DOCSLIB.ORG

Determination of Fatty Acid Synthesis Intermediates in Escherichia Coli and Bacillus Subtilis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Determination of Fatty Acid Synthesis Intermediates in Escherichia Coli and Bacillus Subtilis DETERMINATION OF FATTY ACID SYNTHESIS INTERMEDIATES IN ESCHERICHIA COLI AND BACILLUS SUBTILIS BY SWAMINATH SRINIVAS DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2018 Urbana, Illinois Doctoral Committee: Professor John E. Cronan Jr., Chair Professor James A. Imlay Professor Satish K. Nair Professor Wilfred A. van der Donk ABSTRACT Lipids play crucial roles in maintaining cellular structure and energy storage. Structural lipids in the form of phospholipids constitute almost 10% of the dry cell weight in bacteria with their synthesis requiring 32 moles of ATP per mole of lipid. This significant investment ensures that the flux through the fatty acid biosynthesis (FAS) and related metabolic pathways is very precisely coordinated. A key feature of the FAS pathway is the acyl carrier protein (ACP), which is a small acidic protein that tethers acyl intermediates via a high-energy thioester bond and shuttles them between enzymes. In Escherichia coli, ACP is one of the most abundant soluble proteins with about 60000 copies per cell. Despite being the subject of extensive biochemical and structural studies for several decades, a reliable snapshot of ACP-bound species in any organism under different conditions is unavailable. Previously used methods are severely limited in their capacity to differentiate fatty acid intermediates, suffer from poor reproducibility, require elaborate instrumentation and cannot be used in an ideal setting for determining intracellular fluxes. This dissertation describes a sensitive and facile method to identify and quantify the physiological level of acyl-ACP species in E. coli and Bacillus subtilis under varying conditions of growth. The method primarily relies on an inert strep-tagged ACP which is purified using a two-step purification strategy utilizing volatile buffers of low pH in order to preserve the thioester bond between the acyl group and ACP. Samples obtained via this method were extremely pure and free of unwanted salts. The full length purified strep-tagged ACP were subjected to ESI mass spectrometry. ii Short and medium chain saturated fatty acyl-ACPs as well as long chain saturated and unsaturated fatty acyl-ACP intermediates were observed. Apart from validating widely held ideas about acyl- ACP flux in basic metabolism, acetyl-ACP, a novel intermediate, was also observed in abundance along with much lower levels of holo-ACP than previously thought. Current efforts are focusing on developing and validating a new model for the initiation of fatty acid biosynthesis using a combination of FAS mutants and stable-isotope labelling studies. During our efforts to construct one such mutant, we identified and characterized the function of a FabG temperature-sensitive mutant missing eight residues at its dimerization interface. Surprisingly, this mutant behaved like a point mutant. We identified interactions within the dimerization/tetramerization interface that compensate for each other and showed that the wildtype FabG predominantly exists as a dimer but functions as a tetramer. The mutation also rendered the enzyme extremely sensitive to calcium in vitro and conferred resistance to a calmodulin inhibitor in vivo. In B. subtilis, we have determined that the cellular levels of ACP are ten times lower than those in E. coli. when normalized to the total protein content per cell. Even under our stringent purification conditions, ACP co-purified with several other proteins, most noticeably FabF. We are currently trying to identify if Bacillus ACP is part of a loose complex. Nevertheless, this method has also successfully resolved all expected fatty acid intermediates associated with ACP including the presence of acetyl-ACP as observed in E. coli. iii In addition, I also discuss the development of a new set of vectors with IPTG-dependent origins of replication and a robust CRISPR-Cas9 toolkit for E. coli based on this vector set. iv ACKNOWLEDGEMENTS असतो मा सद्गमय । तमसो मा ज्योतत셍गमय । From ignorance, lead me to the truth. From darkness, lead me to the light I would like to start by thanking John Cronan, my advisor and my role model for his guidance and immense help over these many years. He has been patient and supportive throughout the course of my research and has given me the complete freedom to pursue my own scientific curiosities. His infectious passion and dedication to science has helped me define myself as a scientist. I would like to thank all the members of the Cronan lab, both past and present, for being wonderful colleagues, collaborators and friends. I would especially like to thank Quin Christensen, Alexander Smith, Youjun Feng and Steven Lin for getting me started in lab and for answering innumerable questions patiently. I thank my committee members, Dr. James Imlay, Dr. Satish Nair and Dr. Wilfred van der Donk for their helpful discussion of my work. I also thank Dr. Peter Yau and Dr. Brain Imai from the proteomics facility for helping me understand mass spectrometry and for making this project viable. Finally, I would like to extend a special thanks to my family, especially my parents and my wife for their constant encouragement, understanding and unwavering support from beginning to end. Without you, none of this would have been possible. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION .....................................................................................1 1.1 TYPE II FATTY ACID BIOSYNTHETIC REACTIONS ................................2 1.2 THE ACYL CARRIER PROTEIN ....................................................................5 1.3 INTRACELLULAR TURNOVER OF ACP PROSTHETIC GROUP .............7 1.4 COMPOSITION AND REGULATION OF THE INTRACELLULAR ACP POOL ..............................................................................................................8 1.5 THE INTERACTIVE NATURE OF ACP ......................................................10 1.6 FATTY ACID SYNTHESIS INITIATION AND ITS INHIBITORS ............11 1.7 BETA-KETOACYL-ACP REDUCTASE: FABG ..........................................12 1.8 AIMS AND SCOPE OF THIS THESIS ..........................................................14 1.9 FIGURES .........................................................................................................17 CHAPTER 2: IDENTIFICATION AND CHARACTERIZATION OF FATTY ACID SYNTHESIS INTERMEDIATES IN ESCHERICHIA COLI AND BACILLUS SUBTILIS ...........................................................................................22 2.1 INTRODUCTION ...........................................................................................22 2.2 MATERIALS AND METHODS .....................................................................25 2.2.1 Bacterial Strains, Plasmids and Materials .........................................25 2.2.2 Construction of Strep-tagged ACP ...................................................26 2.2.3 Growth Measurements ......................................................................27 2.2.4 In vitro Fatty Acid Synthesis Assay..................................................27 vi 2.2.5 Radioactive Labeling of Phospholipids and ACP .............................28 2.2.6 Purification of Strep-tagged ACP .....................................................29 2.2.7 Mass Spectral Analysis .....................................................................30 2.3 EXPERIMENTAL RESULTS.........................................................................31 2.3.1 Carboxy-terminal Strep-tagged ACP is Functional in vivo ..............31 2.3.2 Strep-tagged ACP is Easier to Measure and Visualize than its Native Form ...............................................................................................31 2.3.3 Strep-tagged ACPs are Functional in vitro .......................................32 2.3.4 E. coli has Five to Ten-fold more ACP than B. subtilis ....................32 2.3.5 The ACP Interactome of B. subtilis ..................................................33 2.3.6 FabF Tightly Associates with B. subtilis ACP in a Stoichiometric Fashion ..............................................................................33 2.3.7 Purification of Acyl-ACP species from E. coli and B. subtilis .........34 2.3.8 Mass Spectrometric Analysis and Validation ...................................35 2.3.9 The Metabolic Role of Acetyl-ACP .................................................36 2.4 DISCUSSION ..................................................................................................36 2.5 TABLES AND FIGURES ...............................................................................40 CHAPTER 3: AN EIGHT RESIDUE DELETION IN ESCHERICHIA COLI FABG CAUSES TEMPERATURE-SENSITIVE GROWTH AND LIPID SYNTHESIS PLUS RESISTANCE TO THE CALMODULIN INHIBITOR, TRIFLUOPERAZINE .....................................................................................................53 3.1 INTRODUCTION ...........................................................................................53 vii 3.2 MATERIALS AND METHODS .....................................................................55 3.2.1 Bacterial Strains, Plasmids and Materials .........................................55 3.2.2 Construction of E. coli MC1061 fabG∆8 using CRISPR/Cas9 ........56 3.2.3 Structural Modeling and Sequence Alignment .................................57 3.2.4 Expression and Purification of a His-tagged FabGΔ8 Protein
Load more

Recommended publications
  • In Vivo Evaluation of Ixabepilone (BMS247550), a Novel Epothilone B Derivative, Against Pediatric Cancer Models Jennifer K
    Cancer Therapy: Preclinical In vivo Evaluation of Ixabepilone (BMS247550), A Novel Epothilone B Derivative, against Pediatric Cancer Models Jennifer K. Peterson,1Chandra Tucker,1Edward Favours,1PamelaJ. Cheshire,1Jeremy Creech,1 Catherine A. Billups,2 Richard Smykla,3 Francis Y.F. Lee,3 and Peter J. Houghton1 Abstract Purpose:Vinca alkaloids, agents that cause depolymerization of microtubules, are highly active in treatment of many pediatric cancers. In contrast, taxanes, agents that stabilize microtubules, are far less effective against the same cancer types.The purpose of the current study was to evaluate the antitumor activity of ixabepilone, an epothilone B derivative representing a new class of microtubule-stabilizing antimitotic agent in a wide variety of pediatric solid tumor models. Experimental Design: Ixabepilone was administered i.v. every 4 days for three doses to scid mice bearing s.c. human rhabdomyosarcoma (three lines), neuroblastoma (four),Wilms’ tumors (six), osteosarcoma (four), or brain tumors (seven).Tumor diameters were measured weekly, and tumor growth or regressions were determined. Pharmacokinetic studies were done following a single administration of drug at the maximum tolerated dose (MTD) level (10 mg/kg). Results: At the MTD (10 mg/kg), ixabepilone induced objective responses (all tumors in a group achieved z50% volume regression) in three of three rhabdomyosarcoma lines, three of five neuroblastomas, six of seven Wilms’ tumor models, two of six osteosarcoma, and four of eight brain tumor models. However, the dose-response curve was steep with only 2 of 19 tumors models regressing (z50%) at 4.4 mg/kg. In comparison, paclitaxel administered at the MTD on the same schedule failed to induce objective regressions of three tumor lines that were highly sensitive to treatment with ixabepilone.
    [Show full text]
  • 2701.Full-Text.Pdf
    Cancer Therapy: Clinical The Effect of Ketoconazole on the Pharmacokinetics and Pharmacodynamics of Ixabepilone: A First in Class Epothilone B Analogue in Late-Phase Clinical Development Sanjay Goel,1Marvin Cohen,4 S. Nilgu« n C¸ o« mezoglu,4 Lionel Perrin,5 Franc¸ois Andre¤ ,5 DavidJayabalan, 1Lisa Iacono,4 Adriana Comprelli,4 Van T. Ly,4 Donglu Zhang,4 Carrie Xu,4 W. Griffith Humphreys,4 Hayley McDaid,1, 2 Gary Goldberg,1, 3 Susan B. Horwitz,1, 2 andSridhar Mani 1 Abstract Purpose:To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were usedto determine the cytochrome P450 enzyme(s) involvedin the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinet- ics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepi- lone dose administration to 25 mg/m2 when comparedwith single-agent therapy of 40 mg/m 2. Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC0-1.The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral bloodmononuclear cells correlatedwith neutropenia.
    [Show full text]
  • Enabling Direct Compression Tablet Development Of
    ENABLING DIRECT COMPRESSION TABLET DEVELOPMENT OF CELECOXIB THROUGH SOLID STATE ENGINEERING A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Kunlin Wang IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Changquan Calvin Sun, Advisor August 2020 © Kunlin Wang 2020 Acknowledgements Life as a Ph.D. student is a journey filled with gratitude. For my 5 years of Ph.D. study as well as 2 years of undergraduate research at the Sun Lab, the first person I would like to thank is my advisor, Prof. Changquan Calvin Sun. During my undergraduate research in my junior and senior years, he has been a good guide and scientist who triggered my interest in pharmaceutical materials science and lead my way to pursuing a Ph.D. degree in Pharmaceutics. During my graduate study, Dr. Sun has been a great mentor in science as well as a supporter who inspire me for attitude and solutions towards life’s problems. My appreciation also goes to my committee members, Dr. Raj Suryanarayanan, Dr. Timothy Wiedmann, and Dr. Andreas Stein for reviewing my dissertation, offering insightful comments and suggestions, and serving on my Ph. D. exam committees. I have learned the power of logical thinking and analysis from Dr. Sury; the significance of practical thinking from Dr. Wiedmann and the power of getting the big picture from Dr. Stein from a different perspective. I am extremely grateful not only for their time and patience, but for their trust in me as they offer me support in my development as a scientist.
    [Show full text]
  • Medicinova Receives Notice of Allowance for Second Patent Covering MN-166 (Ibudilast) for the Treatment of Glioblastoma
    MediciNova Receives Notice of Allowance for Second Patent Covering MN-166 (ibudilast) for the Treatment of Glioblastoma April 20, 2020 LA JOLLA, Calif., April 20, 2020 (GLOBE NEWSWIRE) -- MediciNova, Inc., a biopharmaceutical company traded on the NASDAQ Global Market (NASDAQ:MNOV) and the JASDAQ Market of the Tokyo Stock Exchange (Code Number: 4875), today announced that it has received a Notice of Allowance from the U.S. Patent and Trademark Office for a pending patent application which covers MN-166 (ibudilast) for the treatment of glioblastoma. This new patent has improved therapeutic claims compared to the first patent which covers MN-166 (ibudilast) for the treatment of glioblastoma, which was granted last year, and has a later expiration date than the first patent. Once issued, the patent maturing from this allowed patent application is expected to expire no earlier than February 2039. The allowed claims cover a method of treating a patient diagnosed with glioblastoma or recurrent glioblastoma, wherein the patient expresses methylated MGMT (O6-methylguanine-DNA methyltransferase), using MN-166 (ibudilast) in combination with one or more other therapeutic agents including temozolomide (TMZ), carmustine, bevacizumab, procarbazine, hydroxyurea, irinotecan, lomustine, nimotuzumab, sirolimus, mipsagargin, cabozantinib, onartuzumab, patupilone (epothilone B), and recombinant oncolytic poliovirus (PVS-RIPO). The allowed claims cover a wide range of doses of MN-166 (ibudilast) during an optionally repeating dosing cycle. The allowed claims also cover different types of glioblastoma including classical glioblastoma, proneural glioblastoma, mesenchymal glioblastoma, and neural glioblastoma. Yuichi Iwaki, MD, PhD, President and Chief Executive Officer of MediciNova, Inc., commented, "We are very pleased to receive notice that this new patent will be granted as it offers better coverage than our first patent covering glioblastoma.
    [Show full text]
  • Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/Mtor Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance
    www.impactjournals.com/oncotarget/ Oncotarget, October, Vol.3, No 10 Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance James A. McCubrey1, Linda S. Steelman1, William H. Chappell1, Stephen L. Abrams1, Richard A. Franklin1, Giuseppe Montalto2, Melchiorre Cervello3, Massimo Libra4, Saverio Candido4, Grazia Malaponte4, Maria C. Mazzarino4, Paolo Fagone4, Ferdinando Nicoletti4, Jörg Bäsecke5, Sanja Mijatovic6, Danijela Maksimovic- Ivanic6, Michele Milella7, Agostino Tafuri8, Francesca Chiarini9, Camilla Evangelisti9, Lucio Cocco10, Alberto M. Martelli9,10 1 Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA 2 Department of Internal Medicine and Specialties, University of Palermo, Palermo, Italy 3 Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, Palermo, Italy 4 Department of Bio-Medical Sciences, University of Catania, Catania, Italy 5 Department of Medicine, University of Göttingen, Göttingen, Germany 6 Department of Immunology, Instititue for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia 7 Regina Elena National Cancer Institute, Rome, Italy 8 Sapienza, University of Rome, Department of Cellular Biotechnology and Hematology, Rome, Italy 9 Institute of Molecular Genetics, National Research Council-Rizzoli Orthopedic Institute, Bologna, Italy. 10 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy Correspondence to: James A. McCubrey, email: [email protected] Keywords: Targeted Therapy, Therapy Resistance, Cancer Stem Cells, Raf, Akt, PI3K, mTOR Received: September 12, 2012, Accepted: October 18, 2012, Published: October 20, 2012 Copyright: © McCubrey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    [Show full text]
  • Interaction of Epothilone Analogs with the Paclitaxel Binding Site: Relationship Between Binding Affinity, Microtubule Stabilization, and Cytotoxicity
    Chemistry & Biology, Vol. 11, 225–236, February, 2004, 2004 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.chembiol.2004.01.014 Interaction of Epothilone Analogs with the Paclitaxel Binding Site: Relationship between Binding Affinity, Microtubule Stabilization, and Cytotoxicity Rube´ n M. Buey,1 J. Fernando Dı´az,1,* sic microtubule-disrupting drugs (colchicine, vinblas- Jose´ M. Andreu,1 Aurora O’Brate,2 tine) block microtubule assembly by binding to the unas- Paraskevi Giannakakou,2 K.C. Nicolaou,3 sembled tubulin dimers and inactivating them, paclitaxel Pradip K. Sasmal,3 Andreas Ritze´ n,3 and docetaxel bind to microtubules, stabilizing them and Kenji Namoto3 and blocking their dynamics [2, 3]. Nevertheless, despite 1Centro de Investigaciones Biolo´ gicas their good activity against ovarian, metastatic breast, Consejo Superior de Investigaciones Cientı´ficas head and neck, and lung cancer [4], paclitaxel has two Ramiro de Maeztu 9 factors that hamper its applicability. First, its low aque- 28040 Madrid ous solubility, and second, the development of pleiotro- Spain pic drug resistance mediated both by the overexpres- 2 Winship Cancer Institute sion of the P-glycoprotein [5, 6] and the presence of Emory University School of Medicine mutations in ␤-tubulin [7, 8]. Atlanta, Georgia 30322 The discovery in recent years of several natural sub- 3 Department of Chemistry and stances with a paclitaxel-like mechanism of action The Skaggs Institute for Chemical Biology (epothilone, discodermolide, laulimalide, eleutherobin, The Scripps Research Institute peloruside, dictyostatin-1, taccalonolide, and jatro- 10550 North Torrey Pines Road phane polyesters; [9–16]) opened new possibilities in La Jolla, California 92037 the field.
    [Show full text]
  • Systemic Epothilone D Improves Hindlimb Function After Spinal Cord Contusion Injury In
    ACCEPTED MANUSCRIPT Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats Beatrice Sandner1 PhD, Radhika Puttagunta1 PhD, Melanie Motsch1, Frank Bradke2 PhD, Jörg Ruschel2 PhD, Armin Blesch1, 3 PhD., Norbert Weidner1, * MD 1Spinal Cord Injury Center, Heidelberg University Hospital, Schlierbacher Landstrasse 200 a, 69118 Heidelberg, Germany 2Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Ludwig- Erhard-Allee 2, 53175 Bonn, Germany 3 Stark Neurosciences Research Institute, Indiana University School of Medicine Department of Neurological Surgery and Goodman Campbell Brain and Spine, 320 West 15th Street, NB 500B, Indianapolis, IN 46202 *Corresponding author: Norbert Weidner, MD Spinal Cord Injury Center Heidelberg UniversityACCEPTED Hospital MANUSCRIPT Schlierbacher Landstrasse 200a 69118 Heidelberg Germany Tel.: +49-6221-5626322 Fax: +49-6221-5626345 ___________________________________________________________________Email: [email protected] This is the author's manuscript of the article published in final edited form as: Sandner, B., Puttagunta, R., Motsch, M., Bradke, F., Ruschel, J., Blesch, A., & Weidner, N. (2018). Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats. Experimental Neurology. https://doi.org/10.1016/j.expneurol.2018.01.018 ACCEPTED MANUSCRIPT Abstract Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery.
    [Show full text]
  • Natural Products. a History of Success and Continuing Promise for Drug Discovery and Development
    Natural Products. A History of Success and Continuing Promise for Drug Discovery and Development Gordon M. Cragg NIH Special Volunteer [email protected] David J. Newman Natural Products Branch Developmental Therapeutics Program National Cancer Institute EARLY DOCUMENTATION OF USE OF MEDICINAL PLANTS http://www.nlm.nih.gov/hmd/collections/archives/index.html • Mesopotamian ~2,600 B. C. E. • Egyptian ~ 1,800 B. C. E. • Chinese – ~1,100 B. C. E. and continuing • Indian ~ 1,000 B. C. E. and continuing • Greek ~ 500 B. C. E. Greco-Roman expertise preserved and coordinated with other traditions by Islamic cultures during the Dark Ages ~ 400-1,100 CE Avicenna. Persian pharmacist, physician, poet, philosopher author: canon medicinae – “final codification of Greco-Roman medicine” Great Moments in Pharmacy Collection APhA Traditional Medicine and Drug Discovery • 80% of the world population resides in developing countries • 80% of people in developing countries utilize plants to meet their primary health care needs • Global pop. ca. 7 billion ca. 4.5 billion people utilize plants to meet their primary health care needs Farnsworth NR, et al. Medicinal Plants in Therapy. Bull. W.H.O. 63:965-981 (1985) Fabricant and Farnsworth, EnViron. Health Perspect. 109, 69-75 (2001) Cordell and Clovard, J. Nat. Prod., 75, 514-525 (2012) Norman Farnsworth 1800s. Discovery of some active principles of major herbal preparations Newman and Cragg. Natural Product Chemistry for Drug Discovery, eds. Buss and Butler, M. S., Royal Soc. Chem., Cambridge, 2010, pp. 3-27 European chemists (apothecaries) revolutionized drug discovery and development. 1817. Sertϋrner reports isolation of morphine from Papaver somniferum.
    [Show full text]
  • Mechanisms to the Human in Life Body
    FROM DEFENSE BACTERIA MECHANISMS TO THE HUMAN IN LIFE BODY FOURTEENTH ANNUAL LSI SYMPOSIUM MAY 21, 2015 Images courtesy of Katherine D. Walton, research investigator, and Deborah Gumucio, professor, Department of Cell and Developmental Biology, U-M Medical School SCHEDULE FORUM HALL, PALMER COMMONS (OVERFLOW SEATING AVAILABLE IN GREAT LAKES NORTH) 8:30 A.M. 1:15 P.M. WELCOME | Alan Saltiel, Ph.D. ROLE OF THE MICROBIOTA IN INFECTION CONTROL Mary Sue Coleman Director of the Life Sciences Institute AND SEQUELAE | Yasmine Belkaid, Ph.D. Chief of Mucosal Immunology Section, Laboratory of 8:35 A.M. Parasitic Diseases, National Institutes of Health, National INTRODUCTION OF THE MARY SUE AND KENNETH Institute of Allergy and Infectious Diseases COLEMAN LIFE SCIENCES LECTURER | Mary Sue Coleman, Ph.D. 1:55 P.M. President Emerita IMMUNE REGULATION OF INTESTINAL HEALTH AND DISEASE | Gregory F. Sonnenberg, Ph.D. 8:50 A.M. Assistant Professor of Microbiology and Immunology in MARY SUE AND KENNETH COLEMAN LIFE SCIENCES Medicine, Weill Cornell Medical College LECTURE: HOMEOSTASIS, INFLAMMATION AND DISEASE | Ruslan Medzhitov, Ph.D. AFTERNOON BREAK David W. Wallace Professor of Immunobiology, Yale School of Medicine; Investigator, Howard Hughes 3:15 P.M. Medical Institute TISSUE CONTROL OF MACROPHAGE HOMEOSTASIS AND FUNCTION | Miriam Merad, M.D., Ph.D. MORNING BREAK Professor of Oncological Science and Medicine; Mount Sinai Chair in Cancer Immunology; Director of Human 10:30 A.M. Immune Monitoring Center, Tisch Cancer Institute, Icahn GENERATION OF A MEMORY OF INFECTION DURING School of Medicine at Mount Sinai CRISPR-CAS IMMUNITY | Luciano Marraffini, Ph.D. Assistant Professor, head of the Laboratory of 3:55 P.M.
    [Show full text]
  • WO 2013/179310 Al 5 December 2013 (05.12.2013) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/179310 Al 5 December 2013 (05.12.2013) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07C 69/76 (2006.01) C07C 69/95 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/IN20 13/000346 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR, 3 1 May 2013 (3 1.05.2013) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 2186/CHE/2012 31 May 2012 (3 1.05.2012) IN (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: MYLAN LABORATORIES LIMITED [IN/ GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, —]; Plot No 564/A/22, Road No 92, Jubilee Hills, Hydera UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, bad 500 034 (IN).
    [Show full text]
  • Patent-Lawyer-Article
    The GLOBAL REACH, LOCAL KNOWLEDGE www.patentlawyermagazine.com Annual 2021 COPYRIGHT CTC LEGAL MEDIA The effect of design space on patent grant and recognition for designs Dr. Yongqiang Qi, Partner and Patent Attorney at Corner Stone, examines the latest judicial interpretation and what it means for design. Protect Patent validity against terms crises Page 56 Page 13 AI patenting Page 18 FFrontront ccover_TPL51_v2aover_TPL51_v2a Alternative.inddAlternative.indd 1 118/12/20208/12/2020 110:060:06 CRIPSR-Cas9 A Nobel Prize, a Global Pandemic, and a Patent Dispute walk into a bar… stop me if you’ve heard this one before Richard Gaugeler, Patent Attorney at Cedar White Bradley, explains how a Noble Prize, the Pandemic and a Patent Dispute are all inextricably linked to the CRISPR-Cas9 Technology. ust as the three individuals who walk into a The functioning of the CRISPR-Cas9 technology bar seemingly appear independent from is as follows: First, the faulty sequence in the Jone another, they nevertheless always turn DNA is identified by a scientist. In this case the out to be inextricably linked through some faulty sequence refers to a defective gene common thread. And our Nobel Prize, Pandemic, which codes for Sickle-Cell anaemia. Second, and Dispute are no different. The thread? Of course, CRISPR uses guideRNA to identify, and bind to I must be talking about CRISPR-Cas9 Technology. the sequence. The guideRNA binds to and The revolutionary gene editing tool that can be used unravels the faulty sequence in the DNA molecule. to make precise incisions in genetic material to Third, Cas9 cuts the faulty sequence to either edit or even delete unwanted genetic code.
    [Show full text]
  • A Common Pharmacophore for Epothilone and Taxanes: Molecular Basis for Drug Resistance Conferred by Tubulin Mutations in Human Cancer Cells
    A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells Paraskevi Giannakakou*†, Rick Gussio‡, Eva Nogales§, Kenneth H. Downing§, Daniel Zaharevitz‡, Birgit Bollbuck¶, George Poyʈ, Dan Sackett**, K. C. Nicolaou¶, and Tito Fojo* *Medicine Branch, National Cancer Institute, and ʈGenetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; ‡Target Structure-Based Drug Discovery Group, Information Technology Branch, and **Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, National Cancer Institute, National Institutes of Health, Frederick, MD 21702; §Life Science Division, Lawrence Berkeley National Laboratory, and Molecular and Cell Biology Department, University of California, Berkeley, CA 94720; and ¶Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 Contributed by Kyriacos C. Nicolaou, December 15, 1999 The epothilones are naturally occurring antimitotic drugs that The PTX-binding site on ␤-tubulin has been identified both share with the taxanes a similar mechanism of action without by electron crystallography, showing PTX bound on the of apparent structural similarity. Although photoaffinity labeling ␣␤-tubulin dimer (10), and by photoaffinity labeling, which has and electron crystallographic studies have identified the taxane- identified amino acids ␤1–31 and ␤217–233 as important areas binding site on ␤-tubulin, similar data are not available for for PTX binding (11, 12). Similar studies, however, for epothi- epothilones. To identify tubulin residues important for epothi- lones are not available. In this study, we present a report of lone binding, we have isolated two epothilone-resistant human epothilone-resistant (EpoR) human cancer cell lines with ovarian carcinoma sublines derived in a single-step selection acquired ␤-tubulin mutations.
    [Show full text]