Gli autori vorrebbero riconoscere i seguenti premi NIH / subawards per il sostegno di questa ricerca: R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, e P51OD011104.

1. Calibrate strumento Mesodissection con 2id software di imaging Il software di imaging 2id verrà indicato come sia il software o il programma per il resto di questo articolo. Questo passo è necessario per monitorare correttamente l&#39;immagine così come allineare più diapositiva frame. <ol><li> Accendere strumento e computer. </li><li> Il software Open e selezionare &quot;strumento di calibrazione&quot;. </li><li> Calibrare viaggio stage utilizzando il joystick. Spostare palco per la posizione in alto a sinistra. Premere il tasto &quot;set&quot; in fase 1 in programma. </li><li> Spostare fase di abbassare giusta posizione. Premere il tasto &quot;set&quot; sotto il punto 2 in programma. </li><li> Calibrare aspirazione premendo prima &quot;Z-button&quot; per sollevare assemblea testa. Premere il tasto &quot;pulse aspirare&quot; e il pulsante &quot;dissezione&quot; sul joystick finché asta di comando del pistone raggiunge prima posizione. Premere il tasto &quot;set&quot; sotto il punto 3 del programma. </li><li> Premere il tasto &quot;Reset aspirato &amp;# 8221; pulsante. Una volta che il pistone si ferma alla posizione di premere il tasto &quot;set&quot; in basso al punto 4. </li><li> Fare clic sulla scheda &quot;scala&quot;. </li><li> Mettere righello calibrazione lato bianco. </li><li> Concentrarsi microscopio, se necessario. </li><li> Disegnare una linea tra le due barre di scala utilizzando il mouse. </li><li> Immettere la distanza in scatola pulsante punto 2. Premere il tasto &quot;Calcola&quot; etichettati. </li><li> Fare clic sulla linguetta &quot;mirino&quot;. Girare la velocità del motore a 1. </li><li> Inserire xScisor nel gruppo della testa. Assemblaggio testata inferiore in asse z posizione pronta premendo il tasto &quot;z&quot;. NOTA: Il gruppo di testa è la grande struttura sulla sommità dello strumento di dissezione. Il xScisor verrà indicato come un po mulino di consumo per il resto di questo articolo. </li><li> Fare clic su &quot;dissezione impegnarsi&quot; sul joystick. NOTA: Questo pulsante si trova sulla parte superiore del joystick. </li><li> Definire centro di rotazione visivamente. Per fare questo, spostare il centro di mirino sul centro di rotazione usando definireparametro d al punto 1 del programma. Utilizzare il su e giù nella &quot;x&quot; e &quot;y bar-asse&quot; per regolare i pixel per allineare mirino. Fare clic sulla scheda &quot;done&quot;. </li><li> Fare clic sull&#39;icona &quot;casa&quot;. NOTA: Questo è rappresentato da un&#39;immagine casa situata in alto a destra. </li></ol> 2. Creare Immagine di riferimento <ol><li> Posizionare H &amp; E vetrino sul palco. Mettere copertura opaca sulla parte superiore della diapositiva. Guidare lo strumento muovendo il joystick per l&#39;area desiderata della diapositiva H &amp; E. Salvare l&#39;immagine generata. NOTA: Sia la scheda &quot;cattura di riferimento&quot; o scheda &quot;sezionare&quot; possono essere utilizzati per completare questo passaggio. </li></ol> 3. Allineare tessuto per dissezione <ol><li> Fare clic sull&#39;opzione &quot;sezionare il tessuto&quot; sullo schermo di casa. </li><li> Compilare l &#39;&quot;operatore&quot;, &quot;numero dissezione di adesione&quot;, &quot;numero di accesso di riferimento&quot;, &quot;xScisor codice a barre&quot;, &amp; #8220; dissezione numero di lotto fluido &quot;,&quot; dimensione xScisor &quot;e&quot; descrizione &quot;nella scheda&quot; setup &quot;. </li><li> Fare clic sulla scheda &quot;trovare tessuti&quot;. </li><li> Importazione di immagini di riferimento precedentemente salvata. </li><li> Inserire un FFPE scivolo senza macchia di 5 micron di spessore sul palco. Spostare palco per la stessa area come quello raffigurato nell&#39;immagine di riferimento. Clicca sulla scheda &quot;Inserisci immagine&quot;. </li><li> Fare clic sulla linguetta &quot;align&quot;. Utilizzare il mouse e le frecce associati per allineare l&#39;immagine di riferimento all&#39;immagine senza macchia. </li></ol> 4. Annota diapositiva <ol><li> Fare clic sulla scheda &quot;annotate&quot;. </li><li> Usate il mouse per disegnare un cerchio attorno all&#39;area desiderata della diapositiva che sarà in paraffina. </li></ol> 5. Caricare consumabili Mill Bit <ol><li> Riempire consumabile bit mulino con tampone desiderato, qui tampone PKD, tirando lo stantuffo su e giù in un movimento fluido più volte. Assicurati di escludere l&#39;ariabolle. </li><li> Sollevare asse Z premendo il tasto &quot;Z&quot;. </li><li> Caricare consumo bit mulino in macchina. Fate questo facendo scorrere la punta mulino consumabile nella parte superiore della macchina in modo che la linea bianca sulle linee strumento con la linea nera sul bit mulino consumabile. Premere il tasto &quot;Reset aspirazione&quot; per consentire stantuffo per essere abbassata nella posizione corretta. NOTA: Dovrebbe essere facile scivolare la punta mulino di consumo. Se non lo è, assicurarsi che le tacche siano allineate correttamente. </li><li> Bassa asse Z premendo nuovamente il tasto &quot;Z&quot;. </li></ol> 6. Dissect Tissue (Figura 4) <ol><li> Apri scheda &quot;sezionare&quot;. </li><li> Girare velocità del motore e aspirazione a 1. </li><li> Casella &quot;spettacolo di monitoraggio&quot;. Nota: Questo consente all&#39;utente di visualizzare l&#39;area sezionato durante il processo di dissezione. </li><li> Una volta pronti a sezionare, continuare a tenere premuto il tasto &quot;impegnarsi&quot;, così come &quot;aspirato &amp; #8221; pulsante mentre si muove joystick in senso antiorario per sezionare il tessuto. </li><li> Continua a sezionare in senso antiorario finché l&#39;aspirato è scheda &quot;pieno&quot; diventa rossa. </li></ol> 7. Svuotare e rimuovere consumabili Mill Bit <ol><li> Mettere una provetta per microcentrifuga 0,5 ml sotto la punta della punta mulino di consumo. </li><li> Pressetta controllo &quot;aspirazione&quot; rapidamente per espellere campione in provetta per microcentrifuga. </li><li> Premere con maggiore forza per espellere usati consumo bit mulino. </li><li> Risciacquare frammento di tessuto tirando indietro lo stantuffo. Spingere lo stantuffo in avanti per esprimere campione rimanente in consumo bit mulino. Il frammento di tessuto sarà ora contenuto all&#39;interno della provetta per microcentrifuga. </li></ol> 8. Lyse campione con proteinasi K digestione da un trattamento termico <ol><li> Aggiungere 70 microlitri TE pH 8,5 contenente 5 mg di proteinasi K per recuperare campione di tessuto. </li><li> Collocare il campione in programmabile riscaldamento-shaker. </li><li> Digestione completa utilizzando programmabile riscaldamento-shaker al seguente impostazione: 60 ˚ C per 30 min a 1.500 giri; 82 ˚ C per 15 min a 1.500 giri; e 25 ˚ C per 1 min a 450 rpm. Il campione è ora pronto per applicazioni a valle. </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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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. 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Gli autori rivelano che non ci sono interessi finanziari concorrenti.

Mesodissection è una tecnica che può essere applicata per l&#39;estrazione di RNA da diapositive lesioni patologiche causate da una vasta gamma di agenti patogeni. È una necessità che l&#39;utente ascolti l&#39;immagine e allinea correttamente l&#39;immagine. Per fare questo, lo strumento deve essere calibrato seguendo le indicazioni sul software di imaging. Quando dissezione, se la slitta e la zona di interesse essere sezionato non si allineano, l&#39;utente deve ricalibrare lo strumento. Un altro punto critico nel...

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

I campioni sono stati tradizionalmente microdissezione manualmente sia da tutto il tessuto o diapositive utilizzando un ago e bisturi. Ciò richiede una netta separazione tra la sezione di tessuto di interesse e dei tessuti circostanti le sezioni 2. Con i progressi attuali nella tecnologia profiling molecolare, vi è stata una crescente necessità di valutare il tessuto a livello cellulare. A causa delle limitazioni di microdissezione manuale, sono stati stabiliti tecniche tra cui LCM per consentire una maggiore precisione di isolamento. Questa tecnica consente al ricercatore di isolare specifiche popolazioni cellulari da vari tipi di cellule e diapositive, che possono poi essere utilizzati per saggi valle come espressione genica. Sebbene molto efficace per saggi a valle, LCM non è senza limitazioni. Primo, LCM è un processo costoso e richiede tempo. Inoltre, a causa della natura instabile di RNA, è spesso difficile da ottenere RNA di alta qualità da campioni LCM 2. A causa della disadvantages di LCM, nuovi progressi nella tecnologia microdissezione sono ancora necessari per renderlo più accessibile a un maggior numero di ricercatori in maniera sensibile conveniente e tempo. Un tale progresso della tecnologia microdissezione ora disponibile è una tecnica nota come mesodissection. In questa tecnica una macchina viene utilizzata per la lavorazione della sezione di tessuto annotato di interesse e aspirati in un po mulino consumabile 3. Quindi, questo esempio può essere aspirato in un tubo di raccolta e utilizzata per applicazioni a valle. I vantaggi di questo sistema sono che è molto meno costoso e delicato momento. Nella nostra esperienza, il sistema consente la dissezione di una sezione di tessuto polmonare granuloma in dieci minuti. D&#39;altra parte, LCM tradizionale sarebbe probabilmente richiederà ore per completare il processo. Il sistema permette all&#39;operatore di caricare una diapositiva di riferimento da utilizzare come confronto così come strumento per l&#39;annotazione. Inoltre, viene generato un report che illustra the area del vetrino che è stato sezionato. Ci sono due svantaggi principali mesodissection. Sebbene efficace a estrarre più celle, è impegnativo per isolare una singola cella. Inoltre, la precisione dell&#39;immagine generata non è chiaro come se si utilizzano altri microscopi di imaging. La tubercolosi (TB) è una importante malattia mortale contagiosa dell&#39;umanità in tutto il mondo e risultati da infezione da Mtb. In una maggioranza di individui esposti ad aerosol di Mtb, l&#39;infezione è latente limitato. In almeno 10 milioni di persone ogni anno, si traduce in malattia tubercolare attiva 4. Durante l&#39;infezione latente, Mtb è contenuto all&#39;interno lesioni polmonari patologiche conosciuti come granulomi. Quindi si è sostenuto che l&#39;esito dell&#39;infezione Mtb viene deciso a livello del granuloma 5. Qui mostriamo come mesodissection può essere utilizzato per microdissect granulomi causati da Mtb. Le slide utilizzatesono da FFPE tessuto polmonare di macachi Rhesus infetti. Ai fini di questa dimostrazione, saremo sezionare granulomi nella loro interezza. Abbiamo inoltre dimostrato che l&#39;RNA può essere estratto dal tessuto recuperato. Questa tecnica può essere applicata a campioni di tessuto da vari altri campioni e poi utilizzato per una varietà di saggi a valle.

<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

Microdissezione è stato utilizzato per l&#39;esame dei tessuti a DNA, RNA e proteina livelli per oltre un decennio. Microscopia laser capture (LCM) è la tecnica di microdissezione più comune usato oggi. In questa tecnica, un laser viene utilizzato per fondere focally una membrana termoplastica che sovrasta una sezione di tessuto disidratato 1. La sezione di tessuto composito viene poi sollevato e separato dalla membrana. Sebbene questa tecnica può essere utilizzata con successo per l&#39;esame dei tessuti, è lunga e costosa. Inoltre, il completamento delle procedure utilizzando questa tecnica richiede l&#39;uso di un laser, limitando così il suo utilizzo. Un nuovo approccio microdissezione più conveniente e pratico chiamato mesodissection è una possibile soluzione per le insidie ​​di LCM. Questa tecnica impiega il sistema MESO-1/MeSectr al mulino tessuto desiderato da un campione di tessuto scorrevole montato mentre contemporaneamente dispensando ed aspirando fluido per recuperare il campione di tessuto desiderato nellaun po &#39;mulino di consumo. Prima che inizi il processo di dissezione, l&#39;utente allinea formalina paraffina fisso incorporato (FFPE) scorrere con un ematossilina e eosina macchiato (H &amp; E) di riferimento diapositiva. Successivamente, l&#39;operatore annota la zona dissezione desiderato e procede a sezionare il segmento appropriato. Il programma genera un&#39;immagine archiviata della dissezione. Il vantaggio principale di mesodissection è la breve durata necessaria per sezionare una diapositiva, stabilendo una media di dieci minuti dal impostato per campionare generazione in questo esperimento. Inoltre, il sistema è molto più conveniente e facile da usare. Un leggero svantaggio è che non è preciso come microscopia laser capture. In questo articolo mostriamo come mesodissection può essere utilizzato per estrarre RNA da diapositive da granulomi FFPE causate da Mycobacterium tuberculosis (Mtb).

Il protocollo di cui sopra mostra come utilizzare una nuova tecnica mesodissection per estrarre l&#39;RNA da diapositive tessuto FFPE. L&#39;efficacia di questo protocollo è mostrato tramite diapositive FFPE di granulomi polmonari da NHP infettati con Mtb in vari stadi infettivi. Figure 1-3 sono immagini dello strumento. Figura 4 mostra come avviene il processo di dissezione e l&#39;immagine risultante generato dal software. Tabella 1 mostra i risultati di est...

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

Microdissezione è stato ampiamente impiegato per l&#39;esame di DNA, RNA e proteine ​​nel tessuto. Microscopia laser capture (LCM) è il metodo più comunemente usato, ma una nuova tecnica di fresatura, mesodissection, è da poco disponibile. Dimostriamo estrazione di RNA da mesodissected in formalina paraffina fissati diapositive tessuti integrati di Mycobacterium tuberculosis granulomi.

Un nuovo approccio Microdissezione per recuperare<em&gt; Mycobacterium tuberculosis</em&gt; Trascrizioni specifici da formalina paraffina fisso incorporato Lung granulomi

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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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 Views.  Tulane National Primate Research Center.  Microdissezione è stato ampiamente impiegato per l'esame di DNA, RNA e proteine ​​nel tessuto. Microscopia laser capture (LCM) è il metodo più comunemente usato, ma una nuova tecnica di fresatura, mesodissection, è da poco disponibile. Dimostriamo estrazione di RNA da mesodissected in formalina paraffina fissati diapositive tessuti integrati di Mycobacterium tuberculosis granulomi. 

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.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

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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Immunologia e infezioni

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.

Crescita Mycobacterium tuberculosis I biofilm

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 modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E 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.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione Microarray

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 rilevamento di batteri all&#39;interno dei tessuti in paraffina utilizzando una sonda DNA Digossina marcato targeting 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 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

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 per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<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 test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<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.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

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.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

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