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

Gli autori desiderano ringraziare tutti i membri dei laboratori del dottor Barletta e il Dott. Powers per gli utili commenti durante lo sviluppo del protocollo. Ringraziamo Wendy Austin per le discussioni utili e correzione di bozze del manoscritto. Il lavoro descritto in questo manoscritto è stato finanziato da borse di studio pilota di semi per ogni investigatore di cui sopra presso la University of Nebraska-Lincoln Center Biologia Redox (genitore sovvenzione # NCRR 2P20RR 017675, D. Becker, PI). Inoltre, ringraziamo il Dr. Ofelia Chacon per fornire fondi da lei R21 sovvenzioni (1R21AI087561-01A1) per le forniture di ricerca e di sostegno stipendio parziale signor Halouska di standardizzare le tecniche NMR riportati nella presente pubblicazione.

1. Protocollo di testo Questo protocollo mette in evidenza l&#39;adattamento della metodologia NMR a M. tubercolosi (Classe III agente). Pertanto, le pratiche di biosicurezza di livello 3 (BSL3) devono essere seguite per lo svolgimento di M. ricerca tubercolosi in un laboratorio certificato annualmente. L&#39;esposizione al laboratorio generati aerosol è il pericolo più importante incontrato da personale che lavora con questi microrganismi. Le seguenti procedure si...

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Un numero significativo di studi ha analizzato i profili di trascrittomica e proteomica di M. tubercolosi sotto una varietà di in vitro e in vivo. 11-16 definitiva, cambiamenti nell&#39;espressione genica e dell&#39;attività enzimatica portare a variazioni nella concentrazione di piccole molecole di peso molecolare. La descrizione completa di questi composti costituisce il metaboloma. Pertanto, gli effetti delle droghe e delle condizioni di crescita variabile sulle vie metaboliche...

<table border="1"><tbody><tr><td> Nome del reagente / attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> ADC Arricchimento </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> Albumina di siero bovino </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Frazione V </td></tr><tr><td> Centrifuga </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Da banco </td></tr><tr><td> Tubi per centrifuga </td><td> Corning </td><td> 430291 </td><td> 50 ml polipropilene sterile </td></tr><tr><td> Fiale criogeniche </td><td> Corning </td><td> 430488 </td><td> 2,0 ml in polipropilene sterile </td></tr><tr><td> Cycloheximide </td><td> AG scientifico </td><td> C-1189 </td><td> Tossico </td></tr><tr><td> D (+) - Glucosio </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Ossido di deuterio </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Beuta </td><td> VWR </td><td> 89095-266 </td><td> Sterile, base piatta, policarbonato, 0,22 micron membrana in PTFE tappo ventilato </td></tr><tr><td> Flash Freeze Flask </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Liofilizzatore </td><td> VWR </td><td> 82019-038 </td><td> 4.5 L da banco </td></tr><tr><td> Glicerina </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Incubatrice </td><td> Nuovo Brunswick </td><td> Innova 40 </td><td> Agitatore da banco </td></tr><tr><td> Lisi Matrix B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> Lysis macchina </td><td> MP Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td> Microcentrifuga </td><td> Eppendorf </td><td> 5415D </td><td> Da banco </td></tr><tr><td> Microcentrifuga </td><td> Beckman Coulter </td><td> Microcentrifuga 22R </td><td> Da banco </td></tr><tr><td> Brodo Middlebrook 7H9 </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> NMR tubi </td><td> Norell </td><td> ST500-7 </td><td> 5 mM </td></tr><tr><td> Arricchimento OADC </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Acido oleico </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Fosfato di potassio bibasico </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Potassio fosfato monobasico </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Rotor - Microcentrifugare 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Sigillata e polipropilene </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Con bio-certificati coperture </td></tr><tr><td> Cloruro di sodio </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Sodio-3-trimethylsilylpropionate-2 ,2,3,3-D4 </td><td> Cambridge Isotope </td><td> DLM-48 </td><td></td></tr><tr><td> Spettrofotometro </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Spettrofotometro Cuvette </td><td> Sagola di salvataggio </td><td> LS-2410 </td><td> 1,5 ml in polistirolo, 2 lati trasparenti </td></tr><tr><td> Siringa </td><td> Becton Dickinson </td><td> 309585 </td><td> Sterile, 3 ml Luer-Lok </td></tr><tr><td> Siringa Filtro </td><td> Nalgene </td><td> 190-2520 </td><td> Acetato di 0,2 micron di cellulosa sterile </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 è una delle principali cause di mortalità degli esseri umani su scala globale. L&#39;emergere di due multi-(MDR) e estesamente-(XDR) ceppi farmaco-resistenti minaccia di far deragliare attuali sforzi di lotta contro la malattia. Pertanto, vi è una necessità urgente di sviluppare farmaci e vaccini che sono più efficaci di quelle attualmente disponibili. Il genoma di M. la tubercolosi è nota da più di 10 anni, ma ci sono importanti lacune nella nostra conoscenza della funzione genica e essenzialità. Molti studi hanno utilizzato da analisi di espressione genica sia a livello trascrittomica e proteomica per determinare gli effetti dei farmaci, ossidanti, e le condizioni di crescita sui modelli globali di espressione genica. In definitiva, la risposta finale di questi cambiamenti si riflette nella composizione metabolico del batterio comprendere alcune migliaia di sostanze a basso peso molecolare. Confrontando i profili metabolici di tipo selvatico e mutanti, sia trattata o treated con un particolare farmaco, può effettivamente permettere l&#39;identificazione di destinazione e può portare allo sviluppo di nuovi inibitori con attività anti-tubercolare. Analogamente, gli effetti di due o più condizioni del metaboloma può anche essere valutato. Risonanza magnetica nucleare (NMR) è una tecnologia potente che viene utilizzato per identificare e quantificare intermedi metabolici. In questo protocollo, le procedure per la preparazione di M. tubercolosi estratti cellulari per l&#39;analisi NMR metabolomica sono descritti. Colture di cellule vengono coltivate in condizioni appropriate e richiesto livello di biosicurezza 3 di contenimento 1, raccolte e sottoposte a lisi meccanica pur mantenendo basse temperature per massimizzare la conservazione dei metaboliti. Lisati cellulari sono recuperati, filtrati sterilizzato, e conservati a temperature bassissime. Aliquote di questi estratti di cellule sono piastrate su agar Middlebrook 7H9 per unità formanti colonia per verificare l&#39;assenza di cellule vitali. Su due mesi di incubazione a 37 ° C, se non vicolonie capaci si osservano, i campioni vengono rimossi dalla struttura di contenimento per il trattamento a valle. Sono estratti liofilizzati, risospese in tampone deuterato e iniettato nello strumento NMR, catturando dati spettroscopici che viene poi sottoposto ad analisi statistica. Le procedure descritte possono essere applicate sia unidimensionale (1D) 1 H NMR e bidimensionale (2D) 1 H-13 C NMR. Questa metodologia fornisce più affidabile di identificazione piccolo peso molecolare metabolita e analisi quantitative più affidabili e sensibili di composizioni estratto cellulare metaboliche rispetto ai metodi cromatografici. Variazioni della procedura descritta in seguito alla fase di lisi cellulare può anche essere adattato per parallela analisi proteomica.

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

Il profilo metabolico di<em&gt; Mycobacterium tuberculosis</em&gt; È determinata dopo la crescita in colture in brodo. Le condizioni possono essere variate per testare gli effetti di integratori alimentari, ossidanti e anti-tubercolosi agenti sul profilo metabolico di questo microrganismo. Procedura per la preparazione estratto è applicabile sia per 1D<sup&gt; 1</sup&gt; H e 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

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

Monitoraggio migrazione delle cellule dendritiche usando<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

Un modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E 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

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione 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

Un nuovo approccio Microdissezione per recuperare<em&gt; Mycobacterium tuberculosis</em&gt; Trascrizioni specifici da formalina paraffina fisso incorporato Lung granulomi

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

Un 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

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

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<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

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<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

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

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