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

Os autores gostariam de agradecer a todos os membros dos laboratórios do Dr. Barletta e Poderes Dr. pelos comentários úteis durante o desenvolvimento do protocolo. Agradecemos Wendy Austin para discussões úteis e revisão do manuscrito. O trabalho descrito neste manuscrito foi financiado por doações piloto de sementes para cada investigador listados acima, da Universidade de Nebraska-Lincoln Center Biologia Redox (pai concessão # NCRR 2P20RR 017675, D. Becker, PI). Além disso, podemos agradecer ao Dr. Ofelia Chacon do fornecimento de fundos a partir de sua concessão R21 (1R21AI087561-01A1) para fontes de pesquisa e de apoio Sr. Halouska do salário parcial de padronizar as técnicas de RMN incluídos nesta publicação.

1. Texto protocolo Este protocolo destaca a adaptação da metodologia de RMN de M. tuberculose (Classe agente III). Portanto, as práticas do Nível de Biossegurança 3 (BSL3) precisam ser seguidas na condução M. pesquisa da tuberculose em um laboratório certificado anualmente. A exposição a aerossóis gerados laboratório é o risco mais importante encontrado por pessoas que trabalham com esses microrganismos. Os seguintes procedimentos são realizados na inst...

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Um número significativo de estudos analisaram os perfis transcriptomic e proteômica de M. tuberculosis, sob uma variedade de ensaios in vitro e in vivo. 11-16 Em última análise, as alterações na expressão de genes e a actividade da enzima levar a variações nas concentrações de pequenas moléculas de peso molecular. A descrição completa destes compostos constitui o metaboloma. Assim, os efeitos da droga e as condições de crescimento diferentes sobre as vias me...

<table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> Enriquecimento ADC </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 Changer Amostra </td><td> Bruker </td><td></td><td></td></tr><tr><td> RMN Bruker Avance </td><td> Bruker </td><td></td><td> 500 MHz </td></tr><tr><td> Albumina de Soro Bovino </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Fração V </td></tr><tr><td> Centrifugador </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Bancada </td></tr><tr><td> Tubos da centrífuga </td><td> Corning </td><td> 430291 </td><td> 50 ml de polipropileno estéril </td></tr><tr><td> Os frascos criogênicos </td><td> Corning </td><td> 430488 </td><td> 2,0 ml de polipropileno estéril </td></tr><tr><td> Cycloheximide </td><td> AG Científico </td><td> C-1189 </td><td> Tóxico </td></tr><tr><td> D (+) - Glucose </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Óxido de deutério </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Garrafa de Erlenmeyer </td><td> VWR </td><td> 89095-266 </td><td> Base, estéril plana, policarbonato, 0,22 um tampa da membrana PTFE ventilada </td></tr><tr><td> Flash Flask Congelar </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Liofilizador </td><td> VWR </td><td> 82019-038 </td><td> 4,5 L de bancada </td></tr><tr><td> Glicerina </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Incubadora </td><td> New Brunswick </td><td> Innova 40 </td><td> Agitador de bancada </td></tr><tr><td> Lise Matrix B </td><td> Biomedicals MP </td><td> 6911-100 </td><td></td></tr><tr><td> Máquina de lise </td><td> Biomedicals MP </td><td> FastPrep-24 </td><td></td></tr><tr><td> Microcentrífuga </td><td> Eppendorf </td><td> 5415D </td><td> Bancada </td></tr><tr><td> Microcentrífuga </td><td> Beckman Coulter </td><td> Microcentrífuga 22R </td><td> Bancada </td></tr><tr><td> Middlebrook 7H9 Broth </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> RMN tubos </td><td> Norell </td><td> ST500-7 </td><td> 5mM </td></tr><tr><td> Enriquecimento OADC </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Ácido oléico </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Fosfato de Potássio Dibásico </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Fosfato de potássio monobásico </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Rotor - Microcentrifugue 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Selado e polipropileno </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Com bio-certificadas capas </td></tr><tr><td> Cloreto de Sódio </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Sódio-3-trimetilsilil-2 ,2,3,3-D4 </td><td> Cambridge Isotope </td><td> DLM-48 </td><td></td></tr><tr><td> Espectrofotômetro </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Cubetas Espectrofotômetro </td><td> Corda salva-vidas </td><td> LS-2410 </td><td> 1,5 ml de poliestireno, 2 lados claros </td></tr><tr><td> Seringa </td><td> Becton Dickinson </td><td> 309585 </td><td> Estéril, 3 ml de Luer-Lok </td></tr><tr><td> Filtrar seringa </td><td> Nalgene </td><td> 190-2520 </td><td> 0,2 mM de acetato de celulose estéril </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 é uma das principais causas de mortalidade em seres humanos em escala global. O surgimento de ambos multi-(MDR) e-(XDR) resistentes à drogas-cepas ameaça descarrilar os esforços atuais de controle da doença. Assim, há uma necessidade urgente de desenvolver drogas e vacinas que são mais eficazes do que aqueles atualmente disponíveis. O genoma de M. tuberculose tem sido conhecida há mais de 10 anos, ainda existem lacunas importantes no conhecimento da função dos genes e essencialidade. Muitos estudos têm utilizado uma vez que a análise da expressão do gene, tanto a nível transcriptomic e proteômica para determinar os efeitos das drogas, oxidantes, e condições de crescimento sobre os padrões globais de expressão do gene. Em última análise, a resposta final destas alterações é reflectido na composição metabólica da bactéria incluindo alguns milhares de pequenos produtos químicos de peso molecular. Comparando os perfis metabólicos de tipo selvagem e estirpes mutantes, quer não tratadas ou treated com um fármaco particular, podem efectivamente permitir a identificação do alvo e pode levar ao desenvolvimento de novos inibidores com actividade anti-tuberculoso. Da mesma forma, os efeitos de duas ou mais condições do metaboloma pode também ser avaliada. Ressonância magnética nuclear (RMN) é uma tecnologia poderosa, que é utilizado para identificar e quantificar os intermediários metabólicos. Neste protocolo, os processos para a preparação de M. extractos de células de tuberculose para análise de RMN são descritos metabolômica. As culturas de células são cultivadas em condições adequadas e exigidas Biossegurança Nível 3 de confinamento, uma colhidas e submetidas a lise mecânica, mantendo a temperaturas frias para maximizar a preservação de metabolitos. Os lisados ​​de células são recuperadas, filtrado esterilizado e armazenado a temperaturas ultra-baixas. Alíquotas destas extractos celulares foram semeadas em agar Middlebrook 7H9 para unidades formadoras de colónias para verificar a ausência de células viáveis. Após dois meses de incubação a 37 ° C, na ausência de vicolónias capazes são observadas, as amostras são removidas da unidade de contenção para o processamento a jusante. Os extratos são liofilizadas, ressuspensas em tampão de deuterado e injectada no instrumento de RMN, a captura de dados espectroscópicos que é então submetida a análise estatística. Os procedimentos descritos podem ser aplicados tanto para unidimensional (1D) 1 H NMR e de duas dimensões (2D) 1 H-13 C análises de RMN. Esta metodologia proporciona mais fiável de identificação de baixo peso molecular e do metabolito mais fiáveis ​​e sensíveis análises quantitativas de composições de extracto de células metabólicas de métodos cromatográficos. Variações do processo descrito a seguir ao passo de lise de células pode também ser adaptado para análise proteômica paralelo.

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

O perfil de metabolômica<em&gt; Mycobacterium tuberculosis</em&gt; É determinada após crescimento em culturas em caldo. As condições podem ser variadas para testar os efeitos de suplementos nutricionais, oxidantes e agentes anti-tuberculose no perfil metabólico deste microrganismo. Procedimento para a preparação de extracto é aplicável tanto para 1D<sup&gt; 1</sup&gt; H e 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; Análises de C NMR.

Preparação de amostras de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos para Estudos Nucleares metabolômica Ressonância Magnética

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

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

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

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

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de 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

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação 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

Uma nova abordagem para Microdissecção Recuperando<em&gt; Mycobacterium tuberculosis</em&gt; transcritos específicos de formalina fixo Parafina incorporado Lung granulomas

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

Um modelo de tecido Pulmão humano 3D para estudos funcionais em<em&gt; Mycobacterium tuberculosis</em&gt; Infecção

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 de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<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

Um ensaio Compatível de alta capacidade para avaliar droga Eficácia contra macrófagos passadas<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

Análise de 18FDG PET-CT Imaging como uma ferramenta para estudar infecção por Mycobacterium tuberculosis e tratamento em primatas não-humanos

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