR Drew Figures: AW, PRR Wrote and Edited Manuscript: AW, LAM, CP, GD, PRR, RWG IV, TJP All authors approved the final version of the manuscript. 1 bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • 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).
    [Show full text]
  • Transcriptome Sequencing and Genome-Wide Association Analyses Reveal Lysosomal Function and Actin Cytoskeleton Remodeling in Schizophrenia and Bipolar Disorder
    Molecular Psychiatry (2015) 20, 563–572 © 2015 Macmillan Publishers Limited All rights reserved 1359-4184/15 www.nature.com/mp ORIGINAL ARTICLE Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder Z Zhao1,6,JXu2,6, J Chen3,6, S Kim4, M Reimers3, S-A Bacanu3,HYu1, C Liu5, J Sun1, Q Wang1, P Jia1,FXu2, Y Zhang2, KS Kendler3, Z Peng2 and X Chen3 Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q ⩽ 0.01, 53 genes; q ⩽ 0.05, 213 genes; q ⩽ 0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (Po10 − 7) and BPD (P = 0.029). To our knowledge, this is the first time that a substantially large number of genes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways.
    [Show full text]
  • IP3-Mediated Gating Mechanism of the IP3 Receptor Revealed by Mutagenesis and X-Ray Crystallography
    IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography Kozo Hamadaa, Hideyuki Miyatakeb, Akiko Terauchia, and Katsuhiko Mikoshibaa,1 aLaboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan; and bNano Medical Engineering Laboratory, RIKEN, Saitama 351–0198, Japan Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 24, 2017 (received for review January 26, 2017) The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated communicate with the channel at a long distance remains ion channel that releases calcium ions (Ca2+) from the endoplasmic unknown, however. reticulum. The IP3-binding sites in the large cytosolic domain are An early gel filtration study of overexpressed rat IP3R1 cyto- 2+ distant from the Ca conducting pore, and the allosteric mecha- solic domain detected an IP3-dependent shift of elution volumes 2+ nism of how IP3 opens the Ca channel remains elusive. Here, we (23), and X-ray crystallography of the IP3-binding domain pro- identify a long-range gating mechanism uncovered by channel vided the first glimpse of IP3-dependent conformational changes mutagenesis and X-ray crystallography of the large cytosolic do- (24). However, the largest IP3R1 fragment solved to date by main of mouse type 1 IP3R in the absence and presence of IP3. X-ray crystallography is limited to the IP3-binding domain in- Analyses of two distinct space group crystals uncovered an IP3- cluding ∼580 residues in IP3R1 (24, 25); thus, the major residual dependent global translocation of the curvature α-helical domain part of the receptor containing more than 1,600 residues in the interfacing with the cytosolic and channel domains.
    [Show full text]
  • Supplementary Table 1. Pain and PTSS Associated Genes (N = 604
    Supplementary Table 1. Pain and PTSS associated genes (n = 604) compiled from three established pain gene databases (PainNetworks,[61] Algynomics,[52] and PainGenes[42]) and one PTSS gene database (PTSDgene[88]). These genes were used in in silico analyses aimed at identifying miRNA that are predicted to preferentially target this list genes vs. a random set of genes (of the same length). ABCC4 ACE2 ACHE ACPP ACSL1 ADAM11 ADAMTS5 ADCY5 ADCYAP1 ADCYAP1R1 ADM ADORA2A ADORA2B ADRA1A ADRA1B ADRA1D ADRA2A ADRA2C ADRB1 ADRB2 ADRB3 ADRBK1 ADRBK2 AGTR2 ALOX12 ANO1 ANO3 APOE APP AQP1 AQP4 ARL5B ARRB1 ARRB2 ASIC1 ASIC2 ATF1 ATF3 ATF6B ATP1A1 ATP1B3 ATP2B1 ATP6V1A ATP6V1B2 ATP6V1G2 AVPR1A AVPR2 BACE1 BAMBI BDKRB2 BDNF BHLHE22 BTG2 CA8 CACNA1A CACNA1B CACNA1C CACNA1E CACNA1G CACNA1H CACNA2D1 CACNA2D2 CACNA2D3 CACNB3 CACNG2 CALB1 CALCRL CALM2 CAMK2A CAMK2B CAMK4 CAT CCK CCKAR CCKBR CCL2 CCL3 CCL4 CCR1 CCR7 CD274 CD38 CD4 CD40 CDH11 CDK5 CDK5R1 CDKN1A CHRM1 CHRM2 CHRM3 CHRM5 CHRNA5 CHRNA7 CHRNB2 CHRNB4 CHUK CLCN6 CLOCK CNGA3 CNR1 COL11A2 COL9A1 COMT COQ10A CPN1 CPS1 CREB1 CRH CRHBP CRHR1 CRHR2 CRIP2 CRYAA CSF2 CSF2RB CSK CSMD1 CSNK1A1 CSNK1E CTSB CTSS CX3CL1 CXCL5 CXCR3 CXCR4 CYBB CYP19A1 CYP2D6 CYP3A4 DAB1 DAO DBH DBI DICER1 DISC1 DLG2 DLG4 DPCR1 DPP4 DRD1 DRD2 DRD3 DRD4 DRGX DTNBP1 DUSP6 ECE2 EDN1 EDNRA EDNRB EFNB1 EFNB2 EGF EGFR EGR1 EGR3 ENPP2 EPB41L2 EPHB1 EPHB2 EPHB3 EPHB4 EPHB6 EPHX2 ERBB2 ERBB4 EREG ESR1 ESR2 ETV1 EZR F2R F2RL1 F2RL2 FAAH FAM19A4 FGF2 FKBP5 FLOT1 FMR1 FOS FOSB FOSL2 FOXN1 FRMPD4 FSTL1 FYN GABARAPL1 GABBR1 GABBR2 GABRA2 GABRA4
    [Show full text]
  • Sodium–Taste Cells Require Skn-1A for Generation and Share Molecular Features with Sweet, Umami, and Bitter Taste Cells
    Research Article: New Research Sensory and Motor Systems Sodium–Taste Cells Require Skn-1a for Generation and Share Molecular Features with Sweet, Umami, and Bitter Taste Cells Makoto Ohmoto,1,2,p Masafumi Jyotaki,1,p J. Kevin Foskett,3,4 and Ichiro Matsumoto1 https://doi.org/10.1523/ENEURO.0385-20.2020 1Monell Chemical Senses Center, Philadelphia, PA 19104, 2Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan, 3Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, and 4Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Abstract Taste buds are maintained via continuous turnover of taste bud cells derived from local epithelial stem cells. A transcription factor Skn-1a (also known as Pou2f3) is required for the generation of sweet, umami (savory), and bitter taste cells that commonly express TRPM5 and CALHM ion channels. Here, we demonstrate that so- dium–taste cells distributed only in the anterior oral epithelia and involved in evoking salty taste also require Skn-1a for their generation. We discovered taste cells in fungiform papillae and soft palate that show similar but not identical molecular feature with sweet, umami, and bitter taste-mediated Type II cells. This novel cell population expresses Plcb2, Itpr3, Calhm3, Skn-1a, and ENaCa (also known as Scnn1a) encoding the putative amiloride-sensitive (AS) salty taste receptor but lacks Trpm5 and Gnat3. Skn-1a-deficient taste buds are pre- dominantly composed of putative non-sensory Type I cells and sour-sensing Type III cells, whereas wild-type taste buds include Type II (i.e., sweet, umami, and bitter taste) cells and sodium–taste cells.
    [Show full text]
  • TRPV2 Calcium Channel Gene Expression and Outcomes in Gastric Cancer Patients: a Clinically Relevant Association
    Journal of Clinical Medicine Article TRPV2 Calcium Channel Gene Expression and Outcomes in Gastric Cancer Patients: A Clinically Relevant Association Pietro Zoppoli 1 , Giovanni Calice 1 , Simona Laurino 1 , Vitalba Ruggieri 1, Francesco La Rocca 1 , Giuseppe La Torre 1, Mario Ciuffi 1, Elena Amendola 2, Ferdinando De Vita 3, Angelica Petrillo 3 , Giuliana Napolitano 2 , Geppino Falco 2,4,* and Sabino Russi 1,* 1 Laboratory of Preclinical and Translational Research, IRCCS—Referral Cancer Center of Basilicata (CROB), 85028 Rionero in Vulture (PZ), Italy; [email protected] (P.Z.); [email protected] (G.C.); [email protected] (S.L.); [email protected] (V.R.); [email protected] (F.L.R.); [email protected] (G.L.T.); mario.ciuffi@crob.it (M.C.) 2 Department of Biology, University of Naples Federico II, 80138 Naples, Italy; [email protected] (E.A.); [email protected] (G.N.) 3 Division of Medical Oncology, Department of Precision Medicine, School of Medicine, University of Study of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; [email protected] (F.D.V.); [email protected] (A.P.) 4 Biogem, Istituto di Biologia e Genetica Molecolare, Via Camporeale, 83031 Ariano Irpino (AV), Italy * Correspondence: [email protected] (G.F.); [email protected] (S.R.); Tel.: +39-081-679092 (G.F.); +39-0972-726239 (S.R.) Received: 16 April 2019; Accepted: 8 May 2019; Published: 11 May 2019 Abstract: Gastric cancer (GC) is characterized by poor efficacy and the modest clinical impact of current therapies. Apoptosis evasion represents a causative factor for treatment failure in GC as in other cancers.
    [Show full text]
  • Ion Channels 3 1
    r r r Cell Signalling Biology Michael J. Berridge Module 3 Ion Channels 3 1 Module 3 Ion Channels Synopsis Ion channels have two main signalling functions: either they can generate second messengers or they can function as effectors by responding to such messengers. Their role in signal generation is mainly centred on the Ca2 + signalling pathway, which has a large number of Ca2+ entry channels and internal Ca2+ release channels, both of which contribute to the generation of Ca2 + signals. Ion channels are also important effectors in that they mediate the action of different intracellular signalling pathways. There are a large number of K+ channels and many of these function in different + aspects of cell signalling. The voltage-dependent K (KV) channels regulate membrane potential and + excitability. The inward rectifier K (Kir) channel family has a number of important groups of channels + + such as the G protein-gated inward rectifier K (GIRK) channels and the ATP-sensitive K (KATP) + + channels. The two-pore domain K (K2P) channels are responsible for the large background K current. Some of the actions of Ca2 + are carried out by Ca2+-sensitive K+ channels and Ca2+-sensitive Cl − channels. The latter are members of a large group of chloride channels and transporters with multiple functions. There is a large family of ATP-binding cassette (ABC) transporters some of which have a signalling role in that they extrude signalling components from the cell. One of the ABC transporters is the cystic − − fibrosis transmembrane conductance regulator (CFTR) that conducts anions (Cl and HCO3 )and contributes to the osmotic gradient for the parallel flow of water in various transporting epithelia.
    [Show full text]
  • Supplementary Table 9. Functional Annotation Clustering Results for the Union (GS3) of the Top Genes from the SNP-Level and Gene-Based Analyses (See ST4)
    Supplementary Table 9. Functional Annotation Clustering Results for the union (GS3) of the top genes from the SNP-level and Gene-based analyses (see ST4) Column Header Key Annotation Cluster Name of cluster, sorted by descending Enrichment score Enrichment Score EASE enrichment score for functional annotation cluster Category Pathway Database Term Pathway name/Identifier Count Number of genes in the submitted list in the specified term % Percentage of identified genes in the submitted list associated with the specified term PValue Significance level associated with the EASE enrichment score for the term Genes List of genes present in the term List Total Number of genes from the submitted list present in the category Pop Hits Number of genes involved in the specified term (category-specific) Pop Total Number of genes in the human genome background (category-specific) Fold Enrichment Ratio of the proportion of count to list total and population hits to population total Bonferroni Bonferroni adjustment of p-value Benjamini Benjamini adjustment of p-value FDR False Discovery Rate of p-value (percent form) Annotation Cluster 1 Enrichment Score: 3.8978262119731335 Category Term Count % PValue Genes List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDR GOTERM_CC_DIRECT GO:0005886~plasma membrane 383 24.33290978 5.74E-05 SLC9A9, XRCC5, HRAS, CHMP3, ATP1B2, EFNA1, OSMR, SLC9A3, EFNA3, UTRN, SYT6, ZNRF2, APP, AT1425 4121 18224 1.18857065 0.038655922 0.038655922 0.086284383 UP_KEYWORDS Membrane 626 39.77128335 1.53E-04 SLC9A9, HRAS,
    [Show full text]