DOCSLIB.ORG

Analysis of ALS and FTLD-U Linked Protein TDP-43 in Drosophila Melanogaster

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Analysis of ALS and FTLD-U linked protein TDP-43 in Drosophila melanogaster Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademische Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Kavita Kaur MSc. aus Amritsar, Indien. Berichter: Prof. Dr. med. J.B. Schulz Prof. Dr. rer. nat. habil Hermann Wagner Tag der Mündlichen Prüfung: 6 Juli 2015 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. Thesis Committee Thesis Committee Members of the thesis committee Supervisor Prof. Dr. med. J.B. Schulz Head, Department of neurology University medical center, RWTH Aachen University Pauwelsstrasse 30 52074 Aachen Prof. Dr. rer. nat. habil Hermann Wagner Institute leader, Chair and institute of biology II (Zoology) Sammelbau 2 Biologie Worringerweg 3 52074 Aachen Prof. Marc Spehr Abteilung Chemosensorik Institute of biology II (Zoology) Sammelbau 2 Biologie Worringerweg 3 52074 Aachen List of publications List of Publications and Author’s contributions Part of this thesis has already been published with authorisation of Prof J.B. Schulz, Head of the Department of Neurology, University Medical Centre RWTH Aachen. Original article:” TDP-43-mediated neuron loss in vivo requires RNA-binding activity Aaron Voigt, David Herholz, Fabienne Fiesel, Kavita Kaur, Daniel Mueller, Peter Karsten, Stephanie S. Weber, Phillipp J. Kahle, Till Marquardt, J.B.Schulz PLoS ONE August 2010 5(8): e 12247 doi10.1317/journal.pone.0012247 Conceived and designed the experiments: Aaron Voigt, David Herholz, Till Marquardt Performed the experiments: Aaron Voigt, David Herholz, Fabienne Fiesel, Kavita Kaur (fly experiments), Daniel Mueller, Peter Karsten, Stephanie S. Weber. Contributed reagents/ materials/ analysis tools: David Herholz, Fabienne Fiesel, Phillipp J. Kahle, J.B. Schulz Poster: “A fly model of TDP-43 proteinopathy” Kavita Kaur, J.B.Schulz, Aaron Voigt Regional Drosophila meeting 2009, Muenster (28 August 2009). Poster Abstract: “Genome-wide screen for modifiers of TDP-43 induced neurodegeneration” Kavita kaur, Peter Karsten, Sabine Hamm, J.B.Schulz and Aaron Voigt. International Society for Neurochemistry 2011, J Neurochem. 118 (Suppl. 1) Poster WE04-14, 165-244 Summary Summary TAR-DNA binding protein (TDP-43) is a multifunctional ribonucleoprotein, which is involved in transcription, splicing and mRNA stablilisation. TDP-43 is predominantly localised in the nucleus but shuttles between nucleus and the cytoplasm. In a pathological situation it is depleted from the nucleus and found mislocalised in the cytoplasm where it is abnormally cleaved, phosphorylated, ubiquitinated and aggregated. TDP-43 has been implicated in wide range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration-U (FTLD-U). The relative impact of the endogenous protein function, alteration and mutations on disease progression or pathology remains unclear. In this study I addressed many questions pertaining to TDP-43 biology with both random and strict comparable expression levels. This study focused on developing and characterising Drosophila models of TDP-43 proteinopathy. In addition, this study investigated the role of ALS/FTLD linked mutations and alterations on neural integrity utilising Drosophila as model system. A direct role of TDP-43 in neurodegeneration is highlighted by the fact that neuronal expression of human TDP-43 in Drosophila causes age- and dose-dependent locomotion deficits and early lethality. Further, targeted expression of TDP-43 to the developing fly eye results in a rough eye phenotype (REP). In this study TDP-43 RNA-binding was found to be absolutely required for toxicity. ALS/FTLD linked mutations and other reported pathological events like mislocalisation or truncation were relatively less toxic when compared to TDP-43 wild-type. Further I performed a large-scale toxicity modifier screen for TDP-43 induced toxicity using REP as readout. I identified novel modifiers of TDP-43-induced toxicity in this genetic screen. The most prominent candidates were RNA/DNA related genes, ubiquitin proteasome pathway related genes and neuron related genes. In addition, I found a novel protein interactor DBNDD2/CK1BP of TDP-43 in a yeast-2- hybrid screen. In the end, I present a very potent model of TDP-43 proteinopathy, which recapitulates a wide range of neuropathological, biochemical and functional features of human TDP-43 proteinopathy. Further working with this model I could uncover many endogenous functions and toxicity modifiers of TDP-43 induced toxicity. This study enhances our knowledge on TDP-43 biology and potentially widens future research strategies. Table of contents Table of contents 1 Introduction ........................................................................................................................... 1 1.1 TDP-43 proteinopathies ..................................................................................................... 1 1.2 Amyotrophic lateral sclerosis (ALS) ................................................................................. 3 1.3 Frontotemporal lobar dementia (FTLD-U) ......................................................................... 5 1.4 TAR-DNA binding protein 43 (TDP-43) .............................................................................. 6 1.4.1 Physiological functions of TDP-43 ...................................................................................... 7 1.4.2 Pathological characteristics of TDP-43 ............................................................................... 8 1.5 Modelling general neurodegeneration in flies ................................................................ 11 1.5.1 The UAS/Gal4 expression system .................................................................................... 11 1.5.2 The rough eye phenotype ................................................................................................ 13 2 Aim of the study .................................................................................................................. 14 3 Material and Methods .......................................................................................................... 15 3.1 Chemicals, buffers and equipment ................................................................................. 15 3.2 Transgenic Drosophila strains and fly keeping conditions ........................................... 18 3.2.1 The UAS/Gal4 expression system and Mating Procedure ................................................ 21 3.2.2 Longevity assays .............................................................................................................. 22 3.2.3 Climbing analyses ............................................................................................................ 23 3.2.4 Screening procedure and rough eye phenotype modification ........................................... 23 3.2.5 Drug administration/pharmacological treatment................................................................ 24 3.3 Protein biochemistry ........................................................................................................ 24 3.3.1 Head lysates preparation ................................................................................................. 24 3.3.2 SDS-polyacrylamide-gel-electrophoresis and Western blot .............................................. 25 3.4 Quantitative PCR analysis. .............................................................................................. 26 3.5 Statistical analysis ........................................................................................................... 27 3.6 Yeast-2-hybrid screen ...................................................................................................... 27 4 Results ................................................................................................................................. 28 4.1 Characterisation of TDP-43 proteinopathy in Drosophila .............................................. 29 4.1.1 GFP-tagged TDP-43 transgenic lines exhibiting Variable expression ............................... 29 4.1.2 Subcellular localisation of TDP-43:GFP tagged variants .................................................. 31 4.1.3 Expressing TDP-43 in eyes results in rough eye phenotype ............................................. 32 4.1.4 Pan-neural TDP-43 expression causes neurodegeneration. ............................................ 34 4.1.5 Expressing TDP-43 in muscles leads to muscle loss ........................................................ 35 4.1.6 TDP-43 toxicity is dose-dependent ................................................................................... 36 4.2 Site directed TDP-43 variants with comparable level of expression ............................. 38 Table of contents 4.2.1 Subcellular localisation of untagged TDP-43 variants in Drosophila ................................. 39 4.2.2 Pan-neural expression of TDP-43 reduces longevity in Drosophila. ................................. 43 4.2.3 Motor neuron specific expression of TDP-43 impaired locomotion ................................... 44 4.2.4 TDP-43 expression leads to loss of neuromuscular function ............................................ 46 4.2.5 Adult- onset model of TDP-43 proteinopathy. ..................................................................
Recommended publications
  • Small Cell Ovarian Carcinoma: Genomic Stability and Responsiveness to Therapeutics

    Small Cell Ovarian Carcinoma: Genomic Stability and Responsiveness to Therapeutics

    Gamwell et al. Orphanet Journal of Rare Diseases 2013, 8:33 http://www.ojrd.com/content/8/1/33 RESEARCH Open Access Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics Lisa F Gamwell1,2, Karen Gambaro3, Maria Merziotis2, Colleen Crane2, Suzanna L Arcand4, Valerie Bourada1,2, Christopher Davis2, Jeremy A Squire6, David G Huntsman7,8, Patricia N Tonin3,4,5 and Barbara C Vanderhyden1,2* Abstract Background: The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer. Method: The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested. Results: BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing.
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals

    Identification of the Binding Partners for Hspb2 and Cryab Reveals

    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
  • 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.
  • Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis

    Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis

    Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis Deepti Verma, Anna-Karin Ekman, Cecilia Bivik Eding and Charlotta Enerbäck The self-archived postprint version of this journal article is available at Linköping University Institutional Repository (DiVA): http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-147791 N.B.: When citing this work, cite the original publication. Verma, D., Ekman, A., Bivik Eding, C., Enerbäck, C., (2018), Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis, Journal of Investigative Dermatology, 138(5), 1088-1093. https://doi.org/10.1016/j.jid.2017.11.036 Original publication available at: https://doi.org/10.1016/j.jid.2017.11.036 Copyright: Elsevier http://www.elsevier.com/ Genome-Wide DNA Methylation Profiling Identifies Differential Methylation in Uninvolved Psoriatic Epidermis Deepti Verma*a, Anna-Karin Ekman*a, Cecilia Bivik Edinga and Charlotta Enerbäcka *Authors contributed equally aIngrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden Corresponding author: Charlotta Enerbäck Ingrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Linköping University SE-581 85 Linköping, Sweden Phone: +46 10 103 7429 E-mail: [email protected] Short title Differential methylation in psoriasis Abbreviations CGI, CpG island; DMS, differentially methylated site; RRBS, reduced representation bisulphite sequencing Keywords (max 6) psoriasis, epidermis, methylation, Wnt, susceptibility, expression 1 ABSTRACT Psoriasis is a chronic inflammatory skin disease with both local and systemic components. Genome-wide approaches have identified more than 60 psoriasis-susceptibility loci, but genes are estimated to explain only one third of the heritability in psoriasis, suggesting additional, yet unidentified, sources of heritability.
  • Tepzz¥ 6Z54za T

    Tepzz¥ 6Z54za T

    (19) TZZ¥ ZZ_T (11) EP 3 260 540 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 27.12.2017 Bulletin 2017/52 C12N 15/113 (2010.01) A61K 9/127 (2006.01) A61K 31/713 (2006.01) C12Q 1/68 (2006.01) (21) Application number: 17000579.7 (22) Date of filing: 12.11.2011 (84) Designated Contracting States: • Sarma, Kavitha AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Philadelphia, PA 19146 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • Borowsky, Mark PL PT RO RS SE SI SK SM TR Needham, MA 02494 (US) • Ohsumi, Toshiro Kendrick (30) Priority: 12.11.2010 US 412862 P Cambridge, MA 02141 (US) 20.12.2010 US 201061425174 P 28.07.2011 US 201161512754 P (74) Representative: Clegg, Richard Ian et al Mewburn Ellis LLP (62) Document number(s) of the earlier application(s) in City Tower accordance with Art. 76 EPC: 40 Basinghall Street 11840099.3 / 2 638 163 London EC2V 5DE (GB) (71) Applicant: The General Hospital Corporation Remarks: Boston, MA 02114 (US) •Thecomplete document including Reference Tables and the Sequence Listing can be downloaded from (72) Inventors: the EPO website • Lee, Jeannie T •This application was filed on 05-04-2017 as a Boston, MA 02114 (US) divisional application to the application mentioned • Zhao, Jing under INID code 62. San Diego, CA 92122 (US) •Claims filed after the date of receipt of the divisional application (Rule 68(4) EPC). (54) POLYCOMB-ASSOCIATED NON-CODING RNAS (57) This invention relates to long non-coding RNAs (IncRNAs), libraries of those ncRNAs that bind chromatin modifiers, such as Polycomb Repressive Complex 2, inhibitory nucleic acids and methods and compositions for targeting IncRNAs.
  • Identification of TMEM131L As a Novel Regulator of Thymocyte Proliferation in Humans

    Identification of TMEM131L As a Novel Regulator of Thymocyte Proliferation in Humans

    Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans This information is current as Nesrine Maharzi, Véronique Parietti, Elisabeth Nelson, of September 25, 2021. Simona Denti, Macarena Robledo-Sarmiento, Niclas Setterblad, Aude Parcelier, Marika Pla, François Sigaux, Jean Claude Gluckman and Bruno Canque J Immunol 2013; 190:6187-6197; Prepublished online 20 May 2013; Downloaded from doi: 10.4049/jimmunol.1300400 http://www.jimmunol.org/content/190/12/6187 Supplementary http://www.jimmunol.org/content/suppl/2013/05/21/jimmunol.130040 http://www.jimmunol.org/ Material 0.DC1 References This article cites 44 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/190/12/6187.full#ref-list-1 Why The JI? Submit online. by guest on September 25, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists,
  • WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, C12Q 1/68 (2018.01) A61P 31/18 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/70 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2018/056167 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 16 October 2018 (16. 10.2018) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/573,025 16 October 2017 (16. 10.2017) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, ΓΕ , IS, IT, LT, LU, LV, (71) Applicant: MASSACHUSETTS INSTITUTE OF MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TECHNOLOGY [US/US]; 77 Massachusetts Avenue, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Cambridge, Massachusetts 02139 (US).
  • Placenta-Derived Exosomes Continuously Increase in Maternal

    Placenta-Derived Exosomes Continuously Increase in Maternal

    Sarker et al. Journal of Translational Medicine 2014, 12:204 http://www.translational-medicine.com/content/12/1/204 RESEARCH Open Access Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy Suchismita Sarker1, Katherin Scholz-Romero1, Alejandra Perez2, Sebastian E Illanes1,2,3, Murray D Mitchell1, Gregory E Rice1,2 and Carlos Salomon1,2* Abstract Background: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies. Methods: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP).
  • Supplementary Methods

    Supplementary Methods

    Supplementary methods Human lung tissues and tissue microarray (TMA) All human tissues were obtained from the Lung Cancer Specialized Program of Research Excellence (SPORE) Tissue Bank at the M.D. Anderson Cancer Center (Houston, TX). A collection of 26 lung adenocarcinomas and 24 non-tumoral paired tissues were snap-frozen and preserved in liquid nitrogen for total RNA extraction. For each tissue sample, the percentage of malignant tissue was calculated and the cellular composition of specimens was determined by histological examination (I.I.W.) following Hematoxylin-Eosin (H&E) staining. All malignant samples retained contained more than 50% tumor cells. Specimens resected from NSCLC stages I-IV patients who had no prior chemotherapy or radiotherapy were used for TMA analysis by immunohistochemistry. Patients who had smoked at least 100 cigarettes in their lifetime were defined as smokers. Samples were fixed in formalin, embedded in paraffin, stained with H&E, and reviewed by an experienced pathologist (I.I.W.). The 413 tissue specimens collected from 283 patients included 62 normal bronchial epithelia, 61 bronchial hyperplasias (Hyp), 15 squamous metaplasias (SqM), 9 squamous dysplasias (Dys), 26 carcinomas in situ (CIS), as well as 98 squamous cell carcinomas (SCC) and 141 adenocarcinomas. Normal bronchial epithelia, hyperplasia, squamous metaplasia, dysplasia, CIS, and SCC were considered to represent different steps in the development of SCCs. All tumors and lesions were classified according to the World Health Organization (WHO) 2004 criteria. The TMAs were prepared with a manual tissue arrayer (Advanced Tissue Arrayer ATA100, Chemicon International, Temecula, CA) using 1-mm-diameter cores in triplicate for tumors and 1.5 to 2-mm cores for normal epithelial and premalignant lesions.
  • Expression Analysis of Genes Located Within the Common Deleted Region

    Expression Analysis of Genes Located Within the Common Deleted Region

    Leukemia Research 84 (2019) 106175 Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Research paper Expression analysis of genes located within the common deleted region of del(20q) in patients with myelodysplastic syndromes T ⁎ Masayuki Shiseki , Mayuko Ishii, Michiko Okada, Mari Ohwashi, Yan-Hua Wang, Satoko Osanai, Kentaro Yoshinaga, Naoki Mori, Toshiko Motoji, Junji Tanaka Department of Hematology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan ARTICLE INFO ABSTRACT Keywords: Deletion of the long arm of chromosome 20 (del(20q)) is observed in 5–10% of patients with myelodysplastic Deletion 20q syndromes (MDS). We examined the expression of 28 genes within the common deleted region (CDR) of del Common deleted region (20q), which we previously determined by a CGH array using clinical samples, in 48 MDS patients with (n = 28) Myelodysplastic syndromes or without (n = 20) chromosome 20 abnormalities and control subjects (n = 10). The expression level of 8 of 28 genes was significantly reduced in MDS patients with chromosome 20 abnormalities compared to that of control subjects. In addition, the expression of BCAS4, ADA, and YWHAB genes was significantly reduced in MDS pa- tients without chromosome 20 abnormalities, which suggests that these three genes were commonly involved in the molecular pathogenesis of MDS. To evaluate the clinical significance, we analyzed the impact of the ex- pression level of each gene on overall survival (OS). According to the Cox proportional hazard model, multi- variate analysis indicated that reduced BCAS4 expression was associated with inferior OS, but the difference was not significant (HR, 3.77; 95% CI, 0.995-17.17; P = 0.0509).
  • Genetic Variability in a Holstein Population Using SNP Markers and Their Use for Monitoring Mating Strategies

    Genetic Variability in a Holstein Population Using SNP Markers and Their Use for Monitoring Mating Strategies

    https://doi.org/10.22319/rmcp.v10i3.4842 Article Genetic variability in a Holstein population using SNP markers and their use for monitoring mating strategies Kathy Scienski a,b,c Angelo Ialacci c Alessandro Bagnato c Davide Reginelli d Marina Durán-Aguilar e Maria Giuseppina Strillacci c* a Texas A&M University, College Station. Interdisciplinary Program in Genetics. Texas, USA. b Texas A&M University. Department of Animal Science, Texas, USA. c Università degli Studi di Milano. Department of Veterinary Medicine, Via Trentacoste 2, 20134 Milano, Italy. d Università degli Studi di Milano. Azienda Agraria Didattico Sperimentale Angelo Menozzi, Landriano, Pavia, Italy. e Universidad Autónoma de Querétaro. Facultad de Ciencias Naturales. Querétaro. México. * Corresponding author: [email protected] Abstract: As genotyping costs continue to decrease, the demand for genotyping has increased among farmers. In most livestock herds, an important issue is controlling the increase in inbreeding coefficient. While this remains a large motive to genotype, producers are often unaware of the other benefits that genotyping could bring. The aim of this study was to demonstrate that SNP chips could be used as an effective herd management tool by utilizing a population of Italian Holstein-Friesian cattle. After filtering, the total number 643 Rev Mex Cienc Pecu 2019;10(3):643-663 of animals and SNPs retained for analyses were 44 and 27,365, respectively. The principal component analyses (PCA) were able to identify a sire and origin-of-sire effect within the herd, while determining that sires do not influence individual genomic selection index values. The inbreeding coefficients calculated from genotypes (FIS) provided a glimpse into the herd’s heterozygosity and determined that the genetic variability is being well maintained.

  • Drosophila and Human Transcriptomic Data Mining Provides Evidence for Therapeutic

    Drosophila and human transcriptomic data mining provides evidence for therapeutic mechanism of pentylenetetrazole in Down syndrome Author Abhay Sharma Institute of Genomics and Integrative Biology Council of Scientific and Industrial Research Delhi University Campus, Mall Road Delhi 110007, India Tel: +91-11-27666156, Fax: +91-11-27662407 Email: [email protected] Nature Precedings : hdl:10101/npre.2010.4330.1 Posted 5 Apr 2010 Running head: Pentylenetetrazole mechanism in Down syndrome 1 Abstract Pentylenetetrazole (PTZ) has recently been found to ameliorate cognitive impairment in rodent models of Down syndrome (DS). The mechanism underlying PTZ’s therapeutic effect is however not clear. Microarray profiling has previously reported differential expression of genes in DS. No mammalian transcriptomic data on PTZ treatment however exists. Nevertheless, a Drosophila model inspired by rodent models of PTZ induced kindling plasticity has recently been described. Microarray profiling has shown PTZ’s downregulatory effect on gene expression in fly heads. In a comparative transcriptomics approach, I have analyzed the available microarray data in order to identify potential mechanism of PTZ action in DS. I find that transcriptomic correlates of chronic PTZ in Drosophila and DS counteract each other. A significant enrichment is observed between PTZ downregulated and DS upregulated genes, and a significant depletion between PTZ downregulated and DS dowwnregulated genes. Further, the common genes in PTZ Nature Precedings : hdl:10101/npre.2010.4330.1 Posted 5 Apr 2010 downregulated and DS upregulated sets show enrichment for MAP kinase pathway. My analysis suggests that downregulation of MAP kinase pathway may mediate therapeutic effect of PTZ in DS. Existing evidence implicating MAP kinase pathway in DS supports this observation.