DOCSLIB.ORG

Identification of an Unusual Glycosyltransferase from a Non

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of an Unusual Glycosyltransferase from a Non Identification of an unusual glycosyltransferase from a non-cultivated microorganism and the construction of an improved Escherichia coli strain harboring the rpoD gene from Clostridium cellulolyticum for metagenome searches Dissertation Zur Erlangung der Würde des Doktors der Naturwissenschaften des Fachbereichs Biologie, der Fakultät für Mathematik, Informatik und Naturwissenschaften, der Universität Hamburg vorgelegt von Julia Jürgensen aus Henstedt-Ulzburg Hamburg 2015 Table of contents I Table of contents 1 Introduction ...................................................................................................... 1 1.1 Flavonoids................................................................................................... 1 1.2 Glycosyltransferases ................................................................................... 3 1.3 Biotechnology ............................................................................................. 4 1.3.1 Biotechnological relevance of glycosyltranferases....................................... 5 1.4 Metagenomics ............................................................................................. 5 1.5 Transcription ............................................................................................... 7 1.6 Phyla ........................................................................................................... 8 1.6.1 Proteobacteria ............................................................................................. 8 1.6.2 Firmicutes ................................................................................................... 9 1.6.2.1 Clostridium cellulolyticum ............................................................................ 9 1.7 Intention of this work ................................................................................... 9 2 Material & Methods ........................................................................................ 10 2.1 Bacterial strains, vectors, primers and constructs...................................... 10 2.2 Media and supplements ............................................................................ 15 2.2.1 Antibiotics and other supplements ............................................................. 16 2.2.2 LB Medium (Sambrook 2001).................................................................... 16 2.2.3 CM3 Medium for Clostridium cellulolyticum ............................................... 16 2.3 Samples and metagenomic libraries.......................................................... 17 2.3.1 Environmental samples ............................................................................. 17 2.3.1.1 Elephant feces .......................................................................................... 18 2.3.1.2 Elbe river sediment ................................................................................... 18 2.3.2 Metagenomic libraries ............................................................................... 18 2.4 Culture conditions ..................................................................................... 18 2.4.1 Cultivation of bacterial strains ................................................................... 18 2.4.2 Determination of cell density ..................................................................... 18 2.4.3 Cell harvesting .......................................................................................... 19 2.4.4 Strain maintenance ................................................................................... 19 2.5 Microscopy ................................................................................................ 19 2.5.1 Standard microscopy ................................................................................ 19 2.6 DNA purification ........................................................................................ 19 2.6.1 Isolation of genomic DNA .......................................................................... 19 2.6.1.1 Isolation of genomic DNA from elephant feces .......................................... 20 2.6.1.2 Isolation of genomic DNA from the Elbe river sediment ............................. 20 2.6.1.3 Isolation of genomic DNA, standard method ............................................. 21 2.6.2 Plasmid isolation “Quick and Dirty” ............................................................ 21 2.6.3 Plasmid isolation with a plasmid mini kit .................................................... 22 2.6.4 Gel extraction of DNA ............................................................................... 22 2.6.5 Purification and concentration of DNA ....................................................... 22 2.6.6 Spectrophotometrical determination of DNA concentration and purity ....... 23 Table of contents II 2.7 RNA purification ........................................................................................ 23 2.7.1 Isolation of total RNA ................................................................................ 23 2.7.2 Purification and concentration of RNA ....................................................... 23 2.7.3 Spectrophotometrical determination of RNA concentration and purity ....... 23 2.8 Agarose gel electrophoresis ...................................................................... 24 2.8.1 Agarose gel electrophoresis for DNA ........................................................ 24 2.8.2 Agarose gel electrophoresis for RNA ........................................................ 24 2.9 Polymerase chain reaction (PCR) ............................................................. 25 2.9.1 PCR primers ............................................................................................. 26 2.9.2 PCR conditions ......................................................................................... 26 2.9.3 PCR volumes ............................................................................................ 27 2.9.4 Direct colony PCR ..................................................................................... 27 2.10 Enzymatic modifications of DNA ............................................................... 27 2.10.1 Site specific digestion of DNA ................................................................... 27 2.10.2 Dephosphorylation of complementary ends............................................... 28 2.10.3 Ligation of DNA ......................................................................................... 28 2.10.3.1 Ligation of PCR products .......................................................................... 28 2.10.3.2 Ligation of fragment with digested ends .................................................... 29 2.11 Transformation .......................................................................................... 29 2.11.1 Heat shock transformation ........................................................................ 29 2.11.1.1 Heat shock transformation of E. coli .......................................................... 29 2.11.1.2 Preparation of chemically competent E. coli cells ...................................... 30 2.11.1.3 Blue-white-screening ................................................................................. 30 2.12 Genome mutation...................................................................................... 31 2.13 Sequencing of DNA ................................................................................... 31 2.13.1 ABI sequencing ......................................................................................... 31 2.13.2 454 sequencing ......................................................................................... 31 2.14 Transcriptomic analysis ............................................................................. 32 2.14.1 Next generation sequencing (Illumina) ...................................................... 32 2.15 Construction of fosmid libraries ................................................................. 32 2.15.1 End-Repair ................................................................................................ 32 2.15.2 Ligation ..................................................................................................... 33 2.15.3 Packaging of fosmid clones ....................................................................... 33 2.15.4 Preparation of phage competent cells ....................................................... 34 2.15.5 Transduction ............................................................................................. 34 2.15.6 Induction ................................................................................................... 34 2.15.7 Storage of metagenomic libraries .............................................................. 34 2.16 Protein biochemical methods .................................................................... 35 2.16.1 Induction ................................................................................................... 35 2.16.2 Preparation of crude cell extracts .............................................................. 35 2.16.3 pET vectors/His-tag affinity columns ......................................................... 36 2.16.3.1 Concentration of eluted protein ................................................................. 36 2.16.4 Protein quantification (Bradford 1967) ....................................................... 36 2.16.5 SDS-polyacrylamide gel
Load more

Recommended publications
  • Oral Ambroxol Increases Brain Glucocerebrosidase Activity in a Nonhuman Primate
    Received: 17 November 2016 | Revised: 24 January 2017 | Accepted: 12 February 2017 DOI 10.1002/syn.21967 SHORT COMMUNICATION Oral ambroxol increases brain glucocerebrosidase activity in a nonhuman primate Anna Migdalska-Richards1 | Wai Kin D. Ko2 | Qin Li2,3 | Erwan Bezard2,3,4,5 | Anthony H. V. Schapira1 1Department of Clinical Neurosciences, Institute of Neurology, University College Abstract London, NW3 2PF, United Kingdom Mutations in the glucocerebrosidase 1 (GBA1) gene are related to both Parkinson disease (PD) and 2Motac Neuroscience, Manchester, United Gaucher disease (GD). In both cases, the condition is associated with deficiency of glucocerebrosi- Kingdom dase (GCase), the enzyme encoded by GBA1. Ambroxol is a small molecule chaperone that has 3 Institute of Laboratory Animal Sciences, been shown in mice to cross the blood-brain barrier, increase GCase activity and reduce alpha- China Academy of Medical Sciences, Beijing synuclein protein levels. In this study, we analyze the effect of ambroxol treatment on GCase City, People’s Republic of China activity in healthy nonhuman primates. We show that daily administration of ambroxol results in 4University de Bordeaux, Institut des Maladies Neurodeg en eratives, UMR 5293, increased brain GCase activity. Our work further indicates that ambroxol should be investigated as Bordeaux F-33000, France a novel therapy for both PD and neuronopathic GD in humans. 5CNRS, Institut des Maladies Neurodeg en eratives, UMR 5293, Bordeaux F-33000, France KEYWORDS Correspondence ambroxol, glucocerebrosidase,
    [Show full text]
  • Fosmid Library End Sequencing Reveals a Rarely Known Genome Structure of Marine Shrimp Penaeus Monodon
    eScholarship Title Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon Permalink https://escholarship.org/uc/item/126680ch Journal BMC Genomics, 12(1) ISSN 1471-2164 Authors Huang, Shiao-Wei Lin, You-Yu You, En-Min et al. Publication Date 2011-05-17 DOI http://dx.doi.org/10.1186/1471-2164-12-242 Supplemental Material https://escholarship.org/uc/item/126680ch#supplemental Peer reviewed eScholarship.org Powered by the California Digital Library University of California Huang et al. BMC Genomics 2011, 12:242 http://www.biomedcentral.com/1471-2164/12/242 RESEARCHARTICLE Open Access Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon Shiao-Wei Huang1, You-Yu Lin1, En-Min You1, Tze-Tze Liu2, Hung-Yu Shu2, Keh-Ming Wu3, Shih-Feng Tsai3, Chu-Fang Lo1, Guang-Hsiung Kou1, Gwo-Chin Ma4, Ming Chen1,4,5, Dongying Wu6,7, Takashi Aoki8, Ikuo Hirono8 and Hon-Tsen Yu1* Abstract Background: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses.
    [Show full text]
  • Numerous Uncharacterized and Highly Divergent Microbes Which Colonize Humans Are Revealed by Circulating Cell-Free DNA
    Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA Mark Kowarskya, Joan Camunas-Solerb, Michael Kerteszb,1, Iwijn De Vlaminckb, Winston Kohb, Wenying Panb, Lance Martinb, Norma F. Neffb,c, Jennifer Okamotob,c, Ronald J. Wongd, Sandhya Kharbandae, Yasser El-Sayedf, Yair Blumenfeldf, David K. Stevensond, Gary M. Shawd, Nathan D. Wolfeg,h, and Stephen R. Quakeb,c,i,2 aDepartment of Physics, Stanford University, Stanford, CA 94305; bDepartment of Bioengineering, Stanford University, Stanford, CA 94305; cChan Zuckerberg Biohub, San Francisco, CA 94158; dDepartment of Pediatrics, Stanford University School of Medicine, Stanford University, Stanford, CA 94305; ePediatric Stem Cell Transplantation, Lucille Packard Children’s Hospital, Stanford University, Stanford, CA 94305; fDivision of Maternal–Fetal Medicine, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305; gMetabiota, San Francisco, CA 94104; hGlobal Viral, San Francisco, CA 94104; and iDepartment of Applied Physics, Stanford University, Stanford, CA 94305 Contributed by Stephen R. Quake, July 12, 2017 (sent for review April 28, 2017; reviewed by Søren Brunak and Eran Segal) Blood circulates throughout the human body and contains mole- the body (18, 19); combining this observation with the average cules drawn from virtually every tissue, including the microbes and genome sizes of a human, bacterium, and virus (Gb, Mb, and viruses which colonize the body. Through massive shotgun sequenc- kb, respectively) suggests that approximately 1% of DNA by ing of circulating cell-free DNA from the blood, we identified mass in a human is derived from nonhost origins. Previous hundreds of new bacteria and viruses which represent previously studies by us and others have shown that indeed approximately unidentified members of the human microbiome.
    [Show full text]
  • A Field Guide to Eukaryotic Transposable Elements
    GE54CH23_Feschotte ARjats.cls September 12, 2020 7:34 Annual Review of Genetics A Field Guide to Eukaryotic Transposable Elements Jonathan N. Wells and Cédric Feschotte Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850; email: [email protected], [email protected] Annu. Rev. Genet. 2020. 54:23.1–23.23 Keywords The Annual Review of Genetics is online at transposons, retrotransposons, transposition mechanisms, transposable genet.annualreviews.org element origins, genome evolution https://doi.org/10.1146/annurev-genet-040620- 022145 Abstract Annu. Rev. Genet. 2020.54. Downloaded from www.annualreviews.org Access provided by Cornell University on 09/26/20. For personal use only. Copyright © 2020 by Annual Reviews. Transposable elements (TEs) are mobile DNA sequences that propagate All rights reserved within genomes. Through diverse invasion strategies, TEs have come to oc- cupy a substantial fraction of nearly all eukaryotic genomes, and they rep- resent a major source of genetic variation and novelty. Here we review the defining features of each major group of eukaryotic TEs and explore their evolutionary origins and relationships. We discuss how the unique biology of different TEs influences their propagation and distribution within and across genomes. Environmental and genetic factors acting at the level of the host species further modulate the activity, diversification, and fate of TEs, producing the dramatic variation in TE content observed across eukaryotes. We argue that cataloging TE diversity and dissecting the idiosyncratic be- havior of individual elements are crucial to expanding our comprehension of their impact on the biology of genomes and the evolution of species. 23.1 Review in Advance first posted on , September 21, 2020.
    [Show full text]
  • GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies
    International Journal of Molecular Sciences Review GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies Andrés Felipe Leal 1 , Eliana Benincore-Flórez 1, Daniela Solano-Galarza 1, Rafael Guillermo Garzón Jaramillo 1 , Olga Yaneth Echeverri-Peña 1, Diego A. Suarez 1,2, Carlos Javier Alméciga-Díaz 1,* and Angela Johana Espejo-Mojica 1,* 1 Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá 110231, Colombia; [email protected] (A.F.L.); [email protected] (E.B.-F.); [email protected] (D.S.-G.); [email protected] (R.G.G.J.); [email protected] (O.Y.E.-P.); [email protected] (D.A.S.) 2 Faculty of Medicine, Universidad Nacional de Colombia, Bogotá 110231, Colombia * Correspondence: [email protected] (C.J.A.-D.); [email protected] (A.J.E.-M.); Tel.: +57-1-3208320 (ext. 4140) (C.J.A.-D.); +57-1-3208320 (ext. 4099) (A.J.E.-M.) Received: 6 July 2020; Accepted: 7 August 2020; Published: 27 August 2020 Abstract: GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay–Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy.
    [Show full text]
  • Genetics and Epigenetics in Parkinson´S Disease
    Genetics and epigenetics in Parkinson´s disease. Luis Navarro Sánchez. Thesis supervisor: Jordi Pérez Tur. Unitat de Genètica Molecular, Institut de Biomedicina de València, CSIC. Doctoral studies: Biotechnology. Facultat de Ciències Biològiques, Universitat de València. Tras preguntas y más preguntas y miedo y nervios y ansiedad y más ansiedad y bloqueos y periodos depresivos y tristeza y mucha, pero mucha, mierda… acabas. Tarde y reventado, pero acabas. Y lo único que quieres es pasar página y que esto no te pase más nunca. ¿Y? Pues que te surgen más preguntas y más miedo y más nervios…. Y te das cuenta de que vives en un puto bucle científico que se está comiendo tu vida. ¿Y? No lo sé, estoy en ello. Terapia y medicación, supongo. Aunque la autodestrucción y los realities son muy apetecibles. En primer lugar, las formalidades. Gracias Jordi por dejarme que trabajara en la UGM. A estas alturas, creo que los dos nos habremos planteado muchas veces si fue una buena decisión, pero ya da igual. A continuación, el momento pasteleo, aunque si no sé dar un abrazo, no penséis que esto me va a salir fluido. Y menos aún, estando sobrio. Y que no se me enfade nadie si no he puesto su nombre o si no he escrito un parrafazo lacrimógeno y azucarado exaltando la belleza de nuestra relación. Si dejo que leas esto, significa que también eres importante para mí. Lo mejor de todo este tiempo sois, de calle, vosotros y vosotras: Bea, Carolina, Eva, Fer, LauraA, LauraG, María, Marta, Pili, Silvia, Vicente/Andreu y VicenteH.
    [Show full text]
  • A Zebrafish Reporter Line Reveals Immune and Neuronal Expression of Endogenous Retrovirus
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.21.427598; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus. Noémie Hamilton1,2*, Amy Clarke1, Hannah Isles1, Euan Carson1, Jean-Pierre Levraud3, Stephen A Renshaw1 1. The Bateson Centre, Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK 2. The Institute of Neuroscience, University of Sheffield, Sheffield, UK 3. Macrophages et Développement de l’Immunité, Institut Pasteur, CNRS UMR3738, 25 rue du docteur Roux, 75015 Paris *Corresponding author: [email protected] Abstract Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons.
    [Show full text]
  • Copycontrol™ Fosmid Autoinduction Solution
    CopyControl™ Fosmid Autoinduction Solution Cat. No. AIS107F Available exclusively thru Lucigen. lucigen.com/epibio www.lucigen.com MA263E CopyControl™ Fosmid Autoinduction Solution • 12/2016 1 MA263E CopyControl™ Fosmid Autoinduction Solution 1. Introduction The CopyControl™ Fosmid Autoinduction Solution is designed to induce CopyControl Fosmid clones and clones retrofitted with the EZ-Tn5™ <oriV/KAN-2> Transposon, grown in TransforMax™ EPI300™ E. coli cells, from single-copy number to a higher-copy number of approximately 50 fosmids per cell. The Fosmid Autoinduction Solution induces expression of a mutant trfA gene contained in the TransforMax EPI300 cells. Expression of trfA gene results in initiation of replication from the oriV high copy origin of replication and subsequent amplification of the CopyControl clones to high copy number. The Fosmid Autoinduction protocol improves upon the existing induction protocol by including the autoinduction supplement in the media prior to culture inoculation, removing the need for time-consuming subculturing and the 2-hour incubation required in the standard induction protocol. The Fosmid Autoinduction Solution also contains cell growth enhancers which boost cell numbers and typically provides higher DNA yields than with the standard CopyControl induction protocol. The hands-off autoinduction protocol makes CopyControl Fosmid Autoinduction solution ideal for high-throughput purification protocols in 96-well format. The autoinduction solution is also compatible with larger scale DNA purifications and can be scaled according to the amount of media used. 2. Product Specifications Storage: Store only at –20°C in a freezer without a defrost cycle. Mix thoroughly after thawing. Size and Formulation: CopyControl Fosmid Autoinduction Solution is available in a 50-ml size concentrate of 500X in sterile water.
    [Show full text]
  • Protocol for Epifos™ Fosmid Library Production
    EpiFOS™ Fosmid Library Production Kit Cat. No. FOS0901 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/EpicentreBio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 149 • 8/2012 1 EPILIT149 Rev. A EpiFOS™ Fosmid Library Production Kit 1. Overview of the EpiFOS Fosmid Library Production Process Fosmid vectors1-3 provide an improved method for cloning and the stable maintenance of cosmid-sized (35-45 kb) libraries in E. coli. The stability of such large constructs in vivo is facilitated by the pEpiFOS™-5 vector that maintains the clones at single copy in the cell. The EpiFOS Fosmid Library Production Kit will produce a complete and unbiased primary fosmid library. The kit utilizes a novel strategy of cloning randomly sheared, end-repaired DNA. Shearing the DNA leads to the generation of highly random DNA fragments in contrast to more biased libraries that result from fragmenting the DNA by partial restriction digests. The steps involved (protocols for steps 2-7 are included in this manual): 1. Purify DNA from the desired source (the kit does not supply materials for this step). 2. Shear the DNA to approximately 40-kb fragments. 3. End-repair the sheared DNA to blunt, 5′-phosphorylated ends. 4. Size-resolve the end-repaired DNA by Low Melting Point (LMP) agarose gel electrophoresis. 5. Purify the blunt-end DNA from the LMP agarose gel. 6. Ligate the blunt-end DNA to the Cloning-Ready pEpiFOS-5 vector. 7. Package the ligated DNA and plate on EPI100™-T1R Plating Strain. Grow clones overnight. pEpiFOS-5 is a 7518 bp.
    [Show full text]
  • Polymorphisms in the Glucocerebrosidase Gene And
    BMC Medical Genetics BioMed Central Technical advance Open Access Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T>C (L444P) Justin T Brown*, Cora Lahey, Walairat Laosinchai-Wolf and Andrew G Hadd Address: Asuragen, Inc., Austin, Texas, USA Email: Justin T Brown* - [email protected]; Cora Lahey - [email protected]; Walairat Laosinchai- Wolf - [email protected]; Andrew G Hadd - [email protected] * Corresponding author Published: 03 August 2006 Received: 17 March 2006 Accepted: 03 August 2006 BMC Medical Genetics 2006, 7:69 doi:10.1186/1471-2350-7-69 This article is available from: http://www.biomedcentral.com/1471-2350/7/69 © 2006 Brown et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Gaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann- Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay. Methods: Biotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin- conjugated fluorophore, the reactions were analyzed by Luminex IS100.
    [Show full text]
  • Analysis of the Binding and Interaction Patterns of 100 Flavonoids with the Pneumococcal Virulent Protein Pneumolysin: an in Silico Virtual Screening Approach
    Available online a t www.scholarsresearchlibrary.com Scholars Research Library Der Pharmacia Lettre, 2016, 8 (16):40-51 (http://scholarsresearchlibrary.com/archive.html) ISSN 0975-5071 USA CODEN: DPLEB4 Analysis of the binding and interaction patterns of 100 flavonoids with the Pneumococcal virulent protein pneumolysin: An in silico virtual screening approach Udhaya Lavinya B., Manisha P., Sangeetha N., Premkumar N., Asha Devi S., Gunaseelan D. and Sabina E. P.* 1School of Biosciences and Technology, VIT University, Vellore - 632014, Tamilnadu, India 2Department of Computer Science, College of Computer Science & Information Systems, JAZAN University, JAZAN-82822-6694, Kingdom of Saudi Arabia. _____________________________________________________________________________________________ ABSTRACT Pneumococcal infection is one of the major causes of morbidity and mortality among children below 2 years of age in under-developed countries. Current study involves the screening and identification of potent inhibitors of the pneumococcal virulence factor pneumolysin. About 100 flavonoids were chosen from scientific literature and docked with pnuemolysin (PDB Id.: 4QQA) using Patch Dockprogram for molecular docking. The results obtained were analysed and the docked structures visualized using LigPlus software. It was found that flavonoids amurensin, diosmin, robinin, rutin, sophoroflavonoloside, spiraeoside and icariin had hydrogen bond interactions with the receptor protein pneumolysin (4QQA). Among others, robinin had the highest score (7710) revealing that it had the best geometrical fit to the receptor molecule forming 12 hydrogen bonds ranging from 0.8-3.3 Å. Keywords : Pneumococci, pneumolysin, flavonoids, antimicrobial, virtual screening _____________________________________________________________________________________________ INTRODUCTION Streptococcus pneumoniae is a gram positive pathogenic bacterium causing opportunistic infections that may be life-threating[1]. Pneumococcus is the causative agent of pneumonia and is the most common agent causing meningitis.
    [Show full text]
  • Bioactive Compounds in Baby Spinach (Spinacia Oleracea L.)
    Bioactive Compounds in Baby Spinach (Spinacia oleracea L.) Effects of Pre- and Postharvest Factors Sara Bergquist Faculty of Landscape Planning, Horticulture and Agricultural Science Department of Crop Science Alnarp Doctoral thesis Swedish University of Agricultural Sciences Alnarp 2006 Acta Universitatis Agriculturae Sueciae 2006: 62 ISSN 1652-6880 ISBN 91-576-7111-7 © 2006 Sara Bergquist, Alnarp Tryck: SLU Service/Repro, Alnarp 2006 Abstract Bergquist, S. 2006. Bioactive compounds in baby spinach (Spinacia oleracea L.). Effects of pre- and postharvest factors. Doctoral dissertation. ISSN 1652-6880, ISBN 91-576-7111-7. A high intake of fruit and vegetables is well known to have positive effects on human health, and has been correlated to a decreased risk of most degenerative diseases of ageing, such as cardiovascular disease, cataracts and several forms of cancer. These protective effects have been attributed to high concentrations of bioactive compounds (ascorbic acid, flavonoids, carotenoids) in fruit and vegetables, partly due to the antioxidative action of some of these compounds. Maintaining a high level of these compounds in fruit and vegetables is therefore desirable. In addition, a high concentration of antioxidants in horticultural produce is believed to improve its storability and reduce the rate of deterioration. This thesis investigated the effects of pre- and postharvest factors on the concentrations of bioactive compounds in baby spinach (Spinacia oleracea L.). The factors studied included sowing time, growth stage at harvest, use of shade nettings and postharvest storage temperature and duration. Bioactive compounds were analysed using reversed-phase HPLC and chlorophylls using a spectrophotometric method. Visual quality of the fresh and stored leaves was scored on a 1-9 scale, where 9 was the best.
    [Show full text]