著者らは、プロトコルを開発しながら有益なコメントを博士バルレッタ博士とパワーズの研究室のすべてのメンバーに感謝したいと思います。我々は、原稿の役立つ議論と校正のためにウェンディオースティンに感謝します。本稿で説明する作業は、ネブラスカ州リンカーンレドックス生物学センター（親助成＃NCRR 2P20RR 017675、D.ベッカー、PI）の大学から上記の各調査官にシードパイロット補助金によって賄われていた。加えて、我々は研究を供給し、このパブリケーションに含まNMR法を標準化するために、氏Halouskaの部分的な給与のサポートのために彼女のR21を助成（1R21AI087561-01A1）から資金を提供するための博士OFELIAチャコンに感謝します。

<ol>
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特別な利害関係は宣言されません。

研究のかなりの数がMのトランスクリプトームとプロテオームのプロファイルを分析してきたin vitroおよび in vivo条件下での様々な環境下で結核 。11月16日最終的には、遺伝子発現と酵素活性の変化は、小分子量の分子の濃度の変動につながる。これらの化合物の完全な説明は、メタボロームを構成している。このように、代謝経路に薬や様々な成長条件の影?...

<table border="1"><tbody><tr><td> 試薬/機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> ADCの充実</td><td> BD BBLミドル</td><td> 212352 </td><td></td></tr><tr><td> BACS-120サンプルチェンジャー</td><td>ブルカー</td><td></td><td></td></tr><tr><td>ブルカーアヴァンセNMR </td><td>ブルカー</td><td></td><td> 500 MHzの</td></tr><tr><td>ウシ血清アルブミン</td><td>フィッシャー·サイエンティフィック</td><td> BP1600-100 </td><td>フラクションV </td></tr><tr><td>遠心</td><td>ベックマン·コールター</td><td>アレグラ、X-15R </td><td>ベンチトップ</td></tr><tr><td>遠心管</td><td>コー​​ニング</td><td> 430291 </td><td> 50ミリリットル滅菌ポリプロピレン</td></tr><tr><td>クライオジェニックバイアル</td><td>コー​​ニング</td><td> 430488 </td><td> 2.0ミリリットル滅菌ポリプロピレン</td></tr><tr><td>シクロヘキサンximide </td><td> AGの科学</td><td> C-1189 </td><td>毒性</td></tr><tr><td> D（+） - グルコース</td><td>アクロス</td><td> 41095-0010 </td><td></td></tr><tr><td>酸化ジュウテリウム</td><td>シグマアルドリッチ</td><td> 617385 </td><td></td></tr><tr><td>三角フラスコ</td><td> VWR </td><td> 89095-266 </td><td>滅菌、フラットベース、ポリカーボネート、0.22μmのPTFE膜ベントキャップ</td></tr><tr><td>フラッシュフリーズフラスコ</td><td> VWR </td><td> 82018-226 </td><td> 750ミリリットル</td></tr><tr><td>凍結乾燥機</td><td> VWR </td><td> 82019-038 </td><td> 4.5 Lのベンチ</td></tr><tr><td>グリセロール</td><td>ギブコBRL </td><td> 15514-029 </td><td></td></tr><tr><td>インキュベーター</td><td>ニューブランズウィック州</td><td>イノーバ40 </td><td>ベンチシェーカー</td></tr><tr><td>行列Bを溶解</td><td> MPバイオメディカル</td><td> 6911-100 </td><td></td></tr><tr><td>溶解機</td><td> MPバイオメディカル</td><td> FastPrep-24 </td><td></td></tr><tr><td>遠心</td><td>エッペンドルフ</td><td> 5415D </td><td>ベンチトップ</td></tr><tr><td>遠心</td><td>ベックマン·コールター</td><td>微量22R </td><td>ベンチトップ</td></tr><tr><td>ミドル7H9ブロス</td><td>ディフコ</td><td> 271310 </td><td></td></tr><tr><td> NMR管</td><td>ノレル</td><td> ST500-7 </td><td> 5mMの</td></tr><tr><td> OADC濃縮</td><td> BD BBLミドル</td><td> 212351 </td><td></td></tr><tr><td>オレイン酸</td><td>シグマ</td><td> O1008 </td><td></td></tr><tr><td>リン酸二カリウム</td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td>リン酸二水素カリウム</td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td>ローター - 微量22R </td><td>ベックマン·コールター</td><td> F241.5P </td><td>密封されたとポリプロピレン</td></tr><tr><td>ローター - アレグラX-15R</td><td>ベックマン·コールター</td><td> SX4750 </td><td>バイオ認定のカバー付き</td></tr><tr><td>塩化ナトリウム</td><td>フィッシャー·サイエンティフィック</td><td> S271-3 </td><td></td></tr><tr><td>ナトリウム3 - トリメチルシリル-2,2,3,3-D4 </td><td>ケンブリッジアイソトープ</td><td> DLM-48 </td><td></td></tr><tr><td>分光光度計</td><td>ベックマン·コールター</td><td> DU-530 </td><td></td></tr><tr><td>分光光度計のキュベット</td><td>命綱</td><td> LS-2410 </td><td> 1.5 mlのポリスチレン、2明確な側面</td></tr><tr><td>注射器</td><td>ベクトン·ディッキンソン</td><td> 309585 </td><td>滅菌、3ミリリットルルアー</td></tr><tr><td>シリンジフィルター</td><td>ナルゲン</td><td> 190-2520 </td><td> 0.2μmの無菌酢酸セルロース</td></tr><tr><td>ツイーン80 </td><td>フィッシャー·サイエンティフィック</td><td> BP338-500 </td><td></td></tr></tbody></table>

sample preparation of mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies

結核菌は、地球規模での人間の死亡率の主要な原因である。両方のマルチ（MDR）と広範囲に（XDR）の薬剤耐性株の出現は、現在の疾病制御の努力を脱線させると脅している。したがって、現時点で入手可能なものよりも有効である薬やワクチンの開発が急務とされている。 Mのゲノム結核は、10年以上のために知られて、まだ遺伝子の機能と本質に関する我々の知識の重要なギャップがあるされています。多くの研究では、以来、遺伝子発現の全体的なパターンに対する薬剤、酸化剤、および増殖条件の影響を決定するためにトランスクリプトームとプロテオームの両方のレベルで遺伝子発現解析を用いてきた。最終的に、これらの変更の最終的な応答は、数千の小さな分子量の化学物質を含む細菌の代謝物に反映されています。未処理またはTRか、野生型と変異株の代謝プロファイルを比較特定の薬物とeated、効果的にターゲットの識別を可能にすることができ、抗結核活性を持つ新規阻害剤の開発につながる可能性があります。同様に、メタボローム上の2つ以上の条件の影響も評価することができる。核磁気共鳴（NMR）は代謝中間体を同定し、定量化するために使用される強力な技術です。このプロトコルでは、Mの準備のための手順NMRメタボローム解析のための結核の細胞抽出物が記載されている。代謝物の保全を最大にするために低温を維持しながら、細胞培養物は、適切な条件と必要な生物学的安全性レベル3封じ込め、1収穫の下で栽培され、機械的な溶解に供される。細胞溶解物を回収し、滅菌濾過し、超低温で保存されています。これらの細胞抽出物からのアリコートを、生細胞の有無を確認するためのコロニー形成単位のためのミドル7H9寒天上にメッキが施されています。 37℃でのインキュベーションの際に二ヶ月℃、無viの場合できるコロニーが観察され、サンプルが下流の処理のための封じ込め施設から削除されます。抽出物は、凍結乾燥重水素化緩衝液に再懸濁し、その後統計的分析に供されている分光データをキャプチャし、NMR装置に注入される。説明する手順は、1次元（1D）1 H NMRおよび2次元（2D）の1 H-13 C NMR分析の両方に適用することができます。この方法論は、クロマトグラフ法よりも信頼性の高い低分子量代謝物の同定と細胞抽出物の代謝物のより信頼性が高く、敏感な定量的な分析を提供しています。細胞溶解工程の後に記載された手順のバリエーションも平行プロテオーム解析に適合させることができる。

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

のメタボロームプロファイル<em&gt;結核菌</em&gt;ブロス培養で成長した後に決定されています。条件は、この微生物の代謝プロファイルに栄養補助剤、酸化剤、抗結核剤の効果をテストするために変化させることができる。抽出物調製のための手順は、1Dの両方に適用されます<sup&gt; 1</sup&gt; Hと2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR分析。

のサンプル調製<em&gt;結核菌</em核磁気共鳴代謝研究のための&gt;抽出

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

sample-preparation-mycobacterium-tuberculosis-extracts-for-nuclear

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Analytical Chemistry

Immunology and Infection

Unit 1

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

Principles of Nuclear Magnetic Resonance

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15.2K ビュー.  University of Nebraska-Lincoln. のメタボロームプロファイル結核菌ブロス培養で成長した後に決定されています。条件は、この微生物の代謝プロファイルに栄養補助剤、酸化剤、抗結核剤の効果をテストするために変化させることができる。抽出物調製のための手順は、1Dの両方に適用されます 1 Hと2D 1 H- 13 C NMR分析。

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

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

免疫・感染学

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

の成長結核バイオフィルム

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

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

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

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

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

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</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

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

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

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 common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

回復への新規マイクロダイセクションアプローチ<em&gt;結核菌</em肺肉芽腫、ホルマリン固定パラフィン包埋から&gt;特異的転写

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

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 understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

上の機能的研究のための3Dヒト肺組織モデル<em&gt;結核菌</em&gt;感染

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

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</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

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</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

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT イメージング勉強結核感染と非ひと霊長類における治療のためのツールとしての解析

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