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

Yazarlar protokolü geliştirirken yararlı yorumlar için Dr Barletta ve Dr Güçlerin laboratuvarlarının tüm üyelerine teşekkür etmek istiyorum. Biz el yazması yararlı tartışmalar ve redaksiyon için Wendy Austin ederim. Bu yazıda anlatılan çalışma Nebraska-Lincoln Redoks Biyolojisi Merkezi (ebeveyn hibe # NCRR 2P20RR 017675, D. Becker, PI) University of Yukarıda listelenen her bir araştırmacı için tohum Pilot hibe tarafından finanse edildi. Ayrıca, araştırma malzemeleri ve bu yayına dahil NMR teknikleri standardize Bay Halouska en kısmi maaş desteği için ona R21 hibe (1R21AI087561-01A1) fon sağlamak için Dr Ofelia Chacon ederim.

1. Protokol Metni Bu protokol M. NMR metodolojisinin adaptasyonu vurgulamaktadır Tüberküloz (Sınıf III ajan). M. yaparken nedenle, Biyogüvenlik Seviyesi 3 (BSL3) uygulamaları takip edilmesi gereken bir yıllık sertifikalı laboratuvarda tüberküloz araştırma. Laboratuvar tarafından oluşturulan aerosoller Maruziyet bu mikroorganizmalar ile çalışan personel tarafından karşılaşılan en önemli tehlikedir. Aşağıdaki yordamlar Kurumumuzda y...

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Çalışmaların önemli bir kısmı M. Transkriptomik ve proteomik profilleri analiz var in vitro ve in vivo koşullarında çeşitli altında tüberküloz. 11-16 Sonuçta, gen ekspresyonu ve enzim aktivitesinde değişiklikler küçük molekül ağırlıklı moleküllerin konsantrasyonları değişikliklere yol açmaktadır. Bu bileşiklerin tam açıklaması metabolome oluşturmaktadır. Böylece, metabolik yolaklar uyuşturucu ve değişen büyüme koşullarına etkiler...

<table border="1"><tbody><tr><td> Reaktif / Ekipman Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> ADC Zenginleştirme </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 Numune Değiştirici </td><td> Bruker </td><td></td><td></td></tr><tr><td> NMR Bruker Avance </td><td> Bruker </td><td></td><td> 500 MHz </td></tr><tr><td> Bovine Albumin Serum </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Fraksiyon V </td></tr><tr><td> Santrifüj </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Tezgahüstü </td></tr><tr><td> Santrifüj Tüpleri </td><td> Corning </td><td> 430291 </td><td> 50 ml steril polipropilen </td></tr><tr><td> Kriyojenik Flakon </td><td> Corning </td><td> 430488 </td><td> 2.0 ml steril polipropilen </td></tr><tr><td> Cycloheximide </td><td> AG Bilimsel </td><td> C-1189 </td><td> Zehirli </td></tr><tr><td> D (+) - Glikoz </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Ağır su </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Erlenmeyer Flask </td><td> VWR </td><td> 89095-266 </td><td> Steril, düz taban, polikarbonat, 0.22 mikron PTFE membran havalandırmalı kapak </td></tr><tr><td> Flaş Freeze Flask </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Kurutucu dondur </td><td> VWR </td><td> 82019-038 </td><td> 4.5 L Benchtop </td></tr><tr><td> Gliserin </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Kuluçka makinesi </td><td> New Brunswick </td><td> Innova 40 </td><td> Tezgahüstü çalkalayıcı </td></tr><tr><td> Parçalama Matris B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> Lizis Makinesi </td><td> MP Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td> Mikrosantrifüj </td><td> Eppendorf </td><td> 5415D </td><td> Tezgahüstü </td></tr><tr><td> Mikrosantrifüj </td><td> Beckman Coulter </td><td> Mikrofuge&#39;de 22R </td><td> Tezgahüstü </td></tr><tr><td> Middlebrook 7H9 Broth </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> NMR tüpler </td><td> Norell </td><td> ST500-7 </td><td> 5mM </td></tr><tr><td> OADC Zenginleştirme </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Oleik Asit </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Potasyum fosfat dibazik </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Potasyum Fosfat mono </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Rotor - mikrofuge&#39;de 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Mühürlü ve polipropilen </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Biyo-sertifikalı kapaklar ile </td></tr><tr><td> Sodyum klorür </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Sodyum-3-trimethylsilylpropionate-2 ,2,3,3-D4 </td><td> Cambridge İzotop </td><td> DLM-48 </td><td></td></tr><tr><td> Spektrofotometre </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Spektrofotometre Küvetleri </td><td> Hayat çizgisi </td><td> LS-2410 </td><td> 1.5 ml polistiren, 2 açık taraf </td></tr><tr><td> Şırınga </td><td> Becton Dickinson </td><td> 309585 </td><td> Steril, 3 ml Luer-Lok </td></tr><tr><td> Şırınga Filtre </td><td> Nalgene </td><td> 190-2520 </td><td> 0.2 um steril selüloz asetat </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 küresel ölçekte insan mortalitenin önemli bir nedenidir. Hem çok (MDR) ve yaygın-(XDR) ilaca dirençli suşların ortaya çıkması mevcut hastalık kontrol çabalarını rayından tehdit ediyor. Böylece, şu anda mevcut olanlardan daha etkili ilaçlar ve aşılar geliştirmek için acil bir ihtiyaç vardır. M. genom tüberküloz fazla 10 yıldır bilinen, fakat gen fonksiyonu ve zorunluluk bilgimizi önemli boşluklar bulunmaktadır olmuştur. Birçok çalışma bu yana gen ifadesinin küresel desenleri ile ilgili ilaçlar, oksidanlar ve büyüme koşullarının etkisini belirlemek amacıyla Transkriptomik ve proteomik düzeyde hem de gen ekspresyon analizi kullandık. Sonuçta, bu değişikliklerin nihai yanıtını birkaç bin küçük moleküler ağırlıklı kimyasal maddeler de dahil olmak üzere bakterinin metabolik kompozisyonu yansıtılır. Tedavi edilmeyen veya tr ya da yabani tip ve mutant suşlarının metabolik profilleri KarşılaştırılmasıBelirli bir ilaç ile eated, etkili bir hedef tespit edilmesini sağlar ve antitüberküloz aktivitesi olan yeni inhibitörlerinin gelişimine neden olabilir. Aynı şekilde, metabolome üzerinde iki ya da daha fazla koşulların etkileri de tespit edilebilir. Nükleer manyetik rezonans (NMR) metabolik ara tanımlamak ve ölçmek için kullanılan güçlü bir teknolojidir. M. hazırlanması için bu protokol, prosedürler NMR metabolomik analiz için tüberküloz hücre ekstreleri tarif edilmektedir. Metabolitlerin korunması maksimize etmek için soğuk sıcaklıkları korurken hücre kültürleri uygun koşullar ve gerekli Biyogüvenlik Seviyesi 3 çevreleme, 1 hasat altında yetiştirilen ve mekanik lizis tabi tutulur. Hücre lizatları kurtarıldı, steril ve ultra-düşük ısılarda saklanan filtrelendi. Bu hücre özleri alikotlar canlı hücrelerinin yokluğu doğrulamak için koloni oluşturan birim için Middlebrook 7H9 agarda kaplanır. 37 inkübasyon iki ay ° C, eğer üzerine hiçbir vimümkün koloniler gözlenir, numune üretim prosesleri için tecrit tesisine kaldırılır. Özler, liyofilize edildi döteryumlanmış tampon içinde tekrar süspanse edilir ve daha sonra istatistiksel analize tabi tutulur spektroskopik verileri yakalamak, NMR aleti olarak enjekte edilir. Anlatılan prosedürler (1D) tek boyutlu 1 H NMR ve iki-boyutlu (2B) 1 H-13 C NMR analizi her ikisi için de uygulanabilir. Bu metodoloji kromatografik yöntemlere göre daha güvenilir küçük molekül ağırlığı metabolit tespiti ve hücre ekstresi metabolik kompozisyonlar daha güvenilir ve duyarlı kantitatif analizler sağlar. Işlem varyasyonları hücre parçalama aşamasını takiben de tarif paralel Proteomik analizleri için adapte edilebilir.

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

Ve metabolomik profili<em&gt; Mycobacterium tuberculosis</em&gt; Broth kültürleri büyüme sonrasında belirlenir. Koşullar bu mikroorganizmanın metabolik profil üzerinde besin takviyeleri, oksidanlar ve anti-tüberküloz ilaçların etkilerini test etmek için değişik olabilir. Özütü hazırlanması için prosedür 1D her ikisi için de geçerlidir<sup&gt; 1</sup&gt; H ve 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR analizleri.

Numune Hazırlama<em&gt; Mycobacterium tuberculosis</emNükleer Manyetik Rezonans metabolomik Araştırmaları&gt; özler

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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Microtiter Plate Format Broth Seyreltme Birinci ve İkinci Line İlaçları Mycobacterium tuberculosis kompleksi Antimikrobiyal Duyarlılık Testi

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

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

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Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon 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

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Kurtarma Bir Roman Mikrodiseksiyon Yaklaşımı<em&gt; Mycobacterium tuberculosis</emAkciğer granülomlara Gömülü Formalinle Sabit Parafin dan&gt; Özel Transkript

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

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Fonksiyonel Araştırmaları 3D İnsan Akciğer Doku Modeli<em&gt; Mycobacterium tuberculosis</em&gt; Enfeksiyon

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 ...

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Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<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

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Bir Yüksek verim Uyumlu Deneyi Makrofaj, pasajlamaya karşı ilaç etkinliğini değerlendirmektir<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

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