Gostaríamos de agradecer Sean Fitzwater e Carmen Giannina Luna Colombo para o segmento de vídeo tuberculose crescimento lapso de tempo. Agradecemos a Marty Roper para ela um feedback completo e excelente durante a edição e co-autoria o Guia do Usuário, a partir do qual o actual protocolo foi tomada principalmente verbatim. Produção deste vídeo foi financiado pelo NIH / Fogarty International Center <a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a> David AJ Moore contribuiu como Research Fellow Wellcome Trust Clínica em Medicina Tropical e Reader em Doenças Infecciosas do Imperial College London (Sociedade número prêmio 078067/Z/05). Mark F. Brady contribuiu como um NIH / Fogarty Fellow Research International Center.

<ol><li> Preparar soluções estoque <ol><li> Fosfato de estoque regulador <ol><li> Misture 950ml de solução de sódio dibásico (9.47g de fosfato de sódio dibásico dissolvido em água destilada 1000ml) com 950ml de potássio solução de fosfato monobásico (9.07g de potássio fosfato monobásico dissolvido em água destilada 1000ml) e agitar; retivesses 50ml de cada solução para ajustar pH se necessário </li><li> Ajustar o pH para 6,8 ± 0,2: adicionar fosfato de sódio dibásico solução para elevar o pH, adicione a solução de fosfato de potássio monobásico de baixar o pH </li><li> Autoclave a 121-124 ° C por 15 minutos para esterilizar </li><li> Placa de uma alíquota de 100μl da solução tampão de fosfato em meio agar nutriente em uma placa de Petri e incubar a 37 ° C por 48 horas para confirmar a esterilidade </li><li> Guarde na geladeira a 2-8 ° C por até um mês Nota: Cada amostra de escarro requer 10ml de solução tampão fosfato </li></ol></li><li> Ações de descontaminação <ol><li> Combine volumes iguais de solução de hidróxido de sódio (8,0 g de hidróxido de sódio dissolvido em água destilada estéril 200ml) e solução de citrato de sódio (5,8 g de citrato de sódio em água destilada estéril 200ml destilada) </li><li> Misture e autoclave a 121-124 ° C por 15 minutos </li><li> Guarde na geladeira a 2-8 ° C por até um mês Nota: Cada amostra de escarro requer 2ml de ações de descontaminação </li></ol></li><li> Cultura de ações de mídia <ol><li> Em 900ml de água destilada estéril, adicionar 3.1ml de glicerol e 1,25 g de casitone e 5,9 g de pó meio 7H9 e misturar com agitação constante até completamente dissolvido </li><li> Autoclave a 121-124 ° C por 15 minutos </li><li> Esfriar e dividir o meio estéril em alíquotas 4.5ml em estéril 16x 100 tubos de vidro milímetros para preparação de amostras, e 10.8ml alíquotas para a preparação de soluções de antibióticos e de controles internos </li><li> Incubar a 37 ° C por 48 horas para verificar a esterilidade (ausência de turbidez) </li><li> Armazenar de 2-8 ° C com tampa bem fechado por até um mês Nota: Cada amostra de escarro requer um tubo contendo 4,5 ml de meio 7H9 </li></ol></li><li> Antibiótico soluções estoque <ol><li> A. Isoniazida estoque (8 mg / ml) <ol><li> Dissolver 20 mg de isoniazida completamente em água destilada estéril, 2,5 ml </li><li> Filtro com filtro de seringa de 0,2 Hm para solvente aquoso </li><li> Loja em alíquotas 20μl em tubos de centrifugação estéril micro a -20 ° C por até 6 meses Nota: Cada alíquota armazenada 20μl é suficiente para até 100 amostras (incluindo o desperdício) </li></ol></li><li> B. A rifampicina estoque (8 mg / ml) </li></ol><ol><li> Dissolver 20mg rifampicina completamente em 1.25ml DMSO </li><li> Adicionar 1.25ml água destilada estéril e misturar </li><li> Filtro com filtro de seringa de 0,2 Hm de solvente orgânico </li><li> Loja em alíquotas 20μl em tubos de microcentrífuga estéril a -20 ° C por até 6 meses Nota: Cada alíquota armazenada 20μl é suficiente para até 100 amostras (incluindo o desperdício) </li></ol></li></ol></li><li> Prepare soluções de trabalho <ol><li> Solução de descontaminação de trabalho <ol><li> Dissolver 0,1 g de cristais de NALC em cada 20ml de solução de descontaminação requerido Nota: Cada amostra necessidades 2ml de solução de descontaminação Nota: Eliminar qualquer solução de descontaminação NaOH-NALC que permanece não utilizado após 24 horas, como NALC perde a sua atividade ao longo do tempo mucolítico </li></ol></li><li> Solução de meios de cultura de trabalho <ol><li> Estabelecidos: <ol><li> 1 tubo com meio 7H9 para cada amostra de escarro para ser processado, além de um tubo adicional para cada placa (para a coluna controle negativo) </li><li> 2 tubos de meio 7H9 para os controles positivo (3 se cepas monoresistant são usados) </li><li> 02/01 tubos com 7H9 10.8ml para a preparação de solução antibiótica </li></ol></li><li> Adicionar 0,5 ml OADC ao 4.5ml de 7H9 em cada tubo de amostra </li><li> Adicionar OADC 1,2 ml para tubos com 7H9 10.8ml </li><li> Separe dois tubos com 5ml 7H9-OADC os controles positivos, e tubo (s) com 12ml 7H9-OADC a ser utilizado para a preparação de solução antibiótica (estes não requerem PANTA) </li><li> PANTA reconstituir e adicionar 0,1 ml para cada tubo de amostra e os tubos de controle negativo (7H9-OADC-PANTA: volume total = 5.1ml) Nota: médio completo com PANTA (7H9-OADC-PANTA) é usado para amostras de escarro e controles negativos; use 7H9-OADC sem PANTA de controlos positivos e para a preparação de solução antibiótica </li></ol></li><li> Antibiótico soluções de trabalho <ol><li> 1. Veja a Figura 1 para as etapas para tornar a solução antibiótica trabalhando em uma placa de 24 poços não utilizados Nota: Para obter resultados precisos de suscetibilidade, as concentrações finais de antibióticos são críticos. O procedimento a seguir é adequado para laboratories equipado com micropipetas. Se micropipetas não estão disponíveis, seringas de tuberculina pode ser usado com uma série de diluições diferentes descritos no Guia do Usuário Apêndice 3 a modsperu.org Nota: Não volte a congelar ou re-utilização de trabalho antibióticos ou soluções de ações como a atividade das drogas podem ser perdidos Descartar todas as soluções não utilizado antibiótico no final do dia de processamento Figura 1.. Tornando as soluções de antibióticos de trabalho (de Guia do Usuário no modsperu.org) </li></ol></li></ol></li><li> Amostra de escarro e placa de preparação <ol><li> Descontaminação <ol><li> 2ml lugar de amostra de escarro em um tubo de centrífuga de 15ml (se for inferior, compõem a 2ml com tampão fosfato; se mais, use apenas 2ml) </li><li> Adicionar 2 ml de solução NaOH-NALC </li><li> Tubo de tampa bem e vortex por 20 segundos; inverter tubo para garantir contatos NaOH-NALC solução a superfície interior do tubo inteiro e tampa </li><li> Deixe repousar por um mínimo de 15 minutos - pode prolongar por alguns minutos se a amostra é particularmente viscoso. Para evitar o excesso de tratamento, não deve exceder 20 minutos </li><li> Encha o tubo de 14ml com tampão fosfato (pH 6,8) para neutralizar alcalinos e encerrar o processo de descontaminação, e misture bem, vire o tubo 4 vezes </li><li> Centrifugar a 3000 g por 15 minutos (ver Apêndice 2 no Guia do Usuário no modsperu.org para a equação de rotações por minuto necessário para atingir 3000 g) </li><li> Deitar fora o sobrenadante cuidadosamente em um recipiente de resíduos líquidos com hipoclorito de sódio 10% ou outro desinfetante adequado e manter o pellet </li></ol></li><li> Preparação da suspensão amostra final e backup <ol><li> Utilizando-7H9-OADC PANTA (a partir do tubo contendo 5.1ml), ressuspender o sedimento em amostra de um volume total de 2ml no tubo de ensaio com uma pipeta Pasteur, misture bem </li><li> Retire 1ml de suspensão da amostra e armazenar em um tubo de microcentrífuga de 2-8 ° C como um backup </li><li> Adicione o segundo 1ml de suspensão de amostra para o tubo com os restantes 7H9-OADC-PANTA; misture bem. Esta é a solução da amostra final pronto para revestimento </li></ol></li><li> Colocando suspensões amostra em 24 poços da placa <ol><li> 900μl lugar da suspensão amostra final em cada um dos quatro poços de uma coluna na placa de 24 poços </li><li> Repita com amostras adicionais até que todas as colunas da placa, com exceção da coluna 3, são preenchidos (ou até que todas as amostras são banhados) </li><li> 900μl lugar de 7H9-OADC meio sem amostra na 4 poços da coluna 3 de cada placa de amostra (negativo controles internos) </li></ol></li><li> Adição de antibióticos para amostras <ol><li> Com uma pipeta multi-canal, preencha cuidadosamente quatro pontas com 100μl dos poços com 7H9-OADC e antibiótico soluções de trabalho (Coluna 2 na diluição de antibióticos placa-ver Figura 1) </li><li> Adicione o alíquotas 100μl para a coluna 1 poços com soluções amostra 900μl sem tocar placa ou amostra </li><li> Repita até que todas as colunas receberam o adicional de 100μl médio (livre de drogas poços) ou soluções de antibiótico (incluindo coluna de controle negativo 3) </li><li> Feche a placa com a sua tampa e coloque em um saco de polietileno lacrado (Ziplock) e selo (saco não for aberto novamente a partir deste ponto) </li><li> Incubar a 37 ° C (CO 2 de enriquecimento não é necessário) </li></ol></li></ol></li><li> Controle de qualidade <ol><li> Controles negativos são executados em cada placa. A presença de qualquer crescimento em qualquer um dos poços na coluna de controle negativo indica a contaminação cruzada, portanto, toda a placa deve ser descartado e as amostras re-plated. </li><li> Os controlos positivos são executados em uma placa separada no final do dia. Uma cepa totalmente drogas suscetíveis é executado em uma coluna e uma cepa resistente à INH e RIF é executado em outra coluna. Se há preocupação sobre a execução de uma cepa MDR, tanto a tensão um monoresistant INH e RIF monoresistant uma cepa pode ser executado no lugar das cepas sensíveis e MDR. Todos os controles positivos deve ter crescimento nos poços livre de drogas para demonstrar que o crescimento apoio da mídia. Para demonstrar que as concentrações da droga estão corretas, a cepa suscetível não deve crescer no INH ou poços RIF, mas a cepa MDR deve crescer também crescer no INH e RIF poços. </li></ol></li><li> Leitura da placa <ol><li> Com o dia do chapeamento como &quot;dia 0&quot;, a leitura da placa começa no dia 5, e se negativo no dia 5 de leitura é feito a cada dia (ou dias alternados, dependendo da carga de trabalho) até o dia 14 e depois a cada 2-3 dias a partir de dia 15 -21. Placas são examinadas em ummicroscópio de luz invertida, com o objetivo de 10x </li><li> A cultura positiva é aquela em que há duas ou mais unidades formadoras de colônia (&gt; 2 CFU) em AMBAS as livre de drogas poços. Micobactérias de crescimento no início se parece com pequenas vírgulas curvas ou espirais (dia 09/05). Formação de colônias geralmente progride para &quot;cordas&quot; ou &quot;emaranhados&quot; (ver imagem na biblioteca modsperu.org) </li><li> No dia em que uma cultura é positiva na 2 livre de drogas poços, os poços de drogas que contêm deve ser lido. Qualquer crescimento na presença de uma droga indica resistência a essa droga. Nota: Se a leitura dos poços de drogas que contêm é feito&gt; uma semana depois de uma cultura positiva é observado nos poços livre de drogas, isso não pode representar a resistência como break-through crescimento é visto ocasionalmente </li><li> As amostras que não são positivos por Dia 21 são consideradas culturas negativas </li><li> Nebulosidade dos poços é resultado de crescimento excessivo de contaminação, enquanto que o crescimento de micobactérias não resulta na nebulosidade </li><li> Após a conclusão de 21 dias as culturas podem ser descartados. Culturas permanecem em seus sacos fechados e são colocados em sacos de autoclave e autoclavados a 121-124 ° C durante 45-60 minutos </li></ol></li></ol>

<ol>
	<li>Arias, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic-observation+drug-susceptibility+assay+for+detection+of+tuberculosis.">Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis.</a> Clin Infect Dis. 44, 674-674 (2007).</li><li>Caviedes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+efficient+detection+and+drug+susceptibility+testing+of+Mycobacterium+tuberculosis+in+sputum+by+microscopic+observation+of+broth+cultures.+The+Tuberculosis+Working+Group+in+Peru.">Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru.</a> J Clin Microbiol. 38, 1203-1203 (2000).</li><li>Caviedes, L., Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+MODS:+a+low-cost,+low-tech+tool+for+high-performance+detection+of+tuberculosis+and+multidrug+resistant+tuberculosis.">Introducing MODS: a low-cost, low-tech tool for high-performance detection of tuberculosis and multidrug resistant tuberculosis.</a> Indian J Med Microbiol. 25, 87-87 (2007).</li><li>Caws, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+MODS+culture+technique+for+the+diagnosis+of+tuberculous+meningitis.">Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis.</a> PLoS ONE. 2, e1173-e1173 (2007).</li><li>Ejigu, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic-observation+drug+susceptibility+assay+provides+rapid+and+reliable+identification+of+MDR-TB.">Microscopic-observation drug susceptibility assay provides rapid and reliable identification of MDR-TB.</a> Int J Tuberc Lung Dis. 12, 332-332 (2008).</li><li>Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+occupational+tuberculosis+in+National+Tuberculosis+Programme+laboratories+in+Korea.">Risk of occupational tuberculosis in National Tuberculosis Programme laboratories in Korea.</a> Int J Tuberc Lung Dis. 11, 138-138 (2007).</li><li>Mello, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic+observation+drug+susceptibility+assay+for+detection+of+Mycobacterium+tuberculosis+resistance+to+isoniazid+or+rifampin.">Clinical evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to isoniazid or rifampin.</a> J Clin Microbiol. 45, 3387-3387 (2007).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+prospects+for+the+MODS+assay+in+multidrug-resistant+tuberculosis+diagnosis.">Future prospects for the MODS assay in multidrug-resistant tuberculosis diagnosis.</a> Future Microbiol. 2, 97-97 (2007).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrequent+MODS+TB+culture+cross-contamination+in+a+high-burden+resource-poor+setting.">Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting.</a> Diagn Microbiol Infect Dis. 56, 35-35 (2006).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic-observation+drug-susceptibility+assay+for+the+diagnosis+of+TB.">Microscopic-observation drug-susceptibility assay for the diagnosis of TB.</a> N Engl J Med. 355, 1539-1539 (2006).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+observation+drug+susceptibility+assay,+a+rapid,+reliable+diagnostic+test+for+multidrug-resistant+tuberculosis+suitable+for+use+in+resource-poor+settings.">Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings.</a> J Clin Microbiol. 42, 4432-4432 (2004).</li><li>Moore, D. A., Roper, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+smear-negative+tuberculosis+in+people+with+HIV/AIDS.">Diagnosis of smear-negative tuberculosis in people with HIV/AIDS.</a> Lancet. 370, 1033-1033 (2007).</li><li>Oberhelman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+recovery+of+Mycobacterium+tuberculosis+from+children+using+the+microscopic+observation+drug+susceptibility+method.">Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method.</a> Pediatrics. 118, e100-e100 (2006).</li><li>Palomino, J. C., Martin, A., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MODS+assay+for+the+diagnosis+of+TB.">MODS assay for the diagnosis of TB.</a> N Engl J Med. 356, 188-189 (2007).</li><li>Park, W. G., Bishai, W. R., Chaisson, R. E., Dorman, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+microscopic+observation+drug+susceptibility+assay+in+drug+susceptibility+testing+for+Mycobacterium+tuberculosis.">Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis.</a> J Clin Microbiol. 40, 4750-4750 (2002).</li><li>Shiferaw, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microscopic+observation+drug+susceptibility+assay+for+detection+of+multidrug-resistant+Mycobacterium+tuberculosis.">Evaluation of microscopic observation drug susceptibility assay for detection of multidrug-resistant Mycobacterium tuberculosis.</a> J Clin Microbiol. 45, 1093-1093 (2007).</li><li>Tovar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+diagnosis+of+pleural+tuberculosis+using+the+microscopic-+observation+drug-susceptibility+technique.">Improved diagnosis of pleural tuberculosis using the microscopic- observation drug-susceptibility technique.</a> Clin Infect Dis. 46, 909-909 (2008).</li><li>Vargas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+sputum-scarce+HIV-associated+pulmonary+tuberculosis+in+Lima,+Peru.">Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru.</a> Lancet. 365, 150-150 (2005).</li></ol>

O ensaio MODS é destinado a ambientes de recursos limitados. Pela primeira vez, MODS traz a capacidade para a detecção rápida de cultura líquido de tuberculose multirresistente e tuberculose resistente a ambientes de recursos limitados em pouco menos de 3 dólares por teste. MODS é um não-proprietário, a metodologia iterativa, ea comunidade MODS está sempre interessada em melhorias que outros laboratórios têm conseguido fazer. Uma preocupação recorrente é a biossegurança da cultura de mídia líquida da tuberculose, p...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Refrigerator/ freezer</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to store pre-prepared broth and antibiotic stocks</td>
</tr>
<tr>
<td>Vortex</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to aid sputum decontamination</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed)</td>
</tr>
<tr>
<td>Incubator (37 degree C)</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for culture; need not be CO2 enriched</td>
</tr>
<tr>
<td>Inverted light microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to read MODS plates</td>
</tr>
<tr>
<td>Autoclave</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to sterilize media, PBS and used plates</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to weigh isoniazid, rifampicin and NALC</td>
</tr>
<tr>
<td>Middlebrook 7H9 broth (Difco)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0713-17-9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Casitone (pancreatic digest casein)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0259&#x2010;17&#x2010;9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Glycerol (glycerin) lyophilized</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G&#x2010;33&#x2010;500</td>
<td>500ml/bottle; culture media base</td>
</tr>
<tr>
<td>PANTA (Antibiotic mixture lyophilized BD) </td>
<td>Reagent</td>
<td>Fisher</td>
<td>B4345114</td>
<td>6 bottles/pack; antibiotic media supplement</td>
</tr>
<tr>
<td>OADC (Middlebrook OADC enrichment BD)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>B11886</td>
<td>10 x 20ml/pack; nutritional media supplement</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (Hibri-Max)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D-2650</td>
<td>100ml/bottle; to prepare rifampicin stock</td>
</tr>
<tr>
<td>Antibiotic stocks: isoniazid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I-3377</td>
<td>50gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Antibiotic stocks: rifampicin</td>
<td>Reagent</td>
<td>Sigma 99</td>
<td>R-3501</td>
<td>1gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Sodium hydroxide (pellets)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>221465</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium citrate (trisodium salt dihydrate)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S-4641</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>N-acetyl-L-cysteine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7250</td>
<td>50gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Potassium Phosphate Monobasic crystal. KH2PO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0662</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium Phosphate Dibasic, anhydrous. Na2HPO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0876</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium hypochlorite</td>
<td>Reagent</td>
<td>household bleach</td>
<td>&nbsp;</td>
<td>to discard contaminated waste</td>
</tr>
<tr>
<td>15ml centrifuge tubes (polypropylene 15ml Falcon 35&#x2010;2096)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>14&#x2010;959&#x2010;49B</td>
<td>500ea/case; for sputum decontamination and concentration</td>
</tr>
<tr>
<td>24 well plates (Plates Tissue 24 wells BD Falcon 35&#x2010;3047)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>50 plates/case; for culture and reading</td>
</tr>
<tr>
<td>Sealable polythene bags 6 X 6 &#x201C; (ziplock)</td>
<td>Consumable</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for biosecurity to contain 24 well plate</td>
</tr>
<tr>
<td>Glass tubes with lid (16 x 100mm and 18 x 145mm)</td>
<td>Consumable</td>
<td>VWR</td>
<td>47729-583</td>
<td>500 tubes/case; to store aliquots of prepared broth</td>
</tr>
<tr>
<td>Screw cap microcentrifuge tubes (1.5ml)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>05&#x2010;669&#x2010;22</td>
<td>1000ea/case; to store aliquots of antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (aqueous solvents) Syringe filter Millex blue</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGL 025 OS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (organic solvents) Syringe filter Millex yellow</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGV 033 RS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>Disposable Pasteur pipettes borosilicate glass 9&#x2033;</td>
<td>Consumable</td>
<td>Fisher</td>
<td>13&#x2010;678&#x2010;20C</td>
<td>720ea/case; to mix PANTA with media mix</td>
</tr>
<tr>
<td>Aerosol barrier tips 1000&#x2010;1300&#x03BC;l</td>
<td>Consumable</td>
<td>Fisher</td>
<td>02&#x2010;707&#x2010;51</td>
<td>1000ea/pk; to dispense media into plate</td>
</tr>
<tr>
<td>USA Scientific Tips One 1&#x2010;200&#x03BC;l yellow tips</td>
<td>Consumable</td>
<td>Fisher</td>
<td>1111&#x2010;0006</td>
<td>1000 tips/bag; to dilute antibiotic stocks</td>
</tr>		</tbody>
		</table>
		</div>

the mods method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Pacientes com tuberculose pulmonar ativa (TB) 15/10 infectar outras pessoas por ano, tornando o diagnóstico de TB ativa essencial para tanto a cura do paciente e prevenção de novas infecções. Além disso, o surgimento da tuberculose resistente a múltiplas drogas (TBMR) significa que a detecção da resistência às drogas é necessário para impedir a disseminação de cepas resistentes. A observação microscópica de drogas susceptibilidade-ensaio (MODS) é uma low-cost, low-tech ferramenta de alto desempenho para detecção de TB e da TBMR. O ensaio MODS é baseada em três princípios: 1) Mycobacterium tuberculosis (MTB) cresce mais rapidamente em meios líquidos do que em meios sólidos 2) o crescimento MTB microscópicas podem ser detectadas anteriormente em meios líquidos de esperar que o aspecto macroscópico das colônias em meios sólidos e que o crescimento é característica de MTB, permitindo que ele seja distinguido de contaminação micobactérias ou fungos ou bactérias atípicas 3) as drogas isoniazida e rifampicina pode ser incorporada no ensaio MODS para permitir a detecção direta simultânea de TBMR, eliminando a necessidade de subcultura para executar um teste de sensibilidade indireto da droga. Concorrentes diagnósticos atuais são dificultados pela baixa sensibilidade com baciloscopia, longos atrasos até o diagnóstico com a cultura meios sólidos, com custos proibitivos de métodos existentes líquido meios de cultura, ea necessidade de fazer subcultura para testes indiretos de drogas susceptibilidade para detectar TBMR. Em contraste, o não-proprietário método MODS tem uma alta sensibilidade para TB e da TBMR, é um método de cultura relativamente rápido, oferece testes de sensibilidade simultânea de drogas para TBMR, e é acessível a locais de recursos limitados em pouco menos de 3 dólares para os testes de TB e TBMR.

Watch this Scientific Journal Video about The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis at JoVE.com

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

A observação microscópica de drogas susceptibilidade-ensaio (MODS) é uma low-cost, low-tech ferramenta de alto desempenho para detecção de tuberculose (TB) e tuberculose multirresistente (TBMR). Este vídeo descreve o MODS líquido método de cultura de mídia.

O método MODS para o diagnóstico da tuberculose e da tuberculose resistente a múltiplas drogas

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

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18.9K visualizações.  The Warren Alpert Medical School of Brown University. A observação microscópica de drogas susceptibilidade-ensaio (MODS) é uma low-cost, low-tech ferramenta de alto desempenho para detecção de tuberculose (TB) e tuberculose multirresistente (TBMR). Este vídeo descreve o MODS líquido método de cultura de mídia.

Vídeo: The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Uma injeção de. Embriões stephensi para gerar malária resistente a mosquitos

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Biologia

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expressão, solubilização, detergente e Purificação de um transportador de membrana, as proteínas MexB resistência a múltiplas drogas

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

O método de cultura de embriões de roedores inteiro usando o Rotator do tipo Sistema de Cultura Garrafa

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Um método simples para criação de imagens Arabidopsis Folhas Usando Perfluorodecalin como um meio de imagem infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método de Avaliação de efeitos de uma gama de comprimentos de onda e intensidades de Terapia da Luz Vermelha / near-infrared sobre o estresse oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...