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

Författarna vill tacka alla medlemmar i laboratorier Dr Barletta och Dr Powers för värdefulla kommentarer och samtidigt utveckla protokollet. Vi tackar Wendy Austin för hjälp diskussioner och korrekturläsning av manuskriptet. Det arbete som beskrivs i detta manuskript har finansierats av bidrag utsäde pilotprojekt till varje utredare ovan från University of Nebraska-Lincoln Redox biologi Center (förälder bidrag # NCRR 2P20RR 017.675, D. Becker, PI). Dessutom tackar vi Dr Ofelia Chacon för att ge pengar från hennes R21 bidrag (1R21AI087561-01A1) för forskning leveranser och Mr Halouska partiella lön stöd för att standardisera NMR-tekniker som ingår i denna publikation.

1. Protokoll text Detta protokoll belyser anpassning av NMR-metod till M. tuberkulos (klass III medel). Därför skyddsnivå 3 (BSL3) metoder måste följas vid genomförandet av M. tuberkulos forskning i ett år certifierat laboratorium. Exponering för laboratorie-genererade aerosoler är den viktigaste risk stött av personal som arbetar med dessa mikroorganismer. Följande förfaranden genomförs på vår institution och variationer kan förekomma utifrån institutionella...

<ol>
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Ett stort antal studier har analyserat transcriptomic och proteomik profiler M. tuberkulos under ett flertal in vitro-och in vivo-betingelser. 11-16 Slutligen, förändringar i genuttryck och enzymaktivitet leda till variationer i koncentrationerna av lågmolekylära molekyler. Den fullständiga beskrivningen av dessa föreningar utgör Metabolome. Således kan effekten av droger och varierande förhållanden tillväxt på metaboliska vägar att följas av metabolomic analys. 17,18</sup...

<table border="1"><tbody><tr><td> Namn på reagens / utrustning </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> ADC Enrichment </td><td> BD BBL Middlebrook </td><td> 212.352 </td><td></td></tr><tr><td> BACS-120 provbytare </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> Bovint serumalbumin </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Fraktion V </td></tr><tr><td> Centrifug </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Bänk </td></tr><tr><td> Centrifugrör </td><td> Corning </td><td> 430.291 </td><td> 50 ml steril polypropen </td></tr><tr><td> Kryogena Flaskorna </td><td> Corning </td><td> 430.488 </td><td> 2,0 ml steril polypropen </td></tr><tr><td> Cycloheximide </td><td> AG Scientific </td><td> C-1189 </td><td> Giftig </td></tr><tr><td> D (+) - Glukos </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Deuteriumoxid </td><td> Sigma Aldrich </td><td> 617.385 </td><td></td></tr><tr><td> Erlenmeyerkolv </td><td> VWR </td><td> 89095-266 </td><td> Steril, platt botten, polykarbonat, 0,22 um PTFE-membran ventilerat lock </td></tr><tr><td> Flash Freeze kolv </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Frys Torktumlare </td><td> VWR </td><td> 82019-038 </td><td> 4,5 L Fasta </td></tr><tr><td> Glycerol </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Inkubator </td><td> New Brunswick </td><td> Innova 40 </td><td> Bänk shaker </td></tr><tr><td> Lysering matris 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> Mikrocentrifug </td><td> Eppendorf </td><td> 5415D </td><td> Bänk </td></tr><tr><td> Mikrocentrifug </td><td> Beckman Coulter </td><td> Microfuge 22R </td><td> Bänk </td></tr><tr><td> Middlebrook 7H9 Broth </td><td> Difco </td><td> 271.310 </td><td></td></tr><tr><td> NMR-rör </td><td> Norell </td><td> ST500-7 </td><td> 5mM </td></tr><tr><td> OADC Anrikning </td><td> BD BBL Middlebrook </td><td> 212.351 </td><td></td></tr><tr><td> Oljesyra </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Dibasiskt kaliumfosfat </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Monobasiskt kaliumfosfat </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> Förseglade och polypropylen </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Med Bio-certifierade omslag </td></tr><tr><td> NATRIUMKLORID </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Natrium-3-rerat-2 ,2,3,3-D4 </td><td> Cambridge Isotope </td><td> DLM-48 </td><td></td></tr><tr><td> Spektrofotometer </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Spektrofotometer Kyvetter </td><td> LIVLINA </td><td> LS-2410 </td><td> 1,5 ml polystyren, 2 tydliga sidor </td></tr><tr><td> Spruta </td><td> Becton Dickinson </td><td> 309.585 </td><td> Sterilt, 3 ml luer-Lok </td></tr><tr><td> Sprutfilter </td><td> Nalgene </td><td> 190-2520 </td><td> 0,2 | im sterilt cellulosaacetat </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 är en viktig orsak till dödsfall hos människor på en global skala. Framväxten av både multi-(MDR) och i stor utsträckning-(XDR) läkemedelsresistenta stammar hotar att spåra ur nuvarande insatser för sjukdomsbekämpning. Således finns det ett akut behov av att utveckla läkemedel och vacciner som är mer effektiva än de som för närvarande finns tillgängliga. Genomet av M. tuberkulos har varit känt i mer än 10 år, men det finns stora luckor i vår kunskap om geners funktion och essentiala. Många studier har sedan använt analys genuttryck på både transcriptomic och proteomik nivåer för att bestämma effekterna av droger, oxidanter och villkor tillväxt på den globala mönster av genuttryck. Ytterst är det slutliga svaret hos dessa förändringar avspeglas i den metaboliska sammansättningen av bakterien inklusive några tusen små molekylvikt kemikalier. Jämföra de metaboliska profilerna för vildtyp och mutanta stammar, antingen obehandlade eller treated med ett särskilt läkemedel, kan effektivt tillåta mål identifiering och kan leda till utveckling av nya inhibitorer med anti-tuberkulos aktivitet. Likaså, kan effekterna av två eller flera förhållanden på Metabolome också bedömas. Kärnmagnetisk resonans (NMR) är en kraftfull teknik som används för att identifiera och kvantifiera metaboliska intermediärer. I detta protokoll förfaranden för framställning av M. tuberkulos cellextrakt för NMR metabolomic analys beskrivs. Cellkulturer odlas under lämpliga förhållanden och som krävs biosäkerhetsnivå 3 inneslutning, skördade 1, och utsattes för mekanisk lys samtidigt kyla för att maximera bevarandet av metaboliter. Cellysaten återvinns, filtreras steriliserades och lagrades vid extremt låga temperaturer. Alikvoter från dessa cellextrakt stryks ut på Middlebrook 7H9 agar för kolonibildande enheter för att kontrollera frånvaron av viabla celler. Vid två månaders inkubation vid 37 ° C, om inget VIsom kan kolonier observeras, prover ur inneslutningen anläggning för efterföljande bearbetning. Extrakten lyofiliseras, återsuspenderas i deutererad buffert och injicerades i NMR-instrument, fånga spektroskopiska data som sedan utsätts för statistisk analys. De beskrivna förfarandena kan tillämpas för både endimensionella (1D) 1 H-NMR och tvådimensionella (2D) 1 H-13 C-NMR-analyser. Denna metod ger mer tillförlitlig liten molekylvikt metabolit identifiering och mer tillförlitliga och känsliga kvantitativa analyser av metabola cellextrakt kompositioner än kromatografiska metoder. Variationer av den procedur som beskrivits efter cellys steget kan också anpassas för parallell proteomic analys.

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

Den metabolomic profil<em&gt; Mycobacterium tuberculosis</em&gt; Bestäms efter tillväxt i buljongkulturer. Förhållanden kan varieras för att testa effekterna av näringstillskott, oxidanter, och anti-tuberkulos medel på den metaboliska profilen för denna mikroorganism. Förfarande för extrakt förberedelse gäller både 1D<sup&gt; 1</sup&gt; H och 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR-analyser.

Provberedning av<em&gt; Mycobacterium tuberculosis</em&gt; Extrakt för Nuclear Magnetic Resonance studier Metabolomic

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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Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Övervakning dendritceller Migration hjälp<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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

En experimentell modell för att studera Tuberculosis-Malaria Coinfection på Natural Överföring av Mycobacterium tuberculosis Och 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

Demonstration av en multiresistent<em&gt; Mycobacterium tuberculosis</em&gt; Amplification 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 förhållningssätt till Återställa<em&gt; Mycobacterium tuberculosis</em&gt; Särskilda Avskrifter från formalinfixerad paraffininbäddade Lung Granulom

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

En 3D Human Lung Tissue Modell för funktionella studier på<em&gt; Mycobacterium tuberculosis</em&gt; Infektion

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

System för Efficacy och cytotoxicitet Screening av inhibitorer Inriktning Intracellulär<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

En hög kapacitet Kompatibel analys för att utvärdera läkemedlets effektivitet mot makrofag passe<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

Analys av 18FDG PET/CT Imaging som ett verktyg för att studera Mycobacterium tuberculosis infektion och behandling i icke-mänskliga primater

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