Os autores gostariam de agradecer aos seguintes prêmios NIH / subawards para o apoio a esta pesquisa: R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, e P51OD011104.

1. Calibrar Mesodissection Instrumento com 2id Software Imaging O software de imagem 2id será referido como o software ou o programa para o restante deste artigo. Esta etapa é necessária para monitorar corretamente a imagem, bem como alinhar vários quadros de slides. <ol><li> Ligue instrumento eo computador. </li><li> Abra o software e selecione &quot;instrumento calibrar&quot;. </li><li> Calibrar viagem etapa, utilizando o joystick. Mover para a posição de fase superior esquerdo. Pressione o botão &quot;set&quot; no passo 1 em programa. </li><li> Mova etapa para baixar posição correta. Pressione o botão &quot;set&quot; no passo 2 no programa. </li><li> Calibrar aspiração pressionando primeiro &quot;botão Z&quot; para levantar a montagem cabeça. Pressione o botão &quot;pulso aspirado&quot; eo botão &quot;dissecação&quot; no joystick até haste de controle êmbolo atinge primeira posição. Pressione o botão &quot;set&quot; na etapa 3 do programa. </li><li> Pressione &quot;aspirado de reset &amp;# 8221; botão. Uma vez que o êmbolo pára no fundo posição imprensa &quot;set&quot; no passo 4. </li><li> Clique na guia &quot;escala&quot;. </li><li> Coloque governante calibração no lado em branco. </li><li> Concentre-se microscópio, se necessário. </li><li> Desenhar linha entre as duas barras de escala usando mouse. </li><li> Introduza a distância na caixa passo 2 Pressione o botão. &quot;Compute&quot; rotulados. </li><li> Clique na aba &quot;mira&quot;. Gire a velocidade do motor a 1. </li><li> Insira xScisor em conjunto da cabeça. Conjunto de cabeça inferior em posição pronta eixo z, premindo o botão &quot;eixo z&quot;. NOTA: O conjunto da cabeça é a grande estrutura na parte superior do instrumento de dissecação. O xScisor será referida como uma ponta de fresagem consumível para o resto do artigo. </li><li> Clique em &quot;dissecação envolver&quot; botão no joystick. NOTA: Este botão está localizado na parte superior do joystick. </li><li> Definir centro de rotação visualmente. Para fazer isso, mover o centro da cruz sobre o centro de rotação usando definird parâmetro no passo 1 do programa. Use o para cima e para baixo setas no &quot;x&quot; e &quot;bares do eixo y&quot; para ajustar os pixels para alinhar mira. Clique na guia &quot;feito&quot;. </li><li> Clique no ícone de &quot;casa&quot;. NOTA: Este é representada uma imagem casa localizada no canto superior direito por. </li></ol> 2. Criar Imagem de referência <ol><li> Coloque H &amp; E de slides no palco. Coloque tampa opaca em cima de slide. Dirija o instrumento, movendo o joystick para a área desejada da H &amp; E slide. Salve a imagem gerada. NOTA: Ou o guia &quot;captura de referência&quot; ou guia &quot;dissecar&quot; pode ser usado para concluir esta etapa. </li></ol> 3. Alinhe Tissue para Dissecção <ol><li> Clique na opção &quot;dissecar tecido&quot; na tela inicial. </li><li> Preencha o &quot;operador&quot;, &quot;número de dissecção adesão&quot;, &quot;referência número de acesso&quot;, &quot;xScisor número de código de barras&quot;, &amp; #8220; dissecção número muito fluida &quot;,&quot; tamanho xScisor &quot;e&quot; descrição &quot;na aba&quot; Configuração &quot;. </li><li> Clique no botão &quot;encontrar tecido&quot; tab. </li><li> Importação salvo anteriormente imagem de referência. </li><li> Coloque um slide de FFPE imaculada de 5 mícrons de espessura no palco. Mover fase para a mesma área que o descrito na imagem de referência. Clique na guia &quot;Inserir imagem&quot;. </li><li> Clique na aba &quot;align&quot;. Use o mouse e as setas associadas para alinhar a imagem de referência para a imagem imaculada. </li></ol> 4. Anotar Deslize <ol><li> Clique na aba &quot;anotar&quot;. </li><li> Use o mouse para desenhar um círculo ao redor da área desejada do slide que será microdissectados. </li></ol> 5. Carregar consumíveis Moinho Bit <ol><li> Preencha consumível pouco moinho com tampão desejado, aqui tampão PKD, puxando o êmbolo para cima e para baixo em um movimento fluido várias vezes. Certifique-se de excluir o arbolhas. </li><li> Levante-eixo Z pressionando botão &quot;eixo Z&quot;. </li><li> Carregar consumível pouco moinho em máquina. Para fazer isso, deslizando a broca de fresagem consumível para a parte superior da máquina, de modo que a linha de branco sobre as linhas de instrumentos com a linha preta na broca de fresagem consumível. Pressione o botão &quot;redefinição de aspiração&quot; para permitir que o êmbolo de ser rebaixada para a posição correta. NOTA: Deve ser fácil para deslizar a pouco moinho consumível. Se não for, verifique se os encaixes estão corretamente alinhados. </li><li> Eixo Z inferior em vez de pressionar o botão &quot;eixo Z&quot;. </li></ol> 6. Dissecar tecidual (Figura 4) <ol><li> Abra guia &quot;dissecar&quot;. </li><li> Vire velocidades do motor e aspiração a 1. </li><li> Marque a caixa &quot;show de rastreamento&quot;. Nota: Isso permite que o usuário visualize a área dissecados durante o processo de dissecação. </li><li> Uma vez pronto para dissecar, continuar a manter o botão &quot;engajar&quot;, bem como &quot;aspirado &amp; #8221; botão enquanto move joystick em sentido anti-horário para dissecar tecidos. </li><li> Continue a dissecar em sentido anti-horário até que o aspirado é guia &quot;completo&quot; fica vermelho. </li></ol> 7. Esvazie e remover consumíveis Moinho Bit <ol><li> Coloque um tubo de microcentrífuga de 0,5 mL sob a ponta da broca de fresagem consumível. </li><li> Haste de controle da &quot;aspiração&quot; rapidamente para ejetar amostra em tubo de microcentrífuga. </li><li> Imprensa com maior força para ejetar usado consumível pouco moinho. </li><li> Enxágüe fragmento de tecido, puxando o êmbolo para trás. Empurre o êmbolo para a frente para expressar amostra restante em consumível pouco moinho. O fragmento de tecido vai agora ser contido no interior do tubo de microcentrífuga. </li></ol> 8. Lyse Amostra com Proteinase K Digestão de um tratamento térmico <ol><li> Adicionar 70 ul de TE, pH 8,5, contendo 5 mg de proteinase K e recuperado da amostra de tecido. </li><li> Colocar a amostra em programável aquecedor-shaker. </li><li> Digestão completa usando programável aquecedor-shaker com a seguinte programação: 60 ˚ C por 30 min a 1.500 rpm; 82 ˚ C durante 15 min a 1500 rpm; e 25 ˚ C durante 1 min a 450 rpm. A amostra está pronto para aplicações a jusante. </li></ol>

<ol>
	<li>Fend, F., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+pathology.">Laser capture microdissection in pathology.</a> J Clin Pathol. 53 (9), 666-672 (2000).</li><li>Esposito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+techniques:+laser+capture+microdissection--increasing+specificity+of+gene+expression+profiling+of+cancer+specimens.">Complementary techniques: laser capture microdissection--increasing specificity of gene expression profiling of cancer specimens.</a> Adv Exp Med Biol. 593, 54-65 (2007).</li><li>Adey, N., Bosh, D., Emery, D., Birch, L., Parry, R. <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+Paraffin+Embedded+Slide+Mounted+Tissue+Sections.">Mesodissection of Paraffin Embedded Slide Mounted Tissue Sections.</a> Journal of Molecular Diagnostics. 14 (6), (2012).</li><li>Fleischmann, R. 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>

Os autores revelam que não existem interesses financeiros concorrentes.

Mesodissection é uma técnica que pode ser aplicada para a extracção de ARN a partir de lâminas de lesões patológicas causadas por uma vasta gama de agentes patogénicos. É uma necessidade que o usuário acompanha a imagem e se alinha a imagem corretamente. Para conseguir isso, o instrumento deve ser calibrado de acordo com as instruções sobre o software de imagem. Ao dissecar, se o slide e área de interesse a ser dissecado não se alinham, o usuário deve recalibrar o instrumento. Outro passo importante no pr...

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

As amostras foram tradicionalmente microdissecado manualmente a partir de qualquer tecido ou conjunto lâminas usando uma agulha e bisturi. Isto exige uma separação clara entre a secção de tecido de interesse e tecidos circundantes pontos 2. Com os avanços na tecnologia actual perfil molecular, tem havido uma necessidade cada vez maior para avaliar tecidos ao nível celular. Devido às limitações de microdissecção manual, foram estabelecidas técnicas, incluindo LCM para permitir uma maior precisão isolamento. Esta técnica permite ao investigador para isolar populações de células específicas a partir de vários tipos de células e de deslizamento, que podem então ser utilizadas para ensaios a jusante, tais como a expressão do gene de perfil. Embora muito eficaz para ensaios a jusante, GCV não é sem limitações. Primeiro, LCM é um processo caro e demorado. Além disso, devido à natureza instável do ARN, que é muitas vezes difícil obter o RNA de alta qualidade a partir de amostras de LCM 2. Devido ao disadvantages de LCM, novos avanços na tecnologia microdissection ainda são necessários para torná-lo mais acessível a um maior número de pesquisadores, de forma sensível a preços acessíveis e de tempo. Um tal avanço da tecnologia microdissection agora está disponível uma técnica conhecida como mesodissection. Nesta técnica uma máquina é usada para moer a secção de tecido anotada de juros e aspira-lo em um consumível pouco moinho 3. Em seguida, essa amostra pode ser aspirado para dentro de um tubo de coleta e usado para aplicações a jusante. As vantagens deste sistema são que é significativamente menos dispendioso e sensíveis ao tempo. Em nossa experiência, o sistema permite a dissecação de uma secção de tecido granuloma pulmonar em dez minutos. Por outro lado, o LCM tradicional seria provável exigir horas para completar o processo. O sistema permite que o operador carregar uma lâmina de referência a ser usado como uma comparação, bem como ferramenta para anotação. Além disso, um relatório é gerado descrevendo ªárea e do slide que foi dissecado. Há duas principais desvantagens para mesodissection. Embora eficaz na extração de várias células, é um desafio para isolar uma única célula. Além disso, a precisão da imagem gerada não é tão clara como se estivesse usando outros microscópios de imagem. A tuberculose (TB) é um grande assassino de doenças infecciosas da humanidade em todo o mundo e resulta de infecção por M. tuberculosis. Na maioria dos indivíduos expostos a aerossóis de Mtb, a infecção é latente limitado. Em pelo menos 10 milhões de pessoas por ano, que resulta em doença tuberculose ativa 4. Durante a infecção latente, Mtb está contido dentro de lesões pulmonares patológicos conhecidos como granulomas. Por isso, tem-se argumentado que o resultado de infecção por M. tuberculosis é decidido ao nível do granuloma 5. Aqui demonstramos como mesodissection pode ser usado para microdissect granulomas provocadas por M. tuberculosis. Os slides utilizadossão de FFPE tecido pulmonar de macacos rhesus infectados. Para o propósito desta demonstração, vamos dissecar granulomas na sua totalidade. Também demonstramos que o RNA pode ser extraído a partir do tecido recuperado. Esta técnica pode ser aplicada a amostras de tecidos de várias outras amostras e, em seguida, utilizados para uma variedade de ensaios a jusante.

<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

Microdissecção foi usado para o exame de tecidos em ADN, ARN, e os níveis de proteína de mais de uma década. Microscopia captura a laser (LCM) é a técnica de microdissecção mais comum usado hoje. Nesta técnica, um laser é usado para fundir focalmente uma membrana termoplástica que se sobrepõe uma secção de tecido 1 desidratado. O compósito secção de tecido é então levantada e separada da membrana. Embora esta técnica pode ser utilizada com sucesso para a análise de tecido, que é demorado e caro. Além disso, a conclusão bem sucedida de procedimentos que utilizam esta técnica requer a utilização de um laser, limitando assim o seu uso. Uma nova abordagem microdissection mais acessível e prático chamado mesodissection é uma possível solução para as armadilhas da LCM. Esta técnica utiliza o sistema de MESO-1/MeSectr moer o tecido desejado a partir de uma amostra de tecido montada deslizante, enquanto simultaneamente a distribuição e aspiração de fluido para recuperar a amostra de tecido desejado emum pouco moinho consumível. Antes do processo de dissecação começa, o usuário alinha a parafina fixado em formalina incorporado (FFPE) deslizar com hematoxilina e eosina (H &amp; E) referência slide. Depois disso, o operador anota a área de dissecção desejada e prossegue para dissecar o segmento apropriado. O programa gera uma imagem arquivada da dissecção. A principal vantagem do mesodissection é a curta duração necessária para dissecar um slide, tendo uma média de 10 minutos de set up para provar geração neste experimento. Além disso, o sistema é muito mais rentável e fácil de usar. Um pequena desvantagem é que não é tão precisa como a microscopia de captura laser. Neste artigo, vamos demonstrar como mesodissection pode ser usado para extrair RNA de slides de granulomas FFPE causada pelo Mycobacterium tuberculosis (Mtb).

O referido protocolo mostra como usar uma nova técnica mesodissection extrair RNA a partir de lâminas de tecido FFPE. Eficácia deste protocolo é mostrado através de slides FFPE de granulomas pulmonares de NHP infectados com Mtb em vários estágios infectantes. Figuras 1-3 são imagens do instrumento. Figura 4 mostra como ocorre o processo de dissecação ea imagem resultante gerado pelo software. Tabela 1 mostra os resultados de extração de RNA com um g...

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

Microdissecção tem sido extensivamente utilizada para a análise de DNA, RNA e proteínas dentro do tecido. Microscopia captura a laser (LCM) é o método mais comumente utilizado, mas uma nova técnica de fresagem, mesodissection, é recentemente disponível. Demonstramos extração de RNA a partir de mesodissected formalina parafina fixo lâminas de tecido embebidos de Mycobacterium tuberculosis granulomas.

Uma nova abordagem para Microdissecção Recuperando<em&gt; Mycobacterium tuberculosis</em&gt; transcritos específicos de formalina fixo Parafina incorporado Lung granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

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 visualizações.  Tulane National Primate Research Center.  Microdissecção tem sido extensivamente utilizada para a análise de DNA, RNA e proteínas dentro do tecido. Microscopia captura a laser (LCM) é o método mais comumente utilizado, mas uma nova técnica de fresagem, mesodissection, é recentemente disponível. Demonstramos extração de RNA a partir de mesodissected formalina parafina fixo lâminas de tecido embebidos de Mycobacterium tuberculosis granulomas. 

Vídeo: 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.

Preparação de amostras de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos para Estudos Nucleares metabolômica Ressonância Magnética

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Imunologia e Infecção

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.

Crescimento de Mycobacterium tuberculosis Biofilmes

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.

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de 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.

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação 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.

A detecção in situ de bactérias dentro dos tecidos de parafina-encaixados Usando um marcado com Digoxina DNA Probe Segmentação 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.

Um modelo de tecido Pulmão humano 3D para estudos funcionais em<em&gt; Mycobacterium tuberculosis</em&gt; Infecção

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 de Eficácia e citotoxicidade Rastreio de inibidores Segmentação 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.

Um ensaio Compatível de alta capacidade para avaliar droga Eficácia contra macrófagos passadas<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.

Análise de 18FDG PET-CT Imaging como uma ferramenta para estudar infecção por Mycobacterium tuberculosis e tratamento em primatas não-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.

Mycobacterium tuberculosis em camundongos com fluorescência de enzima do repórter de imagem

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