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

Авторы хотели бы поблагодарить всех членов лаборатории д-ра Барлетта и д-р держав за полезные замечания при разработке протокола. Мы благодарим Wendy Остина за полезные обсуждения и редактирование рукописи. Работа, описанная в этой рукописи была профинансирована за счет грантов семян пилот каждого следователя, перечисленные выше из Университета Небраски-Линкольна окислительно-восстановительного центра биологии (родитель грант № NCRR 2P20RR 017675, Д. Беккер, PI). Кроме того, мы благодарим доктора Офелия Чакона для предоставления средств из своего гранта R21 (1R21AI087561-01A1) для исследования материалов и частичная поддержка зарплата г-н Halouska, чтобы стандартизировать методы ЯМР включены в эту публикацию.

1. Текст протокола Этот протокол выдвигает на первый план адаптации методологии ЯМР M. туберкулез (класс III агента). Таким образом, уровень биобезопасности 3 (BSL3) практики необходимо следовать при проведении M. туберкулез исследований в ежегодной сертифицированно?...

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Значительное число исследований были проанализированы транскриптомных и протеомных профилей M. туберкулезом в различных в пробирке и в естественных условиях. 11-16 конечном счете, изменения в экспрессии генов и активность фермента приводит к изменениям в концентра?...

<table border="1"><tbody><tr><td> Название Реагенты / Оборудование </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> ADC обогащению </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 образца Changer </td><td> Bruker </td><td></td><td></td></tr><tr><td> Bruker Avance ЯМР </td><td> Bruker </td><td></td><td> 500 МГц </td></tr><tr><td> Бычьего сывороточного альбумина </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Доля V </td></tr><tr><td> Центрифугировать </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Настольные </td></tr><tr><td> Центрифуги трубы </td><td> Гранулирование </td><td> 430291 </td><td> 50 мл стерильной полипропилена </td></tr><tr><td> Криогенная Флаконы </td><td> Гранулирование </td><td> 430488 </td><td> 2,0 мл стерильного полипропилена </td></tr><tr><td> Cycloheximide </td><td> Научно AG </td><td> C-1189 </td><td> Токсичный </td></tr><tr><td> D (+) - Глюкоза </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Оксид дейтерия </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Коническая колба Эрленмейера </td><td> VWR </td><td> 89095-266 </td><td> Стерильные, плоское основание, поликарбонат, 0,22 мкм PTFE мембраны вентилируемый колпачок </td></tr><tr><td> Flash Freeze колба </td><td> VWR </td><td> 82018-226 </td><td> 750 мл </td></tr><tr><td> Замораживание сушилка </td><td> VWR </td><td> 82019-038 </td><td> 4,5 л Настольные </td></tr><tr><td> Глицерин </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Инкубатор </td><td> Нью-Брансуик </td><td> Innova 40 </td><td> Настольный шейкер </td></tr><tr><td> Лизирующий матрицы B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> Лизис машины </td><td> MP Biomedicals </td><td> ФаastPrep-24 </td><td></td></tr><tr><td> Микроцентрифуга </td><td> Eppendorf </td><td> 5415D </td><td> Настольные </td></tr><tr><td> Микроцентрифуга </td><td> Beckman Coulter </td><td> Микроцентрифуге 22R </td><td> Настольные </td></tr><tr><td> Middlebrook 7H9 Broth </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> ЯМР труб </td><td> Norell </td><td> ST500-7 </td><td> 5 мМ </td></tr><tr><td> OADC обогащению </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Олеиновая кислота </td><td> Сигма </td><td> O1008 </td><td></td></tr><tr><td> Калия фосфат двузамещенный </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Фосфат калия однозамещенный </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Ротор - микроцентрифуге 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Герметичные и полипропилена </td></tr><tr><td> Ротор - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> С био-сертифицированную крышки </td></tr><tr><td> Хлористый натрий </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Натрий-3-trimethylsilylpropionate-2 ,2,3,3-D4 </td><td> Cambridge Изотопный </td><td> DLM-48 </td><td></td></tr><tr><td> Спектрофотометр </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Спектрофотометр кювет </td><td> Спасательный трос </td><td> LS-2410 </td><td> 1,5 мл полистирола, 2 ясно сторон </td></tr><tr><td> Шприц </td><td> Becton Dickinson </td><td> 309585 </td><td> Стерильные, 3 мл Luer-Lok </td></tr><tr><td> Шприц фильтр </td><td> Nalgene </td><td> 190-2520 </td><td> 0,2 мкм стерильные ацетата целлюлозы </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 a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

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

Metabolomic профиля<em&gt; Микобактерий туберкулеза</em&gt; Определяется после роста в бульоне культуры. Условия могут быть изменены, чтобы проверить влияние пищевых добавок, окислители, и противотуберкулезных препаратов на метаболический профиль этого микроорганизма. Порядок подготовки экстракт применяют как для 1D<sup&gt; 1</sup&gt; H и 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C-ЯМР-анализа.

Пример подготовки<em&gt; Микобактерий туберкулеза</em&gt; Экстракты для ядерно-магнитного резонанса исследований 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

Principles of Nuclear Magnetic Resonance

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Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Рост Микобактерий туберкулеза Биопленки

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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

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

Мониторинг дендритных клеток с использованием миграции<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Магнитно-резонансная томография

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

Экспериментальная модель для изучения Туберкулез-малярией Коинфекция на естественной передачи Микобактерии туберкулеза И 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

Демонстрация с множественной лекарственной устойчивостью<em&gt; Микобактерии туберкулеза</em&gt; Усиление микрочипов

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

Роман микродиссекция подход к Восстановление<em&gt; Микобактерии туберкулеза</em&gt; Конкретные Стенограммы из фиксированных формалином и залитые парафином легких гранулемы

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

3D-человека легочной ткани Модель функциональных исследований по<em&gt; Микобактерии туберкулеза</em&gt; Инфекция

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

Система для эффективности и цитотоксичности скрининга ингибиторов Таргетинг внутриклеточных<em&gt; Микобактерии туберкулеза</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

Высокая пропускная способность Совместимость анализа для оценки эффективности против наркотиков пассировать макрофагов<em&gt; Микобактерии туберкулеза</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

Анализ 18FDG ПЭТ/КТ изображений как инструмент для обучения микобактерии инфекции и лечение в Non-human приматов

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