DOCSLIB.ORG

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Original Investigation Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics Esther Liu,1 Behram Radmanesh,1 Byungha H. Chung,2 Michael D. Donnan,1 Dan Yi,1 Amal Dadi,1 Kelly D. Smith,3 Jonathan Himmelfarb,2 Mingyao Li,4 Benjamin S. Freedman,2,3 and Jennie Lin 1,5 Abstract Background DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. Methods We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Results Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type–specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Conclusions Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease. KIDNEY360 1: 203–215, 2020. doi: https://doi.org/10.34067/KID.0000422019 Introduction Human kidney organoids derived from induced Apolipoprotein L1 (APOL1)-mediated kidney disease pluripotent stem cells (iPSCs) can be used to model accounts for a portion of the excess risk of CKD and genetic disease mechanisms in the native genomic ESKD among black patients (1,2). The APOL1 high-risk context and cell-type heterogeneity within the kid- genotype, defined as the presence of two risk alleles ney (5–9). Using CRISPR-Cas9–mediated genome (G1 or G2 coding variants), increases the risk of devel- editing, we engineered iPSCs homozygous for the oping CKD, but not all individuals with the high- G1 risk allele and differentiated these cells into risk genotype develop disease (3,4). Much remains three-dimensional (3D) kidney organoids. To eval- unknown regarding mechanisms and modifiers that uate cell type–specific effects of the APOL1 high- render the disease incompletely penetrant, and com- risk genotype, we also performed single-cell RNA plex interactions underlying these mechanisms are sequencing (scRNA-seq), which we and others have difficult to model outside APOL1’snativegenomic previously leveraged to uncover novel biology of locus. As such, current gaps in knowledge may not be how cell-specific phenotypes contribute to kidney fully addressed by induction of transgenic APOL1 development or disease in organoids and other expression in vivo or in vitro. Additionally, because models (10–14). Here we present the application APOL1 is widely expressed across different cell types, of genome-edited, iPSC-derived kidney organoids studying APOL1 risk variants solely within a specific and single-cell transcriptomics to profile APOL1- type of cell (e.g., podocytes) may not fully capture how mediated effects on kidneyorganoidsrelevantto these variants affect the kidney. disease processes. 1Division of Nephrology and Hypertension, Department of Medicine, Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois; 2Division of Nephrology, Department of Medicine, Kidney Research Institute, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington; 3Department of Pathology, University of Washington, Seattle, Washington; 4Department of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania, Philadelphia, Pennsylvania; and 5Section of Nephrology, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois Correspondence: Dr. Jennie Lin, Northwestern University, 303 E Superior Street, Simpson Querrey Biomedical Research Center 8-405, Chicago, IL 60611. Email: [email protected] www.kidney360.org Vol 1 March, 2020 Copyright © 2020 by the American Society of Nephrology 203 204 KIDNEY360 Materials and Methods 10 mg/ml puromycin was added to iPSC culture to select iPSC Culture for cells expressing the donor plasmid (Supplemental iPSC lines previously derived from fibroblasts from a Figure 1A). Seven days later, iPSC colonies were evaluated non-African ancestry donor (1016SevA; Harvard Stem Cell for insertion of HDR template by PCR and Sanger sequenc- Institute) (15–18) and PBMCs from an African ancestry ing validation. After expansion of the successfully knocked-in donor (Penn134-61-26; WiCell) were maintained in feeder- colonies into separate lines, the piggyBac transposase expression free culture on 10-cm dishes coated with 0.5% Geltrex vector (Transposagen Bio) was introduced by electropo- (Gibco) in Modified Tenneille’s Special Recipe 1 (mTeSR1; ration. Additional screening of genotype was performed STEMCELL Technologies), supplemented with 1% penicil- to validate the puromycin cassette excision. lin/streptomycin (Gibco) and 0.02% Plasmocin (Invivogen). fi iPSCs were con rmed to be mycoplasma-free and below iPSC-Derived Kidney Organoid Differentiation passage 48. They were passaged using 1:3 Accutase (STEM- iPSCs were differentiated into kidney organoids follow- CELL Technologies). ing the previously published protocol by Freedman et al. (5) (Figure 1B). Briefly, iPSCs were dissociated with 1:3 Accu- tase and plated onto 24-well plates precoated with 0.5% CRISPR-Cas9 Genome Editing GelTrex in mTeSR1 supplemented with 10 mM Y-27632 APOL1 G1 risk variants (rs73885319 and rs60910145) ROCK Inhibitor (STEMCELL Technologies). After 24 hours, were introduced into the 1016SevA iPSC line through a another layer of GelTrex at 1.5% was added in mTeSR1 genomic footprint-free approach (Figure 1A, Supplemental media. At the end of day 4, the medium was replaced with fl Figure 1A) (19,20). Brie y, the homology-directed repair Advanced RPMI (Gibco) supplemented with 12 mM CHIR- (HDR) template containing the G1 variants was engineered 99021 and 10 ng/ml noggin (STEMCELL Technologies). using the MV-PGK-Puro-TK vector (Transposagen Bio), Approximately 60 hours later, the medium was changed referred to as the PMV vector, which houses a removable to Advanced RPMI with B27 (Gibco). Organoids were cul- fl puromycin selection cassette anked by two homology tured in this medium until collection at day 25. arms. The puromycin cassette is excisable by a piggyBac transposase, leaving only a “TTAA” sequence behind that Induction of APOL1 Expression and Endoplasmic Reticulum can be seamlessly introduced into a coding sequence by Stress carefully choosing sites where the change would be synon- Day 24 G0 and G1 kidney organoids in identically plated ymous. The G1 variants were engineered by two-step PCR wells of a 24-well plate were treated with IFN-g (25 ng/ml; of G0 genomic DNA (Supplemental Figure 1A, Supplemen- PeproTech) for 24 hours to induce APOL1 expression. This tal Table 1) to create the donor template for homology arm dose approximates previous in vitro IFN-g doses used for A, designed to flank the upstream portion of the puromycin macrophage activation (22,23). Endoplasmic reticulum (ER) selection cassette. Arm B, designed to flank the downstream stress was induced by adding 5 mM tunicamycin (Tocris) for end of the selection cassette, was amplified from G0 24 hours. genomic DNA by traditional PCR. Both arm A and arm B underwent separate TOPO TA cloning reactions (Invitro- gen) for insertion into a stable vector for subsequent sub- scRNA-seq cloning into the PMV vector. Stepwise sequential double We performed scRNA-seq on G0 and G1 day-25 kidney restriction-enzyme digests and homology-arm ligations organoids on the 1016SevA background. The organoids were performed on the PMV vector with the following pairs were treated with vehicle, IFN-g, or both IFN-g and tuni- of restriction enzymes: Not1-High Fidelity (HF) and Bbs1- camycin for 24 hours. Organoids were dissociated from the HF, Nco1-HF, and Bsa1-HF (New England Biolabs). The well with TrypLE Express (Gibco) and processed into single- ends of both homology arms bordering the cassette harbor cell suspension by gentle intermittent pipetting while incu- the TTAA piggyBac transposase cut sequence, thus allowing bating in a ThermoMixer (Eppendorf) for up to 15 minutes. 3 for the transposase to excise the cassette from both ends and Single-cell libraries were prepared using the 10 Genomics 9 leave behind the TTAA sequence in a scarless fashion (Sup- Chromium droplet-based platform and the Single Cell 5 3 plemental Figure 1B). To make this genome-editing event Library Construction Kit (10 Genomics), which was 9 footprint-free, we selected a codon site that would allow the chosen to increase read coverage over the 3 chemistry. TTAA nucleotide sequence to be knocked in without alter- At least three technical replicates (different wells from the ing the APOL1 amino acid
Load more

Recommended publications
  • Old Data and Friends Improve with Age: Advancements with the Updated Tools of Genenetwork
    bioRxiv preprint doi: https://doi.org/10.1101/2021.05.24.445383; this version posted May 25, 2021. 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 4.0 International license. Old data and friends improve with age: Advancements with the updated tools of GeneNetwork Alisha Chunduri1, David G. Ashbrook2 1Department of Biotechnology, Chaitanya Bharathi Institute of Technology, Hyderabad 500075, India 2Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, TN 38163, USA Abstract Understanding gene-by-environment interactions is important across biology, particularly behaviour. Families of isogenic strains are excellently placed, as the same genome can be tested in multiple environments. The BXD’s recent expansion to 140 strains makes them the largest family of murine isogenic genomes, and therefore give great power to detect QTL. Indefinite reproducible genometypes can be leveraged; old data can be reanalysed with emerging tools to produce novel biological insights. To highlight the importance of reanalyses, we obtained drug- and behavioural-phenotypes from Philip et al. 2010, and reanalysed their data with new genotypes from sequencing, and new models (GEMMA and R/qtl2). We discover QTL on chromosomes 3, 5, 9, 11, and 14, not found in the original study. We narrowed down the candidate genes based on their ability to alter gene expression and/or protein function, using cis-eQTL analysis, and variants predicted to be deleterious. Co-expression analysis (‘gene friends’) and human PheWAS were used to further narrow candidates.
    [Show full text]
  • Whole-Genome Microarray Detects Deletions and Loss of Heterozygosity of Chromosome 3 Occurring Exclusively in Metastasizing Uveal Melanoma
    Anatomy and Pathology Whole-Genome Microarray Detects Deletions and Loss of Heterozygosity of Chromosome 3 Occurring Exclusively in Metastasizing Uveal Melanoma Sarah L. Lake,1 Sarah E. Coupland,1 Azzam F. G. Taktak,2 and Bertil E. Damato3 PURPOSE. To detect deletions and loss of heterozygosity of disease is fatal in 92% of patients within 2 years of diagnosis. chromosome 3 in a rare subset of fatal, disomy 3 uveal mela- Clinical and histopathologic risk factors for UM metastasis noma (UM), undetectable by fluorescence in situ hybridization include large basal tumor diameter (LBD), ciliary body involve- (FISH). ment, epithelioid cytomorphology, extracellular matrix peri- ϩ ETHODS odic acid-Schiff-positive (PAS ) loops, and high mitotic M . Multiplex ligation-dependent probe amplification 3,4 5 (MLPA) with the P027 UM assay was performed on formalin- count. Prescher et al. showed that a nonrandom genetic fixed, paraffin-embedded (FFPE) whole tumor sections from 19 change, monosomy 3, correlates strongly with metastatic death, and the correlation has since been confirmed by several disomy 3 metastasizing UMs. Whole-genome microarray analy- 3,6–10 ses using a single-nucleotide polymorphism microarray (aSNP) groups. Consequently, fluorescence in situ hybridization were performed on frozen tissue samples from four fatal dis- (FISH) detection of chromosome 3 using a centromeric probe omy 3 metastasizing UMs and three disomy 3 tumors with Ͼ5 became routine practice for UM prognostication; however, 5% years’ metastasis-free survival. to 20% of disomy 3 UM patients unexpectedly develop metas- tases.11 Attempts have therefore been made to identify the RESULTS. Two metastasizing UMs that had been classified as minimal region(s) of deletion on chromosome 3.12–15 Despite disomy 3 by FISH analysis of a small tumor sample were found these studies, little progress has been made in defining the key on MLPA analysis to show monosomy 3.
    [Show full text]
  • Leukaemia Section
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Leukaemia Section Short Communication t(4;9)(q21.22;p24) SEC31A/JAK2 Julie Sanford Biggerstaff Idaho Cytogenetics Diagnostic Laboratory, Boise, ID 83706, USA; [email protected] Published in Atlas Database: April 2017 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0409q21p24ID1682.html Printable original version : http://documents.irevues.inist.fr/bitstream/handle/2042/68905/04-2017-t0409q21p24ID1682.pdf DOI: 10.4267/2042/68905 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2018 Atlas of Genetics and Cytogenetics in Oncology and Haematology Abstract Cytogenetics Review on t(4;9)(q21.22;p24) SEC31A/JAK2, with data on clinics, and the genes involved. Cytogenetics morphological KEYWORDS JAK2 breakapart (commonly available). Chromosome 4; chromosome 9; translocation; SEC31A; JAK2; Hodgkin Lymphoma Genes involved and Clinics and pathology proteins SEC31A (SEC31 homolog A, COPII Disease coat complex component) Hodgkin Lymphoma Location Phenotype/cell stem origin 4q21.22 Hodgkin and Reed-Sternberg cells, which derive DNA/RNA from pre-apoptotic crippled germinal center (GC) B- gene is 72,606 bp with 25 exons; transcribed from cells, are positive for CD30, CD15, CD40 and the - strand; coding region is 62,863 bp with 24 IRF4/MUM1 exons Epidemiology Protein Hodgkin lymphoma itself is common, but this 1166 amnio acids. Protein transport protein SEC31A particular translocation may be rare within the is ubiquitously expressed and forms part of the coat disorder. However, it is not often assayed for; found protein complex II (COPII) which is comprised of at in 2/131 cHL cases examined: a M/31 with nodular least four other proteins in addition to SEC31A this sclerosis cHL, alive 60 mths after diagnosis; and a complex is involved in formation of transport M/83 with lymphocyte-depleted cHL who died at vesicles from the ER to Golgi.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • The Ire1a-XBP1 Pathway Promotes T Helper Cell Differentiation by Resolving Secretory Stress and Accelerating Proliferation
    bioRxiv preprint doi: https://doi.org/10.1101/235010; this version posted December 15, 2017. 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-NC-ND 4.0 International license. The IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation Jhuma Pramanik1, Xi Chen1, Gozde Kar1,2, Tomás Gomes1, Johan Henriksson1, Zhichao Miao1,2, Kedar Natarajan1, Andrew N. J. McKenzie3, Bidesh Mahata1,2*, Sarah A. Teichmann1,2,4* 1. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom 2. EMBL-European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom 3. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 OQH, United Kingdom 4. Theory of Condensed Matter, Cavendish Laboratory, 19 JJ Thomson Ave, Cambridge CB3 0HE, United Kingdom. *To whom correspondence should be addressed: [email protected] and [email protected] Keywords: Th2 lymphocyte, XBP1, Genome wide XBP1 occupancy, Th2 lymphocyte proliferation, ChIP-seq, RNA-seq, Th2 transcriptome Summary The IRE1a-XBP1 pathway, a conserved adaptive mediator of the unfolded protein response, is indispensable for the development of secretory cells. It maintains endoplasmic reticulum homeostasis by facilitating protein folding and enhancing secretory capacity of the cells. Its role in immune cells is emerging. It is involved in dendritic cell, plasma cell and eosinophil development and differentiation. Using genome-wide approaches, integrating ChIPmentation and mRNA-sequencing data, we have elucidated the regulatory circuitry governed by the IRE1a-XBP1 pathway in type-2 T helper cells (Th2).
    [Show full text]
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.
    [Show full text]
  • Cytogenetically Complex SEC31A-ALK Fusions Are Recurrent
    Letters to the Editor A 1.5 30 Takashi Uchiyama from the Deptartment of Hematology/Oncology, ETV6-ABL1 WBC Kyoto University Hospital, Japan for following the patient in Japan, Tony Deblasio for collecting and storing the samples, and Emily 1.0 20 Dolezal for generating the database that facilitated our analysis. ETV6-ABL1 WBC (fold-change in (x109/ L) Correspondence: Stephen D. Nimer, MD, Memorial Sloan transcript level) μ Kettering Cancer Center, 1275 York Avenue, New York, NY 0.5 10 10065, USA. Phone: international +1.646.8883040. Fax: international +1.646.4220246. E-mail: [email protected] Key words: myeloproliferative, neoplasm, imatinib, molecular 0.0 0 response. 2.7.06 5.9.06 1.25.05 9.13.05 5.20.08 4.28.09 Citation: Perna F, Abdel-Wahab O, Levine RL, Jhanwar SC, 08.09.05 11.01.05 Imada K, and Nimer SD. ETV6-ABL1-positive “chronic myeloid 1.5 leukemia”: clinical and molecular response to tyrosine kinase inhibi- B c-myc tion with imatinib. Haematologica 2011; 96(02):342-343. BCL-XL doi:10.3324/haematol.2010.036673 ld1 1.0 NuP98 Fold-change (relative to References HPRT1) 0.5 1. Andreasson P, Johansson B, Carlsson M, Jarlsfelt I, Fioretos T, Mitelman F, et al. BCR/ABL-negative chronic myeloid leukemia with ETV6/ABL fusion. Genes Chromosomes Cancer. 1997;20(3): 0.0 299-304. 2. Barbouti A, Ahlgren T, Johansson B, Hoglund M, Lassen C, 2.7.06 5.9.06 1.25.05 9.13.05 Turesson I, et al. Clinical and genetic studies of ETV6/ABL1-posi- 08.09.05 11.01.05 tive chronic myeloid leukaemia in blast crisis treated with imatinib mesylate.
    [Show full text]
  • Identification of Differentially Expressed Genes in Human Bladder Cancer Through Genome-Wide Gene Expression Profiling
    521-531 24/7/06 18:28 Page 521 ONCOLOGY REPORTS 16: 521-531, 2006 521 Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling KAZUMORI KAWAKAMI1,3, HIDEKI ENOKIDA1, TOKUSHI TACHIWADA1, TAKENARI GOTANDA1, KENGO TSUNEYOSHI1, HIROYUKI KUBO1, KENRYU NISHIYAMA1, MASAKI TAKIGUCHI2, MASAYUKI NAKAGAWA1 and NAOHIKO SEKI3 1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520; Departments of 2Biochemistry and Genetics, and 3Functional Genomics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Received February 15, 2006; Accepted April 27, 2006 Abstract. Large-scale gene expression profiling is an effective CKS2 gene not only as a potential biomarker for diagnosing, strategy for understanding the progression of bladder cancer but also for staging human BC. This is the first report (BC). The aim of this study was to identify genes that are demonstrating that CKS2 expression is strongly correlated expressed differently in the course of BC progression and to with the progression of human BC. establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n=10) or Introduction invasive (n=11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected Bladder cancer (BC) is among the 5 most common to microarray analysis. The validity of the microarray results malignancies worldwide, and the 2nd most common tumor of was verified by real-time RT-PCR. Of the 136 up-regulated the genitourinary tract and the 2nd most common cause of genes we detected, 21 were present in all 14 BCs examined death in patients with cancer of the urinary tract (1-7).
    [Show full text]
  • 1 the TRAPP Complex Mediates Secretion Arrest Induced by Stress Granule Assembly Francesca Zappa1, Cathal Wilson1, Giusepp
    bioRxiv preprint doi: https://doi.org/10.1101/528380; this version posted February 5, 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. The TRAPP complex mediates secretion arrest induced by stress granule assembly Francesca Zappa1, Cathal Wilson1, Giuseppe Di Tullio1, Michele Santoro1, Piero Pucci2, Maria Monti2, Davide D’Amico1, Sandra Pisonero Vaquero1, Rossella De Cegli1, Alessia Romano1, Moin A. Saleem3, Elena Polishchuk1, Mario Failli1, Laura Giaquinto1, Maria Antonietta De Matteis1, 2 1 Telethon Institute of Genetics and Medicine, Pozzuoli (Naples), Italy 2 Federico II University, Naples, Italy 3 Bristol Renal, Bristol Medical School, University of Bristol, UK Correspondence to: [email protected], [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/528380; this version posted February 5, 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. The TRAnsport-Protein-Particle (TRAPP) complex controls multiple membrane trafficking steps and is thus strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified TRAPP as a key component of a branch of the integrated stress response that impinges on the early secretory pathway. TRAPP associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and an arrest of ER export.
    [Show full text]
  • ORM-Like Protein (ORMDL) – Annäherung an Die Funktion Über Die Interaktion
    ORM-like protein (ORMDL) - Annäherung an die Funktion über die Interaktion Julian Klingbeil München 2019 Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig–Maximilians–Universität München Direktor: Prof. Dr. med. Dr. sci. nat. C. Klein ORM-like protein (ORMDL) – Annäherung an die Funktion über die Interaktion Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig–Maximilians–Universität zu München vorgelegt von Julian Malte Klingbeil aus Berlin 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatterin: Prof. Dr. Ania Muntau Mitberichterstatter: Prof. Dr. Katja Radon PD Dr. Anne Hilgendorff Prof. Dr. Jürgen Behr Prof. Dr. Ortrud Steinlein Mitbetreuung durch den promovierten Mitarbeiter: Prof. Dr. Søren Gersting Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 10.10.2019 Inhaltsverzeichnis Zusammenfassung xiii 1 Einleitung 1 1.1 Asthma bronchiale . .1 1.1.1 Epidemiologie, Pathogenese und Klassifikation . .2 1.1.2 Therapie . .4 1.1.3 Gen-Umwelt-Interaktionen und Genomweite Assoziationsstudien5 1.2 Das neue Asthma-Risikogen ORMDL . .8 1.3 Der β2-Adrenorezeptor . 11 1.4 Protein-Protein-Interaktionen . 13 1.4.1 Biolumineszenz-Resonanz-Energie-Transfer . 15 1.5 Zielsetzung der Arbeit . 18 2 Material und Methoden 19 2.1 Material . 19 2.1.1 Laborgeräte . 19 2.1.2 Allgemeine Verbrauchsmaterialien, Chemikalien und Reagenzien . 21 2.1.3 Lösungen, Reagenzien-Kits und Puffer . 26 2.1.4 Medium . 28 2.1.5 Zelllinien und Bakterienstämme . 28 2.1.6 Antikörper . 29 2.1.7 β-Sympathomimetika . 29 2.1.8 Restriktionsenzyme . 30 2.1.9 Vektoren und DNA .
    [Show full text]
  • Identification of Prognosis Biomarkers of Prostatic Cancer in a Cohort Of
    Current Problems in Cancer 43 (2019) 100503 Contents lists available at ScienceDirect Current Problems in Cancer journal homepage: www.elsevier.com/locate/cpcancer Identification of prognosis biomarkers of prostatic cancer in a cohort of 498 patients from TCGA ∗ Zhiqiang Chen , Haiyi Hu Department of Urology, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China a b s t r a c t Objective: Prostatic cancer (PCa) is the first common cancer in male, and the prognostic variables are ben- eficial for clinical trial design and treatment strategies for PCa. This study was performed to identify more potential biomarkers for the prognosis of patients with PCa. Methods and results: The transcriptome data and survival information of a cohort including 498 subjects with PCa were downloaded from TCGA . A total of 4293 differentially expressed genes (DEGs), including 1362 prognosis-related DEGs, were identified in PCa tissues compared with normal tissues. Upregulated genes, including serine/arginine-rich splicing factors (SRSFs; such as SRSF2, SRSF5, SRSF7 and SRSF8 ), and ubiquitin conjugating enzyme E2 (UBE2) members (such as UBE2D2, UBE2G2, UBE2J1 and UBE2E1 ), were identified as negative prognostic biomarkers of PCa, as the high expression of them correlated with poor overall survival of PCa patients. Several downregulated Golgi-ER traffic mediators (such as SEC31A, TMED2, and TMED10 ) were identified as positive prognostic biomarkers of PCa, as the high expression of them correlated with good overall survival of PCa patients. Conclusions: These genes were of great interests in prognosis of PCa, and some of them may be constructive for the augmentation of clinical trial design and treatment strategies for PCa.
    [Show full text]
  • 1 1 2 3 Cell Type-Specific Transcriptomics of Hypothalamic
    1 2 3 4 Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to 5 weight-loss 6 7 Fredrick E. Henry1,†, Ken Sugino1,†, Adam Tozer2, Tiago Branco2, Scott M. Sternson1,* 8 9 1Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 10 20147, USA. 11 2Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, 12 Cambridge CB2 0QH, UK 13 14 †Co-first author 15 *Correspondence to: [email protected] 16 Phone: 571-209-4103 17 18 Authors have no competing interests 19 1 20 Abstract 21 Molecular and cellular processes in neurons are critical for sensing and responding to energy 22 deficit states, such as during weight-loss. AGRP neurons are a key hypothalamic population 23 that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific 24 transcriptomics can be used to identify pathways that counteract weight-loss, and here we 25 report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived 26 young adult mice. For comparison, we also analyzed POMC neurons, an intermingled 27 population that suppresses appetite and body weight. We find that AGRP neurons are 28 considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell 29 type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion 30 channels, neuropeptides, and receptors. Combined with methods to validate and manipulate 31 these pathways, this resource greatly expands molecular insight into neuronal regulation of 32 body weight, and may be useful for devising therapeutic strategies for obesity and eating 33 disorders.
    [Show full text]