Les auteurs tiennent à remercier les NIH prix / sous-subventions suivantes pour le soutien de cette recherche: R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, et P51OD011104.

1. Calibrer Mesodissection Instrument avec 2id logiciel d&#39;imagerie Le logiciel d&#39;imagerie 2id sera appelé soit le logiciel ou le programme pour le reste de cet article. Cette étape est nécessaire pour suivre correctement l&#39;image ainsi que aligner plusieurs cadres de diapositives. <ol><li> Allumez l&#39;instrument et l&#39;ordinateur. </li><li> Ouvrez le logiciel et sélectionnez &quot;instrument d&#39;étalonnage&quot;. </li><li> Calibrer Voyage en scène à l&#39;aide de la manette. Déplacez étape à la position supérieure gauche. Appuyez sur &quot;ensemble&quot; à l&#39;étape 1 dans le programme. </li><li> Déplacez étape pour abaisser la position droite. Appuyez sur &quot;ensemble&quot; à l&#39;étape 2 dans le programme. </li><li> Calibrer aspiration en appuyant d&#39;abord &quot;Z-touche&quot; pour augmenter l&#39;ensemble de tête. Appuyez sur la touche &quot;d&#39;impulsion d&#39;aspiration&quot; et bouton &quot;dissection&quot; sur la manette jusqu&#39;à ce que la tige de commande de piston atteint la position supérieure. Appuyez sur &quot;ensemble&quot; à l&#39;étape 3 de programme. </li><li> Appuyez sur &quot;aspirer réinitialisation et# 8221; bouton. Une fois que le piston s&#39;arrête à la position de fond sur «ensemble» à l&#39;étape 4. </li><li> Cliquez sur l&#39;onglet &quot;échelle&quot;. </li><li> Placez calibrage règle sur le côté vierge. </li><li> Concentrez microscope si nécessaire. </li><li> Tracer une ligne entre les deux barres d&#39;échelle en utilisant la souris. </li><li> Saisissez la distance dans la case de l&#39;étape 2 Appuyez sur le bouton. &quot;Compute&quot; marqué. </li><li> Cliquez sur l&#39;onglet &quot;en forme de croix&quot;. Tourner la vitesse du moteur à une. </li><li> Insérez xScisor dans l&#39;assemblage de la tête. Assemblage de la tête plus basse dans l&#39;axe z position d&#39;attente par le bouton &quot;axe z&quot; en appuyant sur. NOTE: L&#39;ensemble de tête est la grande structure sur le dessus de l&#39;instrument de dissection. Le xScisor sera considéré comme un peu de l&#39;usine de consommables pour le reste de cet article. </li><li> Cliquez sur &quot;dissection s&#39;engager&quot; sur joystick. NOTE: Ce bouton est situé sur le dessus de la manette. </li><li> Définir centre de rotation visuellement. Pour ce faire, déplacez centre de réticule sur le centre de rotation à l&#39;aide de définird paramètre à l&#39;étape 1 du programme. Utilisez le flèches haut et bas dans le &quot;x&quot; et &quot;barres axe y» pour régler les pixels d&#39;aligner croix. Cliquez sur l&#39;onglet &quot;done&quot;. </li><li> Cliquez sur l&#39;icône «maison». NOTE: Ceci est représenté par une image de la maison située dans le coin supérieur droit. </li></ol> 2. Créez Image de référence <ol><li> Placez H &amp; E diaporama sur scène. Placer le couvercle opaque sur le dessus de la lame. Conduisez l&#39;instrument en déplaçant le joystick vers la zone souhaitée de la diapositive H &amp; E. Enregistrer l&#39;image générée. REMARQUE: Soit l&#39;onglet &quot;référence capture&quot; ou l&#39;onglet &quot;disséquer&quot; peuvent être utilisés pour compléter cette étape. </li></ol> 3. Alignez tissus pour dissection <ol><li> Cliquez sur l&#39;option &quot;disséquer les tissus&quot; sur l&#39;écran d&#39;accueil. </li><li> Remplissez la «opérateur», «nombre de dissection de l&#39;adhésion&quot;, &quot;numéro de référence de l&#39;adhésion», «xScisor nombre de codes à barres,&quot; &amp; #8220; dissection numéro de lot fluide &quot;,&quot; taille de xScisor &quot;et&quot; description &quot;dans l&#39;onglet&quot; Configuration &quot;. </li><li> Cliquez sur l&#39;onglet &quot;trouver des tissus&quot;. </li><li> Importer image de référence précédemment enregistré. </li><li> Placez une lame de FFPE tache de 5 microns d&#39;épaisseur sur la scène. Déplacer étape à la même zone que celle présentée dans l&#39;image de référence. Cliquez sur l&#39;onglet &quot;Insérer une image&quot;. </li><li> Cliquez sur l&#39;onglet &quot;align&quot;. Utilisez la souris et les flèches associées à aligner l&#39;image de référence à l&#39;image sans tache. </li></ol> 4. Annoter diaporama <ol><li> Cliquez sur l&#39;onglet &quot;annoter&quot;. </li><li> Utilisez la souris pour dessiner un cercle autour de la zone désirée de la diapositive qui sera microdisséquées. </li></ol> 5. Chargez Mill consommable Bit <ol><li> Remplissez peu de moulin consommable avec le tampon souhaité, ici tampon de PKD, en tirant le piston de haut en bas dans un mouvement fluide à plusieurs reprises. Assurez-vous d&#39;exclure l&#39;airbulles. </li><li> Soulevez l&#39;axe Z en appuyant sur bouton &quot;axe Z&quot;. </li><li> Chargez peu de moulin consommable dans la machine. Pour ce faire, en faisant glisser le peu de moulin consommable dans le haut de la machine de sorte que la ligne blanche sur les lignes de l&#39;instrument avec la ligne noire sur le peu de moulin consommable. Appuyez sur le bouton &quot;reset d&#39;aspiration» pour permettre à piston à être descendu dans la position correcte. REMARQUE: Il devrait être facile de faire glisser le peu de moulin consommable. Si ce n&#39;est pas, assurez-vous que les encoches sont correctement alignés. </li><li> L&#39;axe Z-Bas par le bouton &quot;axe Z&quot; appuyant de nouveau. </li></ol> 6. Disséquer les tissus (Figure 4) <ol><li> Ouvrez l&#39;onglet &quot;disséquer&quot;. </li><li> Tournez vitesses de moteur et d&#39;aspiration à 1. </li><li> Cochez la case &quot;Afficher la poursuite». Remarque: Ceci permet à l&#39;utilisateur de visualiser la zone disséquée pendant le processus de dissection. </li><li> Une fois prêt à disséquer, continuer à tenir le bouton &quot;s&#39;engager&quot; ainsi que &quot;aspirer &amp; #8221; bouton tout en déplaçant le joystick dans le sens antihoraire à disséquer les tissus. </li><li> Continuer à disséquer dans le sens antihoraire jusqu&#39;à ce que la ponction est onglet &quot;complet&quot; devient rouge. </li></ol> 7. Vide et supprimer Mill consommable Bit <ol><li> Placer un tube de microcentrifugation de 0,5 pi sous la pointe de la mèche de broyeur consommable. </li><li> Appuyez sur tige &quot;aspiration&quot; de commande rapidement pour éjecter échantillon dans le tube de centrifugeuse. </li><li> Appuyez avec une force accrue pour éjecter peu utilisé moulin consommable. </li><li> Rincer fragment de tissu en tirant le piston arrière. Pousser le piston pour exprimer échantillon restant dans peu de moulin consommable. Le fragment de tissu est maintenant contenu dans le tube de centrifugeuse. </li></ol> 8. Exemple de Lyse avec la protéinase K digestion Suivi par traitement thermique <ol><li> Ajouter 70 ul de TE pH 8,5, contenant 5 ug de la protéinase K pour un échantillon de tissu récupéré. </li><li> Placer l&#39;échantillon dans programmable chauffe-shaker. </li><li> La digestion complète à l&#39;aide programmable chauffe-shaker avec le réglage suivant: 60 ˚ C pendant 30 min à 1500 tours par minute; 82 ˚ C pendant 15 min à 1500 tours par minute; et 25 ˚ C pendant 1 min à 450 rpm. L&#39;échantillon est maintenant prêt pour les applications en aval. </li></ol>

<ol>
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D., Alland, D., Eisen, J. A., Carpenter, L., White, O., Peterson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+comparison+of+Mycobacterium+tuberculosis+clinical+and+laboratory+strains.">Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.</a> J Bacteriol. 184 (19), 5479-5490 (2002).</li><li>Russell, D. G., Barry 3rd, C. E., Flynn, J. 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. J., Doyle, L. A., Blanchard, J. L., Lackner, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granuloma+correlates+of+protection+against+tuberculosis+and+mechanisms+of+immune+modulation+by+Mycobacterium+tuberculosis.">Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.</a> J Infect Dis. 207 (7), 1115-1127 (2013).</li><li>Kaushal, D., Schroeder, B. G., Tyagi, S., Yoshimatsu, T., Scott, C., Ko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+immunopathology+and+mortality+despite+tissue+persistence+in+a+Mycobacterium+tuberculosis+mutant+lacking+alternative+sigma+factor,+SigH.">Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.</a> Proc Natl Acad Sci U S A. (12), 8330-8335 (2002).</li><li>Mehra, S., Kaushal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+reveals+extended+roles+of+the+Mycobacterium+tuberculosis+stress+response+factor+sigmaH.">Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor sigmaH.</a> J Bacteriol. 191 (12), 3965-3980 (2009).</li><li>Rohde, K. H., Veiga, D. F., Caldwell, S., Balazsi, G., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+the+transcriptional+profiles+and+the+physiological+states+of+Mycobacterium+tuberculosis+during+an+extended+intracellular+infection.">Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection.</a> PLoS Pathog. 8 (6), (2012).</li><li>Fontan, P. A., Voskuil, M. I., Gomez, M., Tan, D., Pardini, M., Manganelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mycobacterium+tuberculosis+sigma+factor+sigmaB+is+required+for+full+response+to+cell+envelope+stress+and+hypoxia+in+vitro,+but+it+is+dispensable+for+in+vivo+growth.">The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.</a> J Bacteriol. 191 (18), 5628-5633 (2009).</li><li>Rustad, T. R., Harrell, M. I., Liao, R., Sherman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+hypoxic+response+of+Mycobacterium+tuberculosis.">The enduring hypoxic response of Mycobacterium tuberculosis.</a> PLoS One. 3 (1), (2008).</li></ol>

Les auteurs décrivent qu&#39;il n&#39;y a pas des intérêts financiers concurrents.

Mesodissection est une technique qui peut être appliquée à l&#39;extraction d&#39;ARN à partir de lames de lésions pathologiques provoquées par une grande variété de pathogènes. C&#39;est une nécessité que l&#39;utilisateur suit l&#39;image et aligne l&#39;image correctement. Pour ce faire, l&#39;instrument doit être étalonné suivant les instructions sur le logiciel d&#39;imagerie. Lors de la dissection, si la lame et de la zone d&#39;intérêt étant disséqué ne correspondent pas, l&#39;utilisateur doit...

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

Les échantillons ont été traditionnellement microdissection soit manuellement à partir d&#39;un tissu ou lames à l&#39;aide d&#39;une aiguille et l&#39;ensemble scalpel. Cela nécessite une séparation claire entre la section de tissu d&#39;intérêt et les tissus environnants sections 2. Avec les progrès actuels de la technologie de profilage moléculaire, il a eu un besoin croissant d&#39;évaluer les tissus au niveau cellulaire. En raison des limitations de microdissection manuel, y compris les techniques LCM ont été établis pour permettre une plus grande précision de l&#39;isolement. Cette technique permet au chercheur d&#39;isoler des populations de cellules spécifiques à partir de divers types de cellules et de diapositives, qui peuvent ensuite être utilisés pour des essais en aval comme profil d&#39;expression génique. Bien que très efficace pour les essais en aval, LCM n&#39;est pas sans limites. Tout d&#39;abord, LCM est un processus long et coûteux. En outre, en raison de la nature instable de l&#39;ARN, il est souvent difficile d&#39;obtenir de l&#39;ARN de haute qualité à partir de deux échantillons LCM. En raison de la disadvantages de LCM, de nouvelles avancées dans la technologie de microdissection sont encore nécessaires pour le rendre plus accessible à un plus grand nombre de chercheurs d&#39;une manière sensible abordable et le temps. Une telle avancée dans la technologie de microdissection maintenant disponible est une technique connue sous le nom mesodissection. Dans cette technique, on utilise une machine à l&#39;usine de la section de tissu annotée d&#39;intérêt et l&#39;aspire dans un peu de moulin consommable 3. Ensuite, cet échantillon peut être aspiré dans un tube de collecte et utilisée pour des applications en aval. Les avantages de ce système est qu&#39;il est beaucoup moins coûteuse en temps et sensible. Dans notre expérience, le système permet la dissection d&#39;une section de tissu de granulome pulmonaire dans dix minutes. D&#39;autre part, LCM traditionnel nécessiterait probablement heures pour terminer le processus. Le système permet à l&#39;opérateur de charger une lame de référence à utiliser à titre de comparaison ainsi que l&#39;outil pour l&#39;annotation. En outre, un rapport est généré ème décrivantzone de e de la lame qui a été disséqué. Il existe deux principaux inconvénients de mesodissection. Bien qu&#39;efficace à extraire des cellules multiples, il est difficile d&#39;isoler une seule cellule. En outre, la précision de l&#39;image générée n&#39;est pas aussi clair que si vous utilisez d&#39;autres microscopes d&#39;imagerie. La tuberculose (TB) est une cause majeure de décès des maladies infectieuses de l&#39;humanité dans le monde entier et les résultats de l&#39;infection par Mtb. Dans la majorité des personnes exposées à des aérosols de Vtt, l&#39;infection est latente limitée. Dans au moins 10 millions de personnes par an, il en résulte la tuberculose active 4. Lors de l&#39;infection latente, Vtt est contenu dans les lésions pulmonaires pathologiques appelés granulomes. Par conséquent, il a été avancé que le résultat de l&#39;infection Vtt est décidé au niveau du granulome 5. Ici, nous montrons comment mesodissection peut être utilisé pour microdissect granulomes provoqués par Mtb. Les diapositives utiliséessont des tissus pulmonaires FFPE de macaques rhésus infectés. Pour les besoins de cette démonstration, nous allons disséquer granulomes dans leur intégralité. Nous démontrons aussi que l&#39;ARN peut être extrait à partir du tissu récupéré. Cette technique peut être appliquée à des échantillons de tissus à partir d&#39;autres échantillons et ensuite utilisé pour une variété de dosages en aval.

<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 a été utilisé pour l&#39;examen des tissus à l&#39;ADN, de l&#39;ARN, et les taux de protéines plus d&#39;une décennie. microscopie de capture laser (LCM) est une technique de microdissection le plus couramment utilisé aujourd&#39;hui. Dans cette technique, on utilise un laser pour faire fondre focalement une membrane thermoplastique qui recouvre une coupe de tissu déshydratée 1. La coupe de tissu composite est alors soulevé et séparé de la membrane. Bien que cette technique peut être utilisée avec succès pour l&#39;examen des tissus, il est long et coûteux. De plus, la réussite des procédures utilisant cette technique nécessite l&#39;utilisation d&#39;un laser, ce qui limite son utilisation. Une nouvelle approche de microdissection plus abordable et pratique appelée mesodissection est une solution possible pour les pièges de la LCM. Cette technique utilise le système à moulin MESO-1/MeSectr le tissu désiré à partir d&#39;un échantillon de tissu monté coulissant tandis que simultanément la distribution et l&#39;aspiration de fluide pour récupérer l&#39;échantillon de tissu désiré enun peu de moulin consommable. Avant le début du processus de dissection, l&#39;utilisateur aligne la formol paraffine fixe intégré (FFPE) glisser avec un hématoxyline et l&#39;éosine souillé (H &amp; E) lame de référence. Par la suite, l&#39;opérateur annote la zone de dissection souhaitée et procède à disséquer le segment approprié. Le programme génère une image archivée de la dissection. Le principal avantage de mesodissection est la courte durée nécessaire pour disséquer une diapositive, en prenant une moyenne de dix minutes de mise en place pour déguster génération dans cette expérience. En outre, le système est considérablement plus économique et facile à utiliser. Un léger inconvénient est qu&#39;il n&#39;est pas aussi précise que la microscopie à capture laser. Dans cet article, nous montrons comment mesodissection peut être utilisé pour extraire l&#39;ARN à partir de diapositives de granulomes FFPE causées par Mycobacterium tuberculosis (Mtb).

Le protocole ci-dessus montre comment utiliser une nouvelle technique de mesodissection pour extraire l&#39;ARN à partir de lames de tissu FFPE. L&#39;efficacité de ce protocole est montré à travers des diapositives FFPE de granulomes pulmonaires du PSN infectées par Mtb à divers stades infectieux. Figures 1-3 sont des images de l&#39;instrument. Figure 4 illustre la façon dont le processus de dissection se produit et l&#39;image résultante générée par le logiciel. <...

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

Microdissection a été largement utilisé pour l&#39;examen de l&#39;ADN, de l&#39;ARN et de la protéine dans le tissu. microscopie de capture laser (LCM) est la méthode la plus couramment utilisée, mais une nouvelle technique de fraisage, mesodissection, est disponible depuis peu. Nous démontrons extraction d&#39;ARN à partir mesodissected formol paraffine fixe des lames de tissus intégrés de granulomes de Mycobacterium tuberculosis.

Une nouvelle approche microdissection de la récupération<em&gt; Mycobacterium tuberculosis</em&gt; Transcriptions spécifiques de formol paraffine fixe embarqué poumon granulomes

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-novel-microdissection-approach-to-recovering-mycobacterium

Research

JoVE Journal

Immunology and Infection

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

7.9K vues.  Tulane National Primate Research Center.  Microdissection a été largement utilisé pour l'examen de l'ADN, de l'ARN et de la protéine dans le tissu. microscopie de capture laser (LCM) est la méthode la plus couramment utilisée, mais une nouvelle technique de fraisage, mesodissection, est disponible depuis peu. Nous démontrons extraction d'ARN à partir mesodissected formol paraffine fixe des lames de tissus intégrés de granulomes de Mycobacterium tuberculosis. 

Vidéo: 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.

Préparation de l&#39;échantillon de<em&gt; Mycobacterium tuberculosis</em&gt; Extraits pour Magnétique Nucléaire études métabolomiques résonance

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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Immunologie et infection

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.

La croissance de la Mycobacterium tuberculosis Les biofilms

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 modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et 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.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

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.

In Situ détection des bactéries dans les tissus inclus en paraffine Utiliser une sonde ADN de digoxine marqué ciblage ARNr 16S

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 modèle de tissu 3D Poumon humain pour les études fonctionnelles sur<em&gt; Mycobacterium tuberculosis</em&gt; 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 ...

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.

Système d&#39;efficacité et de dépistage des inhibiteurs Cytotoxicité ciblage intracellulaire<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 haut-débit Assay Compatible pour évaluer l&#39;efficacité des médicaments contre Macrophage repiquées<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.

Analyse de 18FDG TEP/CT d’imagerie comme un outil pour étudier Mycobacterium tuberculosis Infection et traitement chez les Primates Non humains

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.

Imagerie de Mycobacterium tuberculosis chez la souris avec la journaliste Enzyme Fluorescence

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