فإن الكتاب أود أن أشكر جميع أعضاء مختبرات بارليتا الدكتور والدكتور القوى للتعليقات مفيدة عند وضع البروتوكول. نشكر ويندي لإجراء مناقشات مفيدة أوستن وتصحيح التجارب المطبعية للمخطوطة. وقد تم تمويل هذا العمل المبين في هذا المخطوط من المنح الرائدة البذور لكل محقق المذكورة أعلاه من جامعة نبراسكا لنكولن مركز علم الأحياء الأكسدة (الأم منحة # 017675 NCRR 2P20RR، D. بيكر، PI). وبالإضافة إلى ذلك، نشكر الدكتور أوفيليا شاكون لتوفير الأموال من منحة R21 لها (1R21AI087561-01A1) لوازم البحوث ودعم السيد Halouska في جزء من رواتب توحيد تقنيات NMR المدرجة في هذه النشرة.

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الإعلان عن أي تضارب في المصالح.

وقد حلل عدد كبير من الدراسات لمحات من البروتين وtranscriptomic M. السل في إطار مجموعة من في المختبر والمجراة في ظروف. 11-16 في نهاية المطاف، والتغيرات في التعبير الجيني ونشاط انزيم يؤدي إلى تغيرات في تركيزات الجزيئات الصغيرة الوزن الجزيئي. وصف كامل لهذه المركبا...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف / معدات </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> ADC التخصيب </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 عينة مبدل </td><td> بروكر </td><td></td><td></td></tr><tr><td> بروكر سميت بال المقدمة NMR </td><td> بروكر </td><td></td><td> 500 ميغاهيرتز </td></tr><tr><td> الأبقار مصل الزلال </td><td> فيشر العلمية </td><td> BP1600-100 </td><td> جزء V </td></tr><tr><td> نبذ </td><td> بيكمان كولتر </td><td> أليجرا X-15R </td><td> الفوق </td></tr><tr><td> الطرد المركزي أنابيب </td><td> كورنينج </td><td> 430291 </td><td> 50 مل العقيمة البولي بروبلين </td></tr><tr><td> المبردة قوارير </td><td> كورنينج </td><td> 430488 </td><td> 2.0 مل العقيمة البولي بروبلين </td></tr><tr><td> Cycloheximide </td><td> AG العلمية </td><td> C-1189 </td><td> سام </td></tr><tr><td> D (+) - الجلوكوز </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> الديوتيريوم أوكسيد </td><td> سيغما الدريتش </td><td> 617385 </td><td></td></tr><tr><td> قارورة مخروطي </td><td> VWR </td><td> 89095-266 </td><td> العقيمة، قاعدة مسطحة، والبولي، 0.22 ميكرومتر غطاء غشاء PTFE تنفيس </td></tr><tr><td> فلاش تجميد قارورة </td><td> VWR </td><td> 82018-226 </td><td> 750 مل </td></tr><tr><td> تجميد مجفف </td><td> VWR </td><td> 82019-038 </td><td> 4،5 L الفوق </td></tr><tr><td> الغليسيرول </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> حاضنة </td><td> نيو برونزويك </td><td> إنوفا 40 </td><td> الفوق شاكر </td></tr><tr><td> Lysing مصفوفة B </td><td> النائب Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> تحلل آلة </td><td> النائب Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td> ميكروسنتريفوج </td><td> إيبندورف </td><td> 5415D </td><td> الفوق </td></tr><tr><td> ميكروسنتريفوج </td><td> بيكمان كولتر </td><td> Microfuge 22R </td><td> الفوق </td></tr><tr><td> Middlebrook 7H9 مرق </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> NMR أنابيب </td><td> Norell </td><td> ST500-7 </td><td> 5MM </td></tr><tr><td> OADC التخصيب </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> حمض الأولييك </td><td> سيجما </td><td> O1008 </td><td></td></tr><tr><td> ثنائي القاعدة فوسفات البوتاسيوم </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> أحادى فوسفات البوتاسيوم </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> الدوار - Microfuge 22R </td><td> بيكمان كولتر </td><td> F241.5P </td><td> مختومة والبولي بروبلين </td></tr><tr><td> الدوار - أليجرا X-15R</td><td> بيكمان كولتر </td><td> SX4750 </td><td> مع الحيوي المعتمدة أغطية </td></tr><tr><td> كلوريد الصوديوم </td><td> فيشر العلمية </td><td> S271-3 </td><td></td></tr><tr><td> الصوديوم-3-trimethylsilylpropionate-2-D4 ،2،3،3 </td><td> كامبريدج النظائر </td><td> DLM-48 </td><td></td></tr><tr><td> الطيف </td><td> بيكمان كولتر </td><td> DU-530 </td><td></td></tr><tr><td> الطيف Cuvettes </td><td> حبل النجاة </td><td> LS-2410 </td><td> 1،5 مل البوليسترين، 2 الجانبين واضحة </td></tr><tr><td> محقنة </td><td> بيكتون ديكنسون </td><td> 309585 </td><td> العقيمة، 3 مل، بالتركيبة لوك </td></tr><tr><td> حقنة تصفية </td><td> Nalgene </td><td> 190-2520 </td><td> 0.2 ميكرومتر خلات السليلوز العقيمة </td></tr><tr><td> توين 80 </td><td> فيشر العلمية </td><td> BP338-500 </td><td></td></tr></tbody></table>

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

المتفطرة السلية هو السبب الرئيسي للوفيات في البشر على نطاق عالمي. ظهور متعددة على حد سواء (MDR)، وعلى نطاق واسع-(XDR) سلالات مقاومة للعقاقير تهدد بعرقلة الجهود الحالية لمكافحة الأمراض. وبالتالي، هناك حاجة ملحة لتطوير أدوية واللقاحات التي هي أكثر فعالية من تلك المتاحة حاليا. جينوم M. وقد عرف السل لأكثر من 10 سنوات، ولكن هناك ثغرات هامة في معرفتنا وظيفة الجين والإستخدامات الأساسية. وقد استخدمت العديد من الدراسات منذ الجين التعبير التحليل على الصعيدين transcriptomic والبروتين لتحديد آثار المخدرات، تأكسد، وظروف النمو على الأنماط العالمية في التعبير الجيني. في نهاية المطاف، ينعكس الرد النهائي من هذه التغييرات في تكوين التمثيل الغذائي للبكتيريا بما في ذلك المواد الكيميائية ألف قليل الوزن الجزيئي. مقارنة ملامح التمثيل الغذائي من النوع البري وسلالات متحولة، إما غير المعالجة أو آرeated مع دواء معين، يمكن أن تسمح تحديد الهدف بشكل فعال وربما يؤدي إلى تطوير مثبطات الرواية المضادة للنشاط السل. وبالمثل، يمكن أيضا آثار اثنين أو أكثر من الظروف على أن metabolome المقررة. الرنين النووي المغناطيسي (NMR) هي تقنية قوية التي يتم استخدامها لتحديد وقياس وسيطة الأيض. في هذا البروتوكول والإجراءات لإعداد M. ويرد وصف مقتطفات خلية للتحليل السل metabolomic NMR. تزرع مزارع الخلايا تحت الظروف المناسبة والمطلوبة للسلامة الأحيائية مستوى الاحتواء 3، 1 المقطوع، وتعرض للتحلل الميكانيكية مع الحفاظ على درجات الحرارة الباردة لتحقيق أقصى قدر من الحفاظ الأيضات. يتم استرداد لست] الخلية، التي تمت تصفيتها تعقيمها، وتخزينها في درجات حرارة منخفضة للغاية. ومطلي مأخوذة من هذه المقتطفات الخلية على أجار Middlebrook 7H9 لتشكيل مستعمرة وحدة للتحقق من عدم وجود خلايا قابلة للحياة. على شهرين من الحضانة عند 37 درجة مئوية، إذا لم السادسويلاحظ المستعمرات قادرة، تتم إزالة عينات من منشأة الاحتواء للالتجهيز النهائي. ومجفف بالتجميد مقتطفات، معلق في المخزن المؤقت بالديوتيريوم وحقن في الصك NMR، التقاط البيانات الطيفية التي يتعرض بعد ذلك إلى التحليل الإحصائي. ويمكن تطبيق الإجراءات الموضحة لكلا أحادية البعد (1D) NMR H 1 وثنائية الأبعاد (2D) 1 H-13 C NMR التحليلات. هذه المنهجية توفر أكثر موثوقية الصغيرة تحديد الوزن الجزيئي المستقلب والتحليلات الكمية وأكثر موثوقية من التراكيب الحساسة استخراج الخلايا من الطرق الأيضية الكروماتوغرافي. ويمكن أيضا أشكال مختلفة من الإجراءات الموصوفة في أعقاب خطوة تحلل الخلية تكييفها لتحليل البروتين موازية.

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

الملف الشخصى metabolomic من<em&gt; المتفطرة السلية</em&gt; يتم تحديد بعد النمو في الثقافات مرق. يمكن أن تختلف الشروط لاختبار تأثير من المكملات الغذائية، تأكسد، والأدوية المضادة للسل على الملف الشخصي لهذا التمثيل الغذائي الكائنات الحية الدقيقة. إجراءات إعداد مستخلص ينطبق على حد سواء 1D<sup&gt; 1</sup&gt; H و 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR التحليلات.

تحضير العينة من<em&gt; المتفطرة السلية</em&gt; خلاصات لدراسات النووية المغناطيسي الرنين 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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15.2K Views.  University of Nebraska-Lincoln. الملف الشخصى metabolomic من المتفطرة السلية يتم تحديد بعد النمو في الثقافات مرق. يمكن أن تختلف الشروط لاختبار تأثير من المكملات الغذائية، تأكسد، والأدوية المضادة للسل على الملف الشخصي لهذا التمثيل الغذائي الكائنات الحية الدقيقة. إجراءات إعداد مستخلص ينطبق ?...

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

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.

نمو المتفطرة السلية الأغشية الحيوية

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.

رصد هجرة الخلايا الجذعية باستخدام<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H التصوير بالرنين المغناطيسي

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.

نموذج تجريبي لدراسة الملاريا والسل عدوى مرافقة على انتقال الطبيعية السل المتفطرة و المتصورة 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

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.

مما يدل على متعدد المخدرات مقاومة<em&gt; السل المتفطرة</em&gt; التضخيم ميكروأري

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.

مقاربة الرواية تسليخ مجهري ليتعافى<em&gt; السل المتفطرة</em&gt; نصوص محددة من الفورمالين الثابتة جزءا لا يتجزأ من البارافين الرئة أورام حبيبية

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.

نموذج الأنسجة 3D الرئة الإنسان للدراسات وظيفية على<em&gt; المتفطرة السلية</em&gt; العدوى

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.

نظام لفعالية والسمسة فحص مثبطات استهداف بين الخلايا<em&gt; المتفطرة السلية</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.

وإنتاجية عالية الفحص المدعم لتقييم فعالية الدواء ضد بلعم Passaged<em&gt; المتفطرة السلية</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.

تحليل 18"فدج التصوير" PET/CT كأداة لدراسة بكتريا السل العدوى والعلاج في الإنسان عدم الرئيسات

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

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

تصوير بكتريا السل في الفئران مع "مراسل إنزيم الأسفار"

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...