Name: sample preparation of mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies
Uploaded: 2012-09-03T12:00:00
Description: Watch this Scientific Journal Video about Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies at JoVE.com

De auteurs willen graag alle leden van de laboratoria van Dr Barletta en Dr Powers bedanken voor nuttige opmerkingen bij het ontwikkelen van het protocol. Wij danken Wendy Austin voor nuttige discussies en proeflezen van het manuscript. Het werk beschreven in dit manuscript werd gefinancierd door zaad piloot de subsidie ​​voor elke onderzoeker hierboven genoemde aan de Universiteit van Nebraska-Lincoln Redox Biology Center (ouder subsidie ​​# NCRR 2P20RR 017675, D. Becker, PI). Daarnaast danken wij dr. Ofelia Chacon voor het verstrekken van financiële middelen uit haar R21 subsidie ​​(1R21AI087561-01A1) voor onderzoek leveringen en gedeeltelijke salaris heer Halouska de steun aan NMR-technieken in deze publicatie te standaardiseren.

1. Protocol Tekst Dit protocol benadrukt de aanpassing van NMR methodologie om M. tuberculose (klasse III agent). Daarom Biosafety Level 3 (BSL3) praktijken moeten worden gevolgd bij de uitvoering van M. tuberculose onderzoek in een jaarlijks gecertificeerd laboratorium. Blootstelling aan laboratorium gegenereerde aerosols is het belangrijkste gevaar waarmee Personen die aan deze micro-organismen. De volgende procedures worden uitgevoerd in onze instelling en variaties kunnen...

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Een groot aantal studies hebben de transcriptoom-en proteoomanalyse profielen van M. tuberculose onder verschillende in vitro en in vivo omstandigheden. 11-16 uiteindelijk veranderingen in genexpressie en enzymactiviteit leiden tot variaties in de concentraties van kleine moleculen molecuulgewicht. De volledige beschrijving van deze verbindingen vormt het metaboloom. Aldus kunnen de effecten van drugs en ​​wisselende groeiomstandigheden op metabolisme gevolgd door metabolische a...

<table border="1"><tbody><tr><td> Naam van de Reagens / Equipment </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Reacties </td></tr><tr><td> ADC Verrijking </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 Sample Changer </td><td> Bruker </td><td></td><td></td></tr><tr><td> Bruker Avance NMR </td><td> Bruker </td><td></td><td> 500 MHz </td></tr><tr><td> Bovine Serum Albumine </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Fractie V </td></tr><tr><td> Centrifugeer </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Benchtop </td></tr><tr><td> Centrifuge Tubes </td><td> Corning </td><td> 430291 </td><td> 50 ml steriele polypropyleen </td></tr><tr><td> Cryogene Vials </td><td> Corning </td><td> 430488 </td><td> 2,0 ml steriele polypropyleen </td></tr><tr><td> Cycloheximide </td><td> AG Wetenschappelijke </td><td> C-1189 </td><td> Giftig </td></tr><tr><td> D (+) - Glucose </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Deuteriumoxide </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Erlenmeyer Flask </td><td> VWR </td><td> 89095-266 </td><td> Steriel, vlakke bodem, polycarbonaat, 0,22 pm PTFE membraan ontluchtingsdop </td></tr><tr><td> Flash Freeze Flask </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Freeze Dryer </td><td> VWR </td><td> 82019-038 </td><td> 4,5 L Benchtop </td></tr><tr><td> Glycerol </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Broedmachine </td><td> New Brunswick </td><td> Innova 40 </td><td> Benchtop shaker </td></tr><tr><td> Lyseren Matrix B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> Lysis Machine </td><td> MP Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td> Microcentrifuge </td><td> Eppendorf </td><td> 5415D </td><td> Benchtop </td></tr><tr><td> Microcentrifuge </td><td> Beckman Coulter </td><td> Microfuge 22R </td><td> Benchtop </td></tr><tr><td> Middlebrook 7H9 Broth </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> NMR-buizen </td><td> Norell </td><td> ST500-7 </td><td> 5 mM </td></tr><tr><td> OADC Verrijking </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Oliezuur </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Kalium Dibasisch </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Kalium monobasisch </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Rotor - microfuge 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Afgedichte en polypropyleen </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Met biologische covers </td></tr><tr><td> Natriumchloride </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Natrium-3-trimethylsilylpropionate-2 ,2,3,3-D4 </td><td> Cambridge Isotope </td><td> DLM-48 </td><td></td></tr><tr><td> Spectrofotometer </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Spectrofotometer Cuvetten </td><td> Reddingslijn </td><td> LS-2410 </td><td> 1,5 ml polystyreen, 2 duidelijke kanten </td></tr><tr><td> Injectiespuit </td><td> Becton Dickinson </td><td> 309585 </td><td> Steriele 3 ml Luer-Lok </td></tr><tr><td> Spuitfilter </td><td> Nalgene </td><td> 190-2520 </td><td> 0,2 urn steriel celluloseacetaat </td></tr><tr><td> Tween 80 </td><td> Fisher Scientific </td><td> BP338-500 </td><td></td></tr></tbody></table>

sample preparation of mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies

Mycobacterium tuberculosis is een belangrijke oorzaak van sterfte in de mens op wereldschaal. De opkomst van zowel multi-(MDR) en uitgebreid-(XDR) resistente stammen dreigt te ontsporen huidige ziekte controle-inspanningen. Er is dus een dringende behoefte aan geneesmiddelen en vaccins die effectiever dan de huidige ontwikkelen. Het genoom van M. tuberculose is gekend voor meer dan 10 jaar, maar toch zijn er belangrijke hiaten in onze kennis van gen-functie en wezenlijkheid. Vele studies hebben sindsdien gebruikt genexpressieanalyse zowel het transcriptoom en proteomische niveaus om de effecten van drugs, oxidatiemiddelen en groeiomstandigheden de algemene patronen van genexpressie te bepalen. Uiteindelijk wordt de uiteindelijke reactie van deze veranderingen weerspiegeld in de metabole samenstelling van de bacterie waaronder enkele duizenden kleine molecuul gewicht. Vergelijken van de metabolische profielen van wildtype en mutante stammen, hetzij onbehandeld of treated met een bepaald geneesmiddel kan efficiënt in doelbepaling en kan leiden tot de ontwikkeling van nieuwe remmers met anti-tbc activiteit. Ook kunnen de effecten van twee of meer voorwaarden metaboloom worden beoordeeld. Nucleaire magnetische resonantie (NMR) is een krachtige techniek die wordt gebruikt voor het identificeren en kwantificeren metabole intermediairen. In dit protocol, de procedures voor de opstelling van M. tuberculose celextracten voor NMR metaboloom analyse worden beschreven. Celculturen worden gekweekt onder geschikte omstandigheden en vereist Biosafety Level 3 containment, 1 geoogst en onderworpen aan mechanische lysis behoud koude temperaturen bewaring van metabolieten maximaliseren. Cellysaten worden teruggewonnen, gefiltreerd gesteriliseerd en opgeslagen bij zeer lage temperaturen. Aliquots van deze celextracten worden uitgeplaat op Middlebrook 7H9 agar voor kolonievormende eenheden geen levensvatbare cellen te verifiëren. Na twee maanden incubatie bij 37 ° C, indien geen vikunnen kolonies worden waargenomen, worden monsters uit de opvangvoorziening voor verdere verwerking. Extracten worden gevriesdroogd, geresuspendeerd in gedeutereerd buffer en geïnjecteerd in de NMR instrument, vastleggen spectroscopische gegevens die vervolgens wordt onderworpen aan statistische analyse. De beschreven procedures kunnen worden toegepast voor zowel eendimensionale (1D) 1H NMR en tweedimensionale (2D) 1 H-13 C NMR analyses. Deze methode levert meer betrouwbare kleine molecuulgewicht metaboliet identificatie en meer betrouwbare en gevoelige kwantitatieve analyse van celextract metabolische samenstellingen dan chromatografische methoden. Variaties van de beschreven procedure na cellyse stap kan ook worden aangepast voor parallel proteoomanalyse.

Watch this Scientific Journal Video about Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies at JoVE.com

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

De metaboloom profiel van<em&gt; Mycobacterium tuberculosis</em&gt; Bepaald na groei in bouillon kweken. Voorwaarden kan worden gevarieerd om het effect van voedingssupplementen, oxidanten en anti-tuberculose middelen te testen op de metabolische profiel van dit micro-organisme. Procedure voor het extract voorbereiding is van toepassing voor zowel 1D<sup&gt; 1</sup&gt; H en 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR analyses.

Monstervoorbereiding van<em&gt; Mycobacterium tuberculosis</em&gt; Extracten voor Nuclear Magnetic Resonance metabolietvorming

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

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An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Een experimenteel model om te studeren Tuberculose-Malaria Coinfection op Natuurlijke Transmissie van Mycobacterium tuberculosis En Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Aantonen van een multiresistente<em&gt; Mycobacterium tuberculosis</em&gt; Versterking Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

A Novel Microdissection aanpak voor het herstellen<em&gt; Mycobacterium tuberculosis</em&gt; Specifieke Afschriften van formaline gefixeerde in paraffine ingebedde Lung Granulomen

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Een 3D Human longweefsel Model voor functionele studies op<em&gt; Mycobacterium tuberculosis</em&gt; Infectie

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Systeem voor de werkzaamheid en cytotoxiciteit Screening van inhibitoren voor intracellulaire<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Een High-throughput Compatible Assay te evalueren Drug De werkzaamheid tegen macrofaag gepasseerd<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analyse van 18FDG-PET/CT Imaging als een hulpmiddel voor studeren Mycobacterium tuberculosis infectie en behandeling in niet-menselijke primaten

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...