DOCSLIB.ORG

Chapter I: Introduction

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Chapter I: Introduction DOCTORAL SCHOOL IN BIOLOGY Biology Applied to Human Health XXVII Cycle DUAL RNA-SEQUENCING APPROACH FOR DISSECTING NONTYPEABLE HAEMOPHILUS INFLUENZAE AND HOST CELL TRANSCRIPTOMES Presented by: Buket Baddal Supervisors: Dr. Alfredo Pezzicoli Dr. Marco Soriani Director: Prof. Paolo Visca I dedicate this thesis to my beloved parents who inspired and supported me unconditionally in countless ways. II ABSTRACT Characterization of host-pathogen interactions is critical for the development of next-generation therapies and vaccines. Classical approaches involve the use of transformed cell lines and/or animal models which may not reflect the complexity and response of the human host. The Gram-negative bacterium nontypeable Haemophilus influenzae (NTHi) commonly resides as a commensal in the human nasopharynx from where it can disseminate to local organs to cause a wide spectrum of diseases including otitis media, chronic obstructive pulmonary disease, cystic fibrosis and bronchitis. Successful colonization by NTHi depends on its ability to adhere and adapt to the respiratory tract mucosa, which serves as a frontline defense against respiratory pathogens. In opportunistic infections, colonization is followed by either a paracellular route across the epithelial barrier or invasion of non-phagocytic and epithelial cells. However, the temporal events associated to a successful colonization are far from being fully characterized. Recent improvements in tissue engineering techniques including the development of differentiated primary cell cultures and organotypic 3D cellular models have significantly increased our understanding of microbial pathogenesis by providing physiologically relevant representations of human upper airway tissue. Bridging of these techniques with the currently available next-generation sequencing technologies is a conceptually novel approach for studying infection-linked transcriptome alterations in such systems. Massively parallel cDNA sequencing (RNA-seq) offers the possibility of comprehensive and simultaneous whole genome transcriptional profiling of both host and invading pathogen, and overcomes the existing technical and economical limitations of probe-dependent methods. Taking advantage of the technological advances, we reconstituted the ciliated human bronchial epithelium in vitro using primary bronchial epithelial cells to simultaneously monitor the infection-linked global changes in NTHi and infected host epithelia gene expression by dual RNA-seq. Acquisition of a total of nearly 2,5 billion sequences allowed construction of high-resolution strand-specific transcriptome maps of NTHi during infection of host mucosal surface, and monitoring of metabolic as well as stress-induced host-adaptation strategies of this pathogen. The initial stage of colonization was characterized by the binding of NTHi to cilia. Temporal analysis of host mRNA signatures revealed consequent remodeling of target cell cytoskeleton and junction complexes elicited by bacterial infection, with a profound effect on intermediate filament network of bronchial epithelium. At later stage of infection when bacteria start to internalize, NTHi down-regulated the central metabolism and increased the expression of transporters indicating alterations in the bacterial metabolic regime due to the evolving substrate availability. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms including the transport of exogenous glutathione and the activation of the toxin-antitoxin system. Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs regulated during human host infection in Haemophilus species. Our data by providing a robust and comprehensive catalogue of regulatory and adaptive responses reflecting the complex crosstalk between the host and invading pathogen, may provide important insights into NTHi pathogenesis and the development of efficacious preventive strategies. tissue engineering | host-pathogen interaction | dual RNA-seq | NTHi transcriptome | host response III RIASSUNTO La caratterizzazione delle interazioni ospite-patogeno è fondamentale per lo sviluppo di nuove terapie e vaccini di ultima generazione. Approcci classici prevedono l'utilizzo di linee cellulari trasformate e/o modelli animali che non possono riflettere la complessità e la risposta dell'ospite umano. Il batterio Gram-negativo nontypeable Haemophilus influenzae (NTHi) risiede comunemente come commensale nel rinofaringe umano da dove può diffondere ai tessuti adiacenti provocando un ampio spettro di malattie, tra cui otite media, COPD, fibrosi cistica e bronchite. La capacità di colonizzazione di NTHi dipende dalla sua abilità di aderire ed adattarsi alla mucosa delle vie respiratorie, che ha la funzione di prima linea difensiva contro gli agenti patogeni del tratto respiratorio. Durante le infezioni opportunistiche, la colonizzazione è seguita da un attraversamento paracellulare della barriera epiteliale o dall’invasione di cellule non fagocitiche ed epiteliali. Tuttavia gli eventi temporali associati ad una colonizzazione produttiva non sono stati ancora completamente caratterizzati. Recenti miglioramenti delle tecniche di ingegneria tissutale in vitro, tra cui lo sviluppo di colture cellulari primarie differenziate e modelli cellulari 3D organotipici, hanno aumentato significativamente la nostra comprensione della patogenesi microbica fornendo rappresentazioni fisiologicamente rilevanti di tessuto delle vie aeree superiori umani. La combinazione di queste tecniche con le tecnologie di sequenziamento di nuova generazione è un approccio concettualmente innovativo per studiare le alterazioni del trascrittoma durante un’infezione. Il sequenziamento tramite RNA-seq offre la possibilità di analizzare simultaneamente il trascrittoma di ospite e patogeno, superando le limitazioni tecniche relative alla separazione dei campioni eucariotici da quelli procariotici. In questo studio l'epitelio ciliato bronchiale umano è stato ricostituito in vitro utilizzando cellule epiteliali bronchiali primarie ed usato come modello di infezione per NTHi. Successivamente, tramite dual-RNA sequencing, sono state analizzate le variazioni dell’espressione genica di entrambi ospite e patogeno. L’acquisizione di un totale di quasi 2,5 miliardi di sequenze ha permesso la costruzione di mappe ad alta risoluzione del trascrittoma di NTHi durante l'infezione dell’epitelio, la caratterizzazione dell’adattamento metabolico nonché dei pathways di adattamento allo stress cellulare. Nella fase iniziale della colonizzazione è stata osservata l’adesione di NTHi alle cellule ciliate. L’analisi temporale del trascrittoma dell’epitelio ha evidenziato il rimodellamento del citoscheletro, in particolare come riarrangiamento della rete dei filamenti intermedi. Nelle fasi successive dell’infezione, quando i batteri iniziano ad essere internalizzati, NTHi down- regola il metabolismo centrale e aumenta l'espressione di trasportatori specifici per alcuni substrati disponibili nel nuovo ambiente. Contemporaneamente l'ambiente ossidativo generato dalle cellule infettate induce l’espressione batterica di meccanismi di difesa dallo stress fra cui il trasporto di glutatione esogeno e l'attivazione dei sistemi tossina/anti-tossina. Abbiamo inoltre identificato una serie di nuovi small RNAs batterici fortemente regolati durante l'infezione. Questo lavoro fornisce la caratterizzazione dei trascrittomi dell’ospite e del patogeno nelle prime fasi d’infezione, e suggerisce lo sviluppo di strategie mirate per prevenire le infezioni di NTHi. ingegneria tissutale | interazione ospite-patogeno | dual RNA-seq | NTHi trascrittoma | risposta dell'ospite IV ACKNOWLEDGEMENTS This work was conducted at Novartis Vaccines, Siena in In Vitro Cell Biology Group, headed by Dr. Marco Soriani. I am very thankful for his welcoming in the lab, providing me with the opportunity to work at the frontier of current vaccine development and cutting-edge technologies, but most importantly for always being there to help. I equally thank Prof. Christoph Tang who has awarded me with Marie Curie Fellowship and allowed me to perform my thesis project under the framework of European Community's Seventh Framework Programme “EIMID ITN” [European Institute of Microbiology and Infectious Diseases Initial Training Network, FP7-PEOPLE-2010-264388], as well as his enriching collaboration and hosting me in this laboratory in University of Oxford. I am extremely grateful to my supervisor, Dr. Alfredo Pezzicoli for his guidance over the course of my PhD, for his never-wavering optimism, giving me the initiative to grow as an independent research scientist and above all, for teaching me how much fun microscopy can be. I also thank Prof. Paolo Visca from University of Roma Tre for all the support and valuable advice he has given me during my PhD. I would like to acknowledge all my lab members (past and present) of the In Vitro Cell Biology group at Novartis, particularly Lucia Lapazio, Maria Valeri, Pasquale Marrazzo, Silvia Rossi Paccani and Alessandra Greco for the friendly atmosphere they created in the lab, their scientific feedback, and moral support during tough days. I am especially grateful to my precious friends Christina Merakou, Tanja Dapa, Cristina Faralla, Magdalena Kasendra, Riccardo Barrile and Giacomo Golfieri for their kindhearted help and care, all the fun we had together, keeping
Load more

Recommended publications
  • Screening and Identification of Key Biomarkers in Clear Cell Renal Cell Carcinoma Based on Bioinformatics Analysis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Screening and identification of key biomarkers in clear cell renal cell carcinoma based on bioinformatics analysis Basavaraj Vastrad1, Chanabasayya Vastrad*2 , Iranna Kotturshetti 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. 3. Department of Ayurveda, Rajiv Gandhi Education Society`s Ayurvedic Medical College, Ron, Karnataka 562209, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignancy of the urinary system. The pathogenesis and effective diagnosis of ccRCC have become popular topics for research in the previous decade. In the current study, an integrated bioinformatics analysis was performed to identify core genes associated in ccRCC. An expression dataset (GSE105261) was downloaded from the Gene Expression Omnibus database, and included 26 ccRCC and 9 normal kideny samples. Assessment of the microarray dataset led to the recognition of differentially expressed genes (DEGs), which was subsequently used for pathway and gene ontology (GO) enrichment analysis.
    [Show full text]
  • Namely, Aspartokinase and Aspartate 3-Semialdehyde Dehydrogenase) Are Not Subject to Feedback Inhibition Control by the End Product Methionine
    REGULATION OF HOMOSERINE BIOSYNTHESIS BY L-CYSTEINE, A TERMINA L METABOLITE OF A LINKED PA THWA Y* By PRASANTA DATTA DEPARTMENT OF BIOLOGICAL CHEMISTRY, UNIVERSITY OF MICHIGAN, ANN ARBOR Communicated by Martin D. Kamen, June 23, 1967 In bacteria, the amino acid homoserine is a key branch-point intermediate in the synthesis of several amino acids of the aspartic pathway.' On the one hand, homoserine is converted to threonine (and thus to isoleucine), and through a separate sequence of reactions is transformed to methionine.1 In the latter se- quence, a succinylated product of homoserine is condensed with cysteine to pro- duce cystathionine,2-5 a precursor of methionine. Recent studies (see refs. 6 and 7 for up-to-date reviews on the subject) with several microorganisms have revealed that the synthesis of homoserine is regulated by various combinations of repression and/or feedback inhibition controls of early biosynthetic enzymes of the aspartic pathway by several end-product metabolites. One of these enzymes, homoserine dehydrogenase, catalyzes the pyridine nucleotide-linked reduction of aspartate j3-semialdehyde to homoserine. In the various bacteria that have been examined, this dehydrogenase as well as the two earlier enzymes of the path- way (namely, aspartokinase and aspartate 3-semialdehyde dehydrogenase) are not subject to feedback inhibition control by the end product methionine. This report describes the results of experiments on the control of activity of several bacterial homoserine dehydrogenases by L-cysteine, a terminal metabolite of a linked or connecting pathway, required for the synthesis of cystathionine.2-5 The findings suggest that the size of the homoserine pool in bacterial cells must depend, at least in part, on complex interdependent regulatory interactions of both cysteine and threonine on homoserine dehydrogenase.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002
    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
    [Show full text]
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [Show full text]
  • Miasdb: a Database of Molecular Interactions Associated with Alternative Splicing of Human Pre-Mrnas
    RESEARCH ARTICLE MiasDB: A Database of Molecular Interactions Associated with Alternative Splicing of Human Pre-mRNAs Yongqiang Xing1, Xiujuan Zhao1, Tao Yu2, Dong Liang1, Jun Li1, Guanyun Wei1, Guoqing Liu1, Xiangjun Cui1, Hongyu Zhao1, Lu Cai1* 1 School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, 014010, China, 2 School of Science, Inner Mongolia University of Science and Technology, Baotou, 014010, China a11111 * [email protected] Abstract Alternative splicing (AS) is pervasive in human multi-exon genes and is a major contributor to expansion of the transcriptome and proteome diversity. The accurate recognition of alter- OPEN ACCESS native splice sites is regulated by information contained in networks of protein-protein and Citation: Xing Y, Zhao X, Yu T, Liang D, Li J, Wei G, protein-RNA interactions. However, the mechanisms leading to splice site selection are not et al. (2016) MiasDB: A Database of Molecular fully understood. Although numerous databases have been built to describe AS, molecular Interactions Associated with Alternative Splicing of Human Pre-mRNAs. PLoS ONE 11(5): e0155443. interaction databases associated with AS have only recently emerged. In this study, we doi:10.1371/journal.pone.0155443 present a new database, MiasDB, that provides a description of molecular interactions Editor: Ruben Artero, University of Valencia, SPAIN associated with human AS events. This database covers 938 interactions between human splicing factors, RNA elements, transcription factors, kinases and modified histones for 173 Received: November 19, 2015 human AS events. Every entry includes the interaction partners, interaction type, experi- Accepted: April 28, 2016 mental methods, AS type, tissue specificity or disease-relevant information, a simple Published: May 11, 2016 description of the functionally tested interaction in the AS event and references.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Joseph M. Jez • Department of Biology • Washington University in St
    Joseph M. Jez • Department of Biology • Washington University in St. Louis Joseph M. Jez Professor and Howard Hughes Medical Institute Professor Department of Biology, Washington University in St. Louis One Brookings Drive, Campus Box 1137, St. Louis, MO 63130-4899 Phone: 314-935-3376; Email: [email protected] –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– EDUCATION University of Pennsylvania, Philadelphia, PA Ph.D. Biochemistry & Molecular Biophysics, 1998 Thesis: Steroid Recognition and Engineering of Catalysis in Mammalian Aldo-Keto Reductases Research Advisor: Prof. Trevor M. Penning Penn State University, University Park, PA B.S. Biochemistry (with Honors and English minor), 1992 Research Advisor: Prof. Gregory K. Farber PROFESSIONAL EXPERIENCE Washington University in St. Louis, St. Louis, MO Professor, Department of Biology (2015-current) Co-Director, Plant and Microbial Biosciences Program, Division of Biology & Biomedical Sciences (2013-current) Associate Professor, Department of Biology (2011-2015) Assistant Professor, Department of Biology (2008-2011) Honorary Assistant Professor, Department of Biology (2006-2008) Donald Danforth Plant Science Center, St. Louis, MO Assistant Member & Principal Investigator (2002-2010) Kosan Biosciences, Hayward, CA Scientist, New Technology Group (2001-2002) Supervisor: Dr. Daniel V. Santi The Salk Institute for Biological Studies, La Jolla, CA NIH-NRSA Postdoctoral Research Fellow, Structural Biology Laboratory (1998-2001) Project: Structure, Mechanism,
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • 1 AGING Supplementary Table 2
    SUPPLEMENTARY TABLES Supplementary Table 1. Details of the eight domain chains of KIAA0101. Serial IDENTITY MAX IN COMP- INTERFACE ID POSITION RESOLUTION EXPERIMENT TYPE number START STOP SCORE IDENTITY LEX WITH CAVITY A 4D2G_D 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ B 4D2G_E 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ C 6EHT_D 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ D 6EHT_E 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ E 6GWS_D 41-72 41 72 100 100 3.2Å PCNA X-RAY DIFFRACTION √ F 6GWS_E 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ G 6GWS_F 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ H 6IIW_B 2-11 2 11 100 100 1.699Å UHRF1 X-RAY DIFFRACTION √ www.aging-us.com 1 AGING Supplementary Table 2. Significantly enriched gene ontology (GO) annotations (cellular components) of KIAA0101 in lung adenocarcinoma (LinkedOmics). Leading Description FDR Leading Edge Gene EdgeNum RAD51, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, CENPW, HJURP, NDC80, CDCA5, NCAPH, BUB1, ZWILCH, CENPK, KIF2C, AURKA, CENPN, TOP2A, CENPM, PLK1, ERCC6L, CDT1, CHEK1, SPAG5, CENPH, condensed 66 0 SPC24, NUP37, BLM, CENPE, BUB3, CDK2, FANCD2, CENPO, CENPF, BRCA1, DSN1, chromosome MKI67, NCAPG2, H2AFX, HMGB2, SUV39H1, CBX3, TUBG1, KNTC1, PPP1CC, SMC2, BANF1, NCAPD2, SKA2, NUP107, BRCA2, NUP85, ITGB3BP, SYCE2, TOPBP1, DMC1, SMC4, INCENP. RAD51, OIP5, CDK1, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, ESCO2, CENPW, HJURP, TTK, NDC80, CDCA5, BUB1, ZWILCH, CENPK, KIF2C, AURKA, DSCC1, CENPN, CDCA8, CENPM, PLK1, MCM6, ERCC6L, CDT1, HELLS, CHEK1, SPAG5, CENPH, PCNA, SPC24, CENPI, NUP37, FEN1, chromosomal 94 0 CENPL, BLM, KIF18A, CENPE, MCM4, BUB3, SUV39H2, MCM2, CDK2, PIF1, DNA2, region CENPO, CENPF, CHEK2, DSN1, H2AFX, MCM7, SUV39H1, MTBP, CBX3, RECQL4, KNTC1, PPP1CC, CENPP, CENPQ, PTGES3, NCAPD2, DYNLL1, SKA2, HAT1, NUP107, MCM5, MCM3, MSH2, BRCA2, NUP85, SSB, ITGB3BP, DMC1, INCENP, THOC3, XPO1, APEX1, XRCC5, KIF22, DCLRE1A, SEH1L, XRCC3, NSMCE2, RAD21.
    [Show full text]
  • Of Pseudomonas Aeruginosa Cristian Gustavo Aguilera Rossi1,2, Paulino Gómez-Puertas3 and Juan Alfonso Ayala Serrano2*
    Aguilera Rossi et al. BMC Microbiology (2016) 16:234 DOI 10.1186/s12866-016-0853-x RESEARCH ARTICLE Open Access In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa Cristian Gustavo Aguilera Rossi1,2, Paulino Gómez-Puertas3 and Juan Alfonso Ayala Serrano2* Abstract Background: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now. Results: We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM- PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate.
    [Show full text]
  • Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention?
    cancers Review Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention? Silvia Pietrobono * and Barbara Stecca * Tumor Cell Biology Unit, Core Research Laboratory, Institute for Cancer Research and Prevention (ISPRO), Viale Pieraccini 6, 50139 Florence, Italy * Correspondence: [email protected] (S.P.); [email protected] (B.S.); Tel.: +39-055-7944568 (S.P.); +39-055-7944567 (B.S.) Simple Summary: Sialylation is a post-translational modification that consists in the addition of sialic acid to growing glycan chains on glycoproteins and glycolipids. Aberrant sialylation is an established hallmark of several types of cancer, including breast, ovarian, pancreatic, prostate, colorectal and lung cancers, melanoma and hepatocellular carcinoma. Hypersialylation can be the effect of increased activity of sialyltransferases and results in an excess of negatively charged sialic acid on the surface of cancer cells. Sialic acid accumulation contributes to tumor progression by several paths, including stimulation of tumor invasion and migration, and enhancing immune evasion and tumor cell survival. In this review we explore the mechanisms by which sialyltransferases promote cancer progression. In addition, we provide insights into the possible use of sialyltransferases as biomarkers for cancer and summarize findings on the development of sialyltransferase inhibitors as potential anti-cancer treatments. Abstract: Sialylation is an integral part of cellular function, governing many biological processes Citation: Pietrobono, S.; Stecca, B. including cellular recognition, adhesion, molecular trafficking, signal transduction and endocytosis. Aberrant Sialylation in Cancer: Sialylation is controlled by the levels and the activities of sialyltransferases on glycoproteins and Biomarker and Potential Target for lipids. Altered gene expression of these enzymes in cancer yields to cancer-specific alterations of Therapeutic Intervention? Cancers glycoprotein sialylation.
    [Show full text]
  • Histidinol Dehydrogenase from Neurospora Crassa
    Fungal Genetics Reports Volume 3 Article 1 Histidinol dehydrogenase from Neurospora crassa E. H. Creaser R. B. Drysdale Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Creaser, E. H., and R.B. Drysdale (1963) "Histidinol dehydrogenase from Neurospora crassa," Fungal Genetics Reports: Vol. 3, Article 1. https://doi.org/10.4148/1941-4765.2139 This Research Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact [email protected]. Histidinol dehydrogenase from Neurospora crassa Abstract Histidinol dehydrogenase from Neurospora crassa This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol3/iss1/1 RESEARCH NOTES Creaser, E.H. and R. B. Drysdale. Histidinol It is thought that the locus histidine-3 controls hvo --__ dehydrogenase from Neurosooro w. functions in Neurospora, the first of these being in the early stages ot histidine biosynthesis and largely unknown at present. The second function is to direct the formation of the terminal enzyme in the sequence- histidinol dehydrogenase. We have studied the purification and some properties of this enzyme. The enzyme con be extracted from wet mycelium by grinding with gloss powder or from dried mycelium by extraction with pH 9. I Tris buffer. The extract is treated with 0.05M MnC12 to prec:pitate nucleic acids and unwonted proteins. Ammonium sulphate is added to 50% saturation and the precipitate discorded.
    [Show full text]