Los autores desean agradecer a todos los miembros de los laboratorios del Dr. Barletta y Powers Dr. útiles comentarios durante el desarrollo del protocolo. Damos las gracias a Wendy Austin útil para los debates y correcciones del manuscrito. El trabajo que se describe en este manuscrito fue financiado por becas piloto de semillas para cada investigador mencionado anteriormente de la Universidad de Nebraska-Lincoln Center Biología redox (padre subvención # 017675 2P20RR CNRR, D. Becker, PI). Además, agradecemos a la Dra. Ofelia Chacón para el suministro de fondos de la subvención R21 (1R21AI087561-01A1) para suministros de investigación y de apoyo a sueldo parcial Sr. Halouska para estandarizar técnicas de RMN incluyen en esta publicación.

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+extraction+method+for+mycobacterial+metabolome+analysis.">Development of an extraction method for mycobacterial metabolome analysis.</a> J. Pharm. Biomed. Anal. 41, 196-200 (2006).</li><li>de Carvalho, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+of+Mycobacterium+tuberculosis+reveals+compartmentalized+co-catabolism+of+carbon+substrates.">Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates.</a> Chem. Biol. 17, 1122-1131 (2010).</li><li>de Carvalho, L. 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U.S.A. 107, 9819-9824 (2010).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+metabolism+in+Mycobacterium+smegmatis+during+the+transition+from+O2-rich+to+O2-poor+conditions+as+studied+by+isotopomer-assisted+metabolite+analysis.">Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis.</a> Biotechnol. Lett. 31, 1233-1240 (2009).</li><li>Kweon, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polyomic+approach+to+elucidate+the+fluoranthene-degradative+pathway+in+Mycobacterium+vanbaalenii+PYR-1.">A polyomic approach to elucidate the fluoranthene-degradative pathway in Mycobacterium vanbaalenii PYR-1.</a> J. Bacteriol. 189, 4635-4647 (2007).</li><li>Hasan, M. 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No hay conflictos de interés declarado.

Un gran número de estudios han analizado los perfiles transcriptomic y proteómicos de M. tuberculosis bajo una variedad de ensayos in vitro e in vivo. 11-16 En última instancia, los cambios en la expresión génica y la actividad enzimática conducen a variaciones en las concentraciones de las pequeñas moléculas de peso molecular. La descripción completa de estos compuestos constituye el metaboloma. Por lo tanto, los efectos de las drogas y las diferentes condiciones ...

<table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> ADC Enriquecimiento </td><td> BD BBL Middlebrook </td><td> 212352 </td><td></td></tr><tr><td> BACS-120 Cambiador de muestras </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> Albúmina de suero bovino </td><td> Fisher Scientific </td><td> BP1600-100 </td><td> Fracción V </td></tr><tr><td> Centrífugo </td><td> Beckman Coulter </td><td> Allegra X-15R </td><td> Benchtop </td></tr><tr><td> Tubos de centrífuga </td><td> Corning </td><td> 430291 </td><td> 50 ml estéril de polipropileno </td></tr><tr><td> Viales criogénicos </td><td> Corning </td><td> 430488 </td><td> 2,0 ml estéril de polipropileno </td></tr><tr><td> Cycloheximide </td><td> AG Scientific </td><td> C-1189 </td><td> Tóxico </td></tr><tr><td> D (+) - Glucosa </td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td> Óxido de deuterio </td><td> Sigma Aldrich </td><td> 617385 </td><td></td></tr><tr><td> Erlenmeyer Flask </td><td> VWR </td><td> 89095-266 </td><td> Base de estéril, planas, policarbonato, 0,22 m tapa de la membrana PTFE con ventilación </td></tr><tr><td> Frasco de Flash Freeze </td><td> VWR </td><td> 82018-226 </td><td> 750 ml </td></tr><tr><td> Secador en frío </td><td> VWR </td><td> 82019-038 </td><td> 4,5 l de sobremesa </td></tr><tr><td> Glicerol </td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td> Incubadora </td><td> New Brunswick </td><td> Innova 40 </td><td> Benchtop agitador </td></tr><tr><td> Lisis Matriz B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td> Lisis de la máquina </td><td> MP Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td> Microcentrífuga </td><td> Eppendorf </td><td> 5415D </td><td> Benchtop </td></tr><tr><td> Microcentrífuga </td><td> Beckman Coulter </td><td> Microfuge 22R </td><td> Benchtop </td></tr><tr><td> Middlebrook 7H9 caldo </td><td> Difco </td><td> 271310 </td><td></td></tr><tr><td> Tubos de RMN </td><td> Norell </td><td> ST500-7 </td><td> 5 mM </td></tr><tr><td> El enriquecimiento OADC </td><td> BD BBL Middlebrook </td><td> 212351 </td><td></td></tr><tr><td> Ácido Oleico </td><td> Sigma </td><td> O1008 </td><td></td></tr><tr><td> Fosfato de potasio dibásico </td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td> Fosfato de potasio monobásico </td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> Rotor - Microfuge 22R </td><td> Beckman Coulter </td><td> F241.5P </td><td> Sellado y polipropileno </td></tr><tr><td> Rotor - Allegra X-15R</td><td> Beckman Coulter </td><td> SX4750 </td><td> Con bio-certificados cubiertas </td></tr><tr><td> Cloruro de Sodio </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Sodio-3-trimethylsilylpropionate-2 ,2,3,3-D4 </td><td> Cambridge Isótopos </td><td> DLM-48 </td><td></td></tr><tr><td> Espectrofotómetro </td><td> Beckman Coulter </td><td> DU-530 </td><td></td></tr><tr><td> Cubetas </td><td> Línea de vida </td><td> LS-2410 </td><td> 1,5 ml de poliestireno, 2 lados claros </td></tr><tr><td> Jeringa </td><td> Becton Dickinson </td><td> 309585 </td><td> Estéril, 3 ml Luer-Lok </td></tr><tr><td> Filtro de jeringa </td><td> Nalgene </td><td> 190-2520 </td><td> 0,2 micras acetato de celulosa 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 es la principal causa de mortalidad en los seres humanos a escala mundial. La aparición de ambos múltiples (MDR) y ampliamente-(XDR) de las cepas resistentes a los medicamentos amenaza con descarrilar los esfuerzos actuales de control de la enfermedad. Por lo tanto, existe una necesidad urgente de desarrollar fármacos y vacunas que son más eficaces que los disponibles actualmente. El genoma de M. tuberculosis se conoce desde hace más de 10 años, sin embargo hay importantes lagunas en nuestro conocimiento de la función de los genes y la esencialidad. Muchos estudios han utilizado desde entonces el análisis de la expresión génica a nivel transcriptomic y proteómicos para determinar los efectos de las drogas, oxidantes, y las condiciones de crecimiento en los patrones globales de expresión génica. En última instancia, la respuesta final de estos cambios se refleja en la composición metabólico de la bacteria incluyendo unos pocos miles de productos químicos de bajo peso molecular. Comparando los perfiles metabólicos de tipo salvaje y cepas mutantes, o bien no tratados o treated con un fármaco particular, puede efectivamente permitir la identificación del blanco y puede conducir al desarrollo de nuevos inhibidores con actividad antituberculosa. Del mismo modo, los efectos de dos o más condiciones en el metaboloma también se puede evaluar. Resonancia magnética nuclear (RMN) es una poderosa tecnología que se utiliza para identificar y cuantificar los intermediarios metabólicos. En este protocolo, los procedimientos para la preparación de M. extractos celulares tuberculosis para análisis de RMN se describen metabolómico. Los cultivos de células se cultivan en condiciones apropiadas y contención requerido nivel de bioseguridad 3, 1 recolectadas, y se sometieron a lisis mecánica, manteniendo las temperaturas frías para maximizar la conservación de los metabolitos. Los lisados ​​celulares se recuperó, se filtró esterilizada, y se almacena a temperaturas ultra-bajas. Partes alícuotas de estos extractos celulares se sembraron en agar Middlebrook 7H9 para unidades formadoras de colonias para verificar la ausencia de células viables. Tras dos meses de incubación a 37 ° C, si no vicolonias capaces se observan, las muestras se retiran de la instalación de contención para el procesamiento aguas abajo. Los extractos se liofilizaron, se resuspendieron en tampón deuterado y se inyectó en el instrumento de RMN, la captura de los datos espectroscópicos que se somete después a análisis estadístico. Los procedimientos descritos se pueden aplicar por tanto de una dimensión (1D) de RMN 1 H y de dos dimensiones (2D) 1 H-13 C análisis de RMN. Esta metodología proporciona más fiable identificación de pequeño peso molecular metabolito y análisis cuantitativos más fiables y sensibles de las composiciones de extracto de células metabólicas que los métodos cromatográficos. Las variaciones del procedimiento descrito después de la etapa de lisis celular también se puede adaptar para el análisis proteómico 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

El perfil de metabolómica<em&gt; Mycobacterium tuberculosis</em&gt; Se determina después de crecimiento en cultivos de caldo. Las condiciones se pueden variar para probar los efectos de los suplementos nutricionales, agentes oxidantes, y agentes contra la tuberculosis en el perfil metabólico de este microorganismo. Procedimiento para la preparación del extracto es aplicable tanto para 1D<sup&gt; 1</sup&gt; H y 2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C análisis de RMN.

Preparación de la muestra de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos de Estudios de Resonancia Magnética Nuclear metabolómica

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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15.3K vistas.  University of Nebraska-Lincoln. El perfil de metabolómica Mycobacterium tuberculosis Se determina después de crecimiento en cultivos de caldo. Las condiciones se pueden variar para probar los efectos de los suplementos nutricionales, agentes oxidantes, y agentes contra la tuberculosis en el perfil metabólico de este microorganismo. Procedimiento para la preparación del extracto es aplicable tanto para 1D 1 H y 2D 1 H- 13 C análisis d...

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.

Crecimiento de Mycobacterium tuberculosis Las biopelículas

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

Investigación

Inmunología e Infección

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.

De las Migraciones células dendríticas mediante resonancia 19F / 1H Magnetic

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.

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y 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

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.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

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.

Un modelo de tejido 3D Pulmón humano de Estudios funcionales en<em&gt; Mycobacterium tuberculosis</em&gt; Infección

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.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización 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

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.

Un ensayo compatible de alto rendimiento para evaluar la eficacia del fármaco contra los macrófagos a pases<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

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.

Análisis de 18FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

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.

Proyección de imagen de tuberculosis del Mycobacterium en ratones con reportero enzima fluorescencia

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

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modelado de la Tuberculosis de Mycobacterium marinum infección pez cebra adulto

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

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Crecimiento de Mycobacterium tuberculosis Las biopelículas

De las Migraciones células dendríticas mediante resonancia 19F / 1H Magnetic

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y Plasmodium berghei

Demostración de un resistente a múltiples fármacos

Un modelo de tejido 3D Pulmón humano de Estudios funcionales en

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular

Un ensayo compatible de alto rendimiento para evaluar la eficacia del fármaco contra los macrófagos a pases

Análisis de ¹⁸FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

Proyección de imagen de tuberculosis del Mycobacterium en ratones con reportero enzima fluorescencia

Modelado de la Tuberculosis de Mycobacterium marinum infección pez cebra adulto