Los autores desean agradecer a las siguientes NIH premios / sub-premios para el apoyo de esta investigación: R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, y P51OD011104.

1. Calibrar Instrumento Mesodissection con 2ID Imaging Software El software de imágenes 2ID se conoce como el software o el programa para el resto de este artículo. Este paso es necesario para seguir correctamente la imagen, así como alinear varios marcos de diapositivas. <ol><li> Encienda el instrumento y el ordenador. </li><li> Abra el software y seleccione &quot;instrumento de calibración&quot;. </li><li> Calibrar viajes etapa con el joystick. Mueva el escenario a la posición superior izquierda. Pulse el botón &quot;set&quot; en el paso 1 en el programa. </li><li> Mueva el escenario para bajar la posición correcta. Pulse el botón &quot;set&quot; en el paso 2 en el programa. </li><li> Calibrar la aspiración pulsando primero &quot;Z-button&quot; para recaudar conjunto de la cabeza. Pulse el botón &quot;pulso aspirado&quot; y el botón &quot;disección&quot; en la palanca de mando hasta que la varilla de control de émbolo alcanza la primera posición. Pulse el botón &quot;set&quot; en el paso 3 del programa. </li><li> Pulse el botón &quot;reinicio aspirado y# 8221; botón. Una vez que el émbolo se detiene en la posición de la prensa &quot;conjunto&quot; de abajo en el paso 4. </li><li> Haga clic en la pestaña de &quot;escala&quot;. </li><li> Coloque la regla de calibración en el lado blanco. </li><li> Centrarse microscopio si es necesario. </li><li> Dibuja una línea entre las dos barras de escala a través del ratón. </li><li> Introduzca la distancia en el recuadro paso 2 botón. Presione &quot;compute&quot; etiquetados. </li><li> Haga clic en la pestaña de &quot;punto de mira&quot;. Gire la velocidad del motor a 1. </li><li> Inserte xScisor en el conjunto de cabeza. Conjunto de la cabeza inferior en el eje z posición lista pulsando el botón &quot;eje Z&quot;. NOTA: El conjunto de la cabeza es la gran estructura en la parte superior del instrumento de disección. El xScisor se conoce como un poco molino de consumible para el resto de este artículo. </li><li> Haga clic en el botón &quot;disección de participar&quot; en la palanca de mando. NOTA: Este botón se encuentra en la parte superior de la palanca de mando. </li><li> Definir centro de rotación visual. Para ello, mueva centro del punto de mira sobre el centro de giro con definird parámetro en el paso 1 del programa. Use la flechas arriba y abajo en la &quot;x&quot; y los bares &quot;eje y&quot; para ajustar los píxeles para alinear en forma de cruz. Haga clic en la pestaña de &quot;hecho&quot;. </li><li> Haga clic en el icono de &quot;casa&quot;. NOTA: Esto se representa una imagen de la casa ubicada en la esquina superior derecha cerca. </li></ol> 2. Crear Imagen de referencia <ol><li> Coloque H &amp; E diapositivas en el escenario. Coloque la tapa opaca en la parte superior de la diapositiva. Conducir el instrumento moviendo el joystick hacia la zona deseada de la corredera H &amp; E. Guarda la imagen generada. NOTA: Cualquiera de la pestaña &quot;Referencia de captura&quot; o pestaña &quot;diseccionar&quot; se puede utilizar para completar este paso. </li></ol> 3. Alinee Paño para Disección <ol><li> Haga clic en la opción &quot;diseccionar tejido&quot; en la pantalla principal. </li><li> Rellene el &quot;operador&quot;, &quot;número de la disección de la adhesión&quot;, &quot;el número de acceso de referencia&quot;, &quot;número de código de barras xScisor,&quot; &amp; #8220; disección número de lote fluida &quot;,&quot; Tamaño xScisor &quot;y&quot; Descripción &quot;en la pestaña&quot; Configuración &quot;. </li><li> Haga clic en la pestaña de &quot;encontrar el tejido&quot;. </li><li> Importación de imágenes de referencia previamente guardados. </li><li> Coloque una diapositiva FFPE sin manchas de 5 micras de espesor en el escenario. Mueva etapa a la misma zona que el representado en la imagen de referencia. Haga clic en la pestaña &quot;Insertar imagen&quot;. </li><li> Haga clic en la pestaña &quot;align&quot;. Usa el ratón y las flechas asociadas para alinear la imagen de referencia a la imagen sin mancha. </li></ol> 4. Anotar Slide <ol><li> Haga clic en la pestaña de &quot;anotar&quot;. </li><li> Utilice el ratón para dibujar un círculo alrededor del área deseada de la diapositiva que se microdissected. </li></ol> 5. Cargue consumible Mill Bit <ol><li> Rellene consumible poco molino con tampón deseado, aquí búfer PKD, tirando del émbolo hacia arriba y hacia abajo en un movimiento fluido varias veces. Asegúrese de excluir el aireburbujas. </li><li> Levante el eje Z por &quot;botón del eje Z&quot; presionando. </li><li> Cargar consumible poco molino en la máquina. Para ello, deslizando la fresa de consumibles en la parte superior de la máquina para que la línea blanca en las líneas de instrumentos con la línea de negro en la fresa de los consumibles. Pulse el botón de &quot;reinicio de aspiración&quot; para permitir que el émbolo sea bajado a la posición correcta. NOTA: Debe ser fácil para deslizar la fresa de los consumibles. Si no es así, asegúrese de que los indicadores estén alineados correctamente. </li><li> Bajo el eje Z pulsando de nuevo el botón &quot;eje Z&quot;. </li></ol> 6. Disección de tejidos (Figura 4) <ol><li> Abra la pestaña &quot;diseccionar&quot;. </li><li> Gire a velocidades de motor y aspiración a 1. </li><li> Marque la casilla &quot;espectáculo de seguimiento&quot;. Nota: Esto permite al usuario visualizar el área diseccionado durante el proceso de disección. </li><li> Una vez listo para diseccionar, continúe sosteniendo el botón &quot;participar&quot;, así como &quot;el aspirado y #8221; botón mientras se mueve la palanca de mando en una dirección en sentido antihorario para diseccionar el tejido. </li><li> Continuar para diseccionar en una dirección hacia la izquierda hasta el aspirado es pestaña &quot;lleno&quot; se vuelve rojo. </li></ol> 7. Vaciar y quitar consumible Mill Bit <ol><li> Coloque un tubo de microcentrífuga de 0,5 l bajo la punta de la fresa de los consumibles. </li><li> Varilla Press control &quot;aspiración&quot; rápidamente para expulsar la muestra en tubo de microcentrífuga. </li><li> Pulse con el aumento de la fuerza para expulsar usado fresa de consumible. </li><li> Enjuague fragmento de tejido tirando del émbolo hacia atrás. Empuje el émbolo hacia adelante para expresar la muestra restante en consumibles poco molino. El fragmento de tejido será ahora contenido dentro del tubo de microcentrífuga. </li></ol> 8. Muestra Lyse con proteinasa K digestión por un tratamiento térmico <ol><li> Añadir 70 l de TE pH 8,5 que contiene 5 g de proteinasa K a la muestra de tejido recuperada. </li><li> Coloque la muestra en programable calentador de agitador. </li><li> La digestión completa usando programable calentador-agitador a la siguiente configuración: 60 ˚ C durante 30 minutos a 1500 rpm; 82 ˚ C durante 15 min a 1500 rpm; y 25 ˚ C durante 1 minuto a 450 rpm. La muestra está ahora lista para aplicaciones posteriores. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis:+what+we+don't+know+can,+and+does,+hurt+us.">Tuberculosis: what we don't know can, and does, hurt us.</a> Science. 328 (5980), 852-856 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+fibrous+tissue.">Mesodissection of fibrous tissue.</a> AvanSciBio. , (2013).</li><li>Paige, C., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penitentiary+or+penthouse+condo:+the+tuberculous+granuloma+from+the+microbe's+point+of+view.">Penitentiary or penthouse condo: the tuberculous granuloma from the microbe's point of view.</a> Cell Microbiol. 12 (3), 301-309 (2010).</li><li>Mehra, S., Alvarez, X., Didier, P. 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Los autores dan a conocer que no hay intereses financieros en competencia.

Mesodissection es una técnica que puede aplicarse a la extracción de RNA a partir de diapositivas de las lesiones patológicas causadas por una amplia gama de patógenos. Es una necesidad que el usuario sigue el imagen y se alinea la imagen correctamente. Para lograr esto, el instrumento debe ser calibrado siguiendo las instrucciones en el software de imágenes. Cuando la disección, si la diapositiva y área de interés siendo diseccionado no se alinean, el usuario debe volver a calibrar el instrumento. Otro paso cr?...

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>. The corresponding author was changed from:
Teresa A. Hudock
to:
Deepak Kaushal

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>.

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

Las muestras han sido tradicionalmente microdissected manualmente, ya sea todo el tejido o diapositivas utilizando una aguja y bisturí. Para ello se requiere una clara separación entre la sección de tejido de interés y los tejidos circundantes secciones 2. Con los avances actuales en la tecnología de perfil molecular, ha habido una creciente necesidad de evaluar tejido a nivel celular. Debido a las limitaciones de microdisección manual, incluyendo técnicas de LCM se establecieron para permitir una mayor precisión de aislamiento. Esta técnica permite al investigador para aislar poblaciones de células específicas de diversos tipos de células y de diapositivas, que luego se pueden utilizar para ensayos de aguas abajo, tales como perfiles de expresión génica. Aunque es muy eficaz para los ensayos posteriores, LCM no está exenta de limitaciones. En primer lugar, LCM es un proceso costoso y consume mucho tiempo. Además, debido a la naturaleza inestable de ARN, que es a menudo un reto para obtener ARN de alta calidad a partir de muestras LCM 2. Debido a la disadvantages de LCM, siguen siendo necesarios nuevos avances en la tecnología de microdisección para hacerlo más accesible a un mayor número de investigadores de una manera sensible asequible y tiempo. Uno de estos avances en la tecnología de microdisección ahora disponible es una técnica conocida como mesodissection. En esta técnica se utiliza una máquina para moler la sección de tejido anotada de interés y aspira en una fresa de consumibles 3. A continuación, esta muestra puede ser aspirado por un tubo de recogida y se utiliza para aplicaciones posteriores. Las ventajas de este sistema son que es significativamente menos costoso y sensible al tiempo. En nuestra experiencia, el sistema permite la disección de una sección de tejido granuloma pulmonar en diez minutos. Por otro lado, LCM tradicional requeriría probablemente horas para completar el proceso. El sistema permite al operador cargar un portaobjetos de referencia a utilizar como una comparación, así como herramienta para la anotación. Además se genera un informe esbozando ªárea e de la diapositiva que se ha diseccionado. Hay dos desventajas principales de mesodissection. Aunque es eficaz en la extracción de varias celdas, es difícil aislar una sola célula. Además, la precisión de la imagen generada no es tan clara como si el uso de otros microscopios de imagen. La tuberculosis (TB) es una importante causa de muerte de la enfermedad infecciosa de la humanidad en todo el mundo y es consecuencia de la infección por Mtb. En la mayoría de los individuos expuestos a aerosoles de Mtb, la infección está latente limitado. En por lo menos 10 millones de personas anualmente, da lugar a la enfermedad de la TB activa 4. Durante la infección latente, Mtb está contenida dentro de las lesiones pulmonares patológicos conocidos como granulomas. Por lo tanto, se ha argumentado que el resultado de la infección por Mtb se decide a nivel del granuloma 5. Aquí demostramos cómo mesodissection se puede utilizar para microdissect granulomas causados ​​por Mtb. Las diapositivas utilizadasson de FFPE tejido pulmonar de los macacos rhesus infectados. Para el propósito de esta demostración, vamos a diseccionar granulomas en su totalidad. También demuestran que el ARN se puede extraer de los tejidos recuperados. Esta técnica se puede aplicar a muestras de tejido de varios otros especímenes y luego se usa para una variedad de ensayos de aguas abajo.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">MeSectr</td>
			<td style="width:113px;">AvanSci Bio</td>
			<td style="width:108px;"></td>
			<td style="width:195px;">Mesodissector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">400 nm xScisors</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Other sizes available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">THOR</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Programmable Heater-Shaker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop2000</td>
			<td>ThermoScientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy FFPE extraction kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">73504</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovation RNA-Seq FFPE System&#160;</td>
			<td>Nugen</td>
			<td align="right">7150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">28104</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas

Microdissection se ha utilizado para el examen de los tejidos en el ADN, el ARN y los niveles de proteína durante más de una década. Microscopía de captura láser (LCM) es la técnica de microdisección más común usado hoy en día. En esta técnica, se utiliza un láser para fundir focalmente una membrana termoplástica que se superpone a una sección de tejido deshidratado 1. La sección de tejido de material compuesto entonces se levanta y se separa de la membrana. Aunque esta técnica puede utilizarse con éxito para el examen del tejido, que consume tiempo y es caro. Además, la finalización con éxito de los procedimientos que utilizan esta técnica requiere el uso de un láser, lo que limita su uso. Un nuevo enfoque de microdisección más asequible y práctica llamada mesodissection es una posible solución a las trampas de la LCM. Esta técnica emplea el sistema de MESO-1/MeSectr moler el tejido deseado a partir de una muestra de tejido montada deslizante, mientras que al mismo tiempo la dispensación y aspiración de fluido para recuperar la muestra de tejido deseada enun poco molino de consumible. Antes de que comience el proceso de disección, el usuario alinea la parafina fijado en formol incorporado (FFPE) deslice con hematoxilina y eosina manchado (H &amp; E) Tobogán de referencia. A partir de entonces, el operador anota el área de la disección deseada y procede a diseccionar el segmento apropiado. El programa genera una imagen archivada de la disección. La principal ventaja de mesodissection es la corta duración necesaria para diseccionar una diapositiva, teniendo un promedio de diez minutos de configurar para probar la generación en este experimento. Además, el sistema es significativamente más rentable y fácil de usar. Una ligera desventaja es que no es tan precisa como la microscopía de captura por láser. En este artículo se demuestra cómo mesodissection puede utilizarse para extraer el ARN a partir de diapositivas de granulomas FFPE causadas por Mycobacterium tuberculosis (Mtb).

El protocolo mencionado se muestra cómo utilizar una nueva técnica mesodissection para extraer el ARN a partir de diapositivas de tejidos FFPE. La eficacia de este protocolo se muestra a través de diapositivas FFPE de granulomas pulmonares de NHP infectados con Mtb en varias fases infecciosas. Figuras 1-3 son imágenes del instrumento. Figura 4 representa cómo se produce el proceso de disección y la imagen resultante generada por el software. Tabla 1 muest...

Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

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

Microdisección se ha empleado ampliamente para el examen de ADN, ARN, y proteínas dentro del tejido. Microscopía de captura láser (LCM) es el método más comúnmente utilizado, pero una nueva técnica de fresado, mesodissection, es recientemente disponible. Demostramos la extracción de RNA de mesodissected formalina parafina fijo diapositivas de tejidos embebidos de granulomas de Mycobacterium tuberculosis.

Un nuevo enfoque para la recuperación Microdissection<em&gt; Mycobacterium tuberculosis</em&gt; Las transcripciones específicas de formalina parafina fijo Embedded pulmón 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 ...

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Immunology and Infection

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

7.9K vistas.  Tulane National Primate Research Center.  Microdisección se ha empleado ampliamente para el examen de ADN, ARN, y proteínas dentro del tejido. Microscopía de captura láser (LCM) es el método más comúnmente utilizado, pero una nueva técnica de fresado, mesodissection, es recientemente disponible. Demostramos la extracción de RNA de mesodissected formalina parafina fijo diapositivas de tejidos embebidos de granulomas de Mycobacterium tuberculosis. 

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

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

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

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

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

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) ...

Investigación

Inmunología e Infección

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 ...

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 ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

La detección in situ de las bacterias dentro de los tejidos embebidos en parafina utilizando un ADN sonda digoxina marcada focalización 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

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 ...