Vorremmo riconoscere Sean Fitzwater e Carmen Giannina Luna Colombo per il segmento video di tubercolosi crescita time-lapse. Ringraziamo Marty Roper per lei un feedback accurato ed eccellente durante il montaggio e co-autore del Manuale d&#39;uso, da cui è tratto l&#39;attuale protocollo per lo più alla lettera. La produzione di questo video è stato finanziato dal NIH / Fogarty International Center <a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a> David AJ Moore ha contribuito come Research Fellow Wellcome Trust clinica in Medicina Tropicale e Reader in Malattie Infettive presso l&#39;Imperial College di Londra (Fellowship Premio numero 078067/Z/05). Mark F. Brady ha contribuito come NIH / Research Fellow Fogarty International Center.

<ol><li> Preparare soluzioni di <ol><li> Tampone fosfato magazzino <ol><li> Mescolare 950ml di soluzione di sodio bibasico (9.47g di fosfato di sodio dibasico sciolto in 1000 ml di acqua distillata) con 950ml di soluzione di potassio fosfato monobasico (9.07g di fosfato monobasico di potassio disciolti in 1000 ml di acqua distillata) e mescolare; trattenere 50ml di ciascuna soluzione per regolare pH, se necessario, </li><li> Regolare il pH a 6,8 ± 0,2: aggiungere soluzione di fosfato di sodio bibasico per aumentare il pH, aggiungere la soluzione di potassio fosfato monobasico per abbassare il pH </li><li> Autoclave a 121-124 ° C per 15 minuti per sterilizzare </li><li> Piastra un&#39;aliquota 100μl della soluzione tampone fosfato su agar nutriente in piastre di Petri e incubare a 37 ° C per 48 ore per confermare la sterilità </li><li> Conservare in frigorifero a 2-8 ° C fino a un mese Nota: ogni campione di espettorato richiede 10 ml di tampone fosfato </li></ol></li><li> Decontaminazione magazzino <ol><li> Combina volumi uguali di soluzione di idrossido di sodio (8.0g di idrossido di sodio sciolto in 200 ml di acqua distillata sterile) e citrato di sodio (5,8 g di sodio citrato in 200ml di acqua distillata sterile distillata) </li><li> Mescolare e autoclave a 121-124 ° C per 15 minuti </li><li> Conservare in frigorifero a 2-8 ° C fino a un mese Nota: ogni campione di espettorato richiede 2 ml di brodo di decontaminazione </li></ol></li><li> Cultura dei media magazzino <ol><li> In 900 ml di acqua distillata sterile aggiungere 3.1ml di glicerolo e 1.25g di casitone e di 5.9g di polvere medio 7H9; mescolare con agitazione costante fino a completo scioglimento </li><li> Autoclave a 121-124 ° C per 15 minuti </li><li> Raffreddare e dividere il medium sterile in aliquote a 4,5 ml sterili 16x tubi di 100 mm di vetro per la preparazione dei campioni, e 10.8ml aliquote per la preparazione di soluzioni di antibiotici e controlli interni </li><li> Incubare a 37 ° C per 48 ore per verificare la sterilità (mancanza di torbidità) </li><li> Conservare a 2-8 ° C con tappo ben chiuso per un massimo di un mese Nota: ogni campione di espettorato richiede una provetta contenente 4,5 ml di medium 7H9 </li></ol></li><li> Soluzioni stock antibiotico <ol><li> A. isoniazide magazzino (8 mg / ml) <ol><li> Sciogliere 20 mg di isoniazide completamente in 2,5 ml di acqua distillata sterile </li><li> Filtrare con filtro per siringa 0.2μm solvente acquoso </li><li> Conservare in aliquote 20μl in sterili micro tubi di centrifugazione a -20 ° C fino a 6 mesi Nota: ogni aliquota memorizzata 20μl è sufficiente per un massimo di 100 campioni (tra cui gli sprechi) </li></ol></li><li> B. Rifampicina magazzino (8 mg / ml) </li></ol><ol><li> Sciogliere rifampicina 20mg completamente in 1.25ml DMSO </li><li> Aggiungi 1.25ml acqua sterile distillata e mescolare </li><li> Filtrare con filtro siringa 0.2μm per solventi organici </li><li> Conservare in aliquote 20μl in microprovette sterili a -20 ° C fino a 6 mesi Nota: ogni aliquota memorizzata 20μl è sufficiente per un massimo di 100 campioni (tra cui gli sprechi) </li></ol></li></ol></li><li> Preparare le soluzioni di lavoro <ol><li> Decontaminazione soluzione di lavoro <ol><li> Sciogliere 0,1 g di cristalli NALC in ogni 20ml di soluzione di decontaminazione richiesto Nota: Ogni campione deve 2 ml di soluzione di decontaminazione Nota: Eliminare le NaOH-NALC soluzione di decontaminazione che rimane inutilizzato dopo 24 ore come NALC perde la sua attività mucolitica nel tempo </li></ol></li><li> Soluzione cultura mediatica di lavoro <ol><li> Di cui: <ol><li> 1 tubo di media 7H9 per ogni campione di espettorato per essere trasformata, maggiorato di 1 tubo aggiuntivo per ogni piatto (per la colonna di controllo negativo) </li><li> 2 tubi di medio 7H9 per i controlli positivi (3 se ceppi monoresistant sono utilizzati) </li><li> 1-2 tubi con 7H9 10.8ml per la preparazione di una soluzione antibiotica </li></ol></li><li> Aggiungere 0,5 ml OADC al 4,5 ml di 7H9 in ogni provetta </li><li> Aggiungi OADC 1,2 ml di tubi con 10.8ml 7H9 </li><li> Mettere da parte 2 tubi con 5ml 7H9-OADC per i controlli positivi, e tubo (s) con 12ml 7H9-OADC da utilizzare per la preparazione di una soluzione antibiotica (questi programmi non richiedono PANTA) </li><li> Ricostituire PANTA e aggiungere 0,1 ml per ogni provetta e per i tubi di controllo negativo (7H9-OADC-PANTA: volume = 5.1ml) Nota: media Completo di PANTA (7H9-OADC-PANTA) viene utilizzato per campioni di espettorato e controlli negativi; uso 7H9-OADC senza PANTA per i controlli positivi e per la preparazione di una soluzione antibiotica </li></ol></li><li> Antibiotico soluzioni di lavoro <ol><li> 1. Vedere la Figura 1 per la procedura per rendere la soluzione antibiotica che lavorano in un inutilizzato 24-pozzetti Nota: Per ottenere risultati accurati suscettibilità, le concentrazioni finali antibiotico sono critici. La seguente procedura è adatto per laboratories dotato di micropipette. Se non sono disponibili micropipette, siringhe tubercolina può essere utilizzato con una diversa serie di diluizioni descritto nel Manuale dell&#39;utente Appendice 3 a modsperu.org Nota: non ri-congelare o riutilizzo di lavoro antibiotico o soluzioni stock dell&#39;attività del farmaco può essere perso Eliminare tutte le soluzioni non utilizzate antibiotici alla fine del trattamento in giornata, figura 1.. Rendere le soluzioni antibiotiche di lavoro (da Guida dell&#39;utente al modsperu.org) </li></ol></li></ol></li><li> L&#39;espettorato del campione e la piastra di preparazione <ol><li> Decontaminazione <ol><li> 2ml luogo di campione di espettorato in un tubo da centrifuga 15 ml (se meno, portare a 2 ml con tampone fosfato, se più, utilizzare solo 2 ml) </li><li> Aggiungere 2 ml di NaOH-NALC soluzione </li><li> Tappo e tubo vortex per 20 secondi; invertire tubo per assicurare NaOH-NALC contatti soluzione l&#39;intera superficie interna del tubo e coperchio </li><li> Lasciate riposare per un minimo di 15 minuti - può prolungare di qualche minuto, se il campione è particolarmente viscoso. Per evitare un eccesso di trattamento, non dovrebbe superare i 20 minuti </li><li> Riempire il tubo 14 ml con tampone fosfato (pH 6,8) per neutralizzare alcali e terminare il processo di decontaminazione, e mescolare bene invertendo il tubo di 4 volte </li><li> Centrifugare a 3000 g per 15 minuti (vedi appendice 2 nella Guida al modsperu.org per l&#39;equazione di rotazioni al minuto necessario per raggiungere 3000 g) </li><li> Versare cautamente fuori surnatante in un contenitore di rifiuti liquidi con ipoclorito di sodio al 10% o altro disinfettante appropriato e conservare il pellet </li></ol></li><li> Preparazione della sospensione campione finale e di backup <ol><li> Utilizzando 7H9-OADC-PANTA (dal tubo contenente 5.1ml), risospendere il campione di pellet in un volume totale di 2 ml in provetta con una pipetta Pasteur, mescolare bene </li><li> Togliere 1 ml di sospensione dei campioni e conservare in una provetta a 2-8 ° C come backup </li><li> Aggiungere il 1ml seconda sospensione del campione per il tubo con il restante 7H9-OADC-PANTA, mescolare bene. Questa è la soluzione campione finale pronto per la placcatura </li></ol></li><li> Posizionamento sospensioni campione nel 24-pozzetti <ol><li> 900μl luogo della sospensione campione finale in ciascuno dei 4 pozzetti di una colonna del 24-pozzetti </li><li> Ripetere con campioni supplementari fino a quando tutte le colonne del piatto, ad eccezione di Colonna 3, sono piene (o fino a quando tutti i campioni sono placcati) </li><li> 900μl Luogo di 7H9-OADC mezzo senza il campione in 4 pozzi di colonna 3 di ogni piatto del campione (negativo dei controlli interni) </li></ol></li><li> L&#39;aggiunta di antibiotici ai campioni <ol><li> Utilizzando una pipetta multicanale, con attenzione riempire 4 punte con 100μl dai pozzi con le soluzioni di lavoro 7H9-OADC e antibiotico (colonna 2 nella diluizione antibiotico piastra-vedi Figura 1) </li><li> Aggiungere le aliquote 100μl al 1 pozzi colonna con soluzioni campione 900μl senza toccare piastra o campione </li><li> Ripetere fino a quando tutte le colonne hanno ricevuto il 100μl aggiuntivo di media (drug-free pozzi) o soluzioni antibiotiche (compresa colonna di controllo negativo 3) </li><li> Chiudere la piastra con il coperchio e mettere in un sigillabile politene (Ziplock) sacchetto e guarnizione (borsa non si apre di nuovo da questo punto in poi) </li><li> Incubare a 37 ° C (CO 2 arricchimento non è necessario) </li></ol></li></ol></li><li> Controllo qualità <ol><li> Controlli negativi sono eseguiti su ogni piatto. La presenza di una crescita in uno dei pozzi nella colonna di controllo negativo indica la contaminazione crociata, quindi l&#39;intera piastra deve essere scartata ed i campioni ri-plated. </li><li> I controlli positivi sono eseguiti su un piatto a parte alla fine della giornata. Un pieno di droga sensibili ceppo viene eseguito in una colonna e un ceppo resistente a INH e RIF viene eseguito in un&#39;altra colonna. Se vi è preoccupazione per l&#39;esecuzione un ceppo MDR, sia un ceppo INH monoresistant e un ceppo RIF monoresistant può essere eseguito al posto dei ceppi sensibili e MDR. Tutti i controlli positivi dovrebbero avere una crescita nel drug-free pozzi per dimostrare che la crescita dei supporti. Per dimostrare che le concentrazioni di farmaco siano corrette, il ceppo sensibili non dovrebbero crescere in INH o RIF pozzi, ma il ceppo MDR dovrebbe crescere anche crescere nella INH e RIF pozzi. </li></ol></li><li> Piastra di lettura <ol><li> Con il giorno della placcatura come &quot;giorno 0&quot;, lettura targhe inizia il 5 ° giorno, e se negativo al giorno 5 lettura avviene ogni giorno (o giorni alterni a seconda del carico di lavoro) fino al giorno 14 e poi ogni 2-3 giorni dal giorno 15 -21. Le piastre sono esaminati in unainvertito microscopio ottico con l&#39;obiettivo 10x </li><li> Una cultura positiva è quella in cui ci sono due o più unità formanti colonia (&gt; 2 CFU) in entrambi i farmaci senza pozzi. All&#39;inizio la crescita dei micobatteri sembra piccolo virgole curve o spirali (giorni 5-9). Formazione di colonie di solito progredisce a &quot;corde&quot; o &quot;grovigli&quot; (vedi libreria di immagini su modsperu.org) </li><li> Il giorno una cultura positiva nei 2 drug-free pozzi, il farmaco contenente i pozzi devono essere lette. Qualsiasi crescita in presenza di un farmaco indica che la resistenza al farmaco. Nota: Se la lettura del farmaco contenente pozzi viene fatto&gt; 1 settimana dopo una cultura positiva è notato nel drug-free pozzi, questo non può rappresentare la resistenza di rottura attraverso la crescita è occasionalmente </li><li> I campioni che non sono positivi dopo 21 giorni sono considerati colture negative </li><li> Nuvolosità dei pozzi è il risultato della crescita eccessiva contaminazione, mentre la crescita dei micobatteri non determina nuvolosità </li><li> Al termine di 21 giorni culture può essere scartata. Culture rimanere nel loro sacchetti sigillati e sono disposti in sacchetti autoclave e sterilizzati in autoclave a 121-124 ° C per 45-60 minuti </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>

Il saggio MODS è rivolto a risorse limitate. Per la prima volta, MODS porta la capacità per una rapida individuazione coltura liquida di tubercolosi e di tubercolosi multiresistente a risorse limitate a poco meno di $ 3 per prova. MODS è un non-proprietarie, iterativo metodologia, e la comunità MODS è sempre interessata a miglioramenti che altri laboratori sono riusciti a fare. Una preoccupazione ricorrente è la biosicurezza di liquido cultura mediatica di tubercolosi, perché i liquidi possono essere versato o aerosol. Credia...

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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

I pazienti con tubercolosi polmonare attiva (TB) infettare altre 10-15 persone l&#39;anno, rendendo la diagnosi di TBC attiva essenziale sia per curare il paziente e prevenire nuove infezioni. Inoltre, la comparsa di tubercolosi multiresistente (MDRTB) significa che il rilevamento della resistenza ai farmaci è necessario per fermare la diffusione di ceppi resistenti ai farmaci. L&#39;osservazione microscopica-droga suscettibilità (MODS) è un test a basso costo, low-tech strumento ad alte prestazioni di rilevamento della tubercolosi e MDRTB. Il saggio MODS si basa su tre principi: 1) Mycobacterium tuberculosis (MTB) cresce più velocemente in mezzi liquidi che su terreni solidi 2) la crescita MTB microscopici possono essere rilevati in precedenza in mezzi liquidi che aspettare l&#39;aspetto macroscopico delle colonie su terreni solidi, e che la crescita è caratteristica della MTB, che permette di distinguerlo da micobatteri atipici contaminazioni batteriche o fungine o 3) i farmaci isoniazide e rifampicina può essere incorporato nel saggio MODS per consentire la simultanea rilevazione diretta di MDRTB, ovviando alla necessità di una sottocultura di effettuare indiretta farmaco test di sensibilità. Concorrenti diagnostica attuali sono ostacolati da una bassa sensibilità con striscio dell&#39;espettorato, ritardo prolungato fino diagnosi con una solida cultura dei media, dei costi proibitivi con gli attuali metodi di liquido cultura dei media, e la necessità di fare sottocultura per i test indiretti sensibilità ai farmaci per rilevare MDRTB. Al contrario, il non proprietario metodo MODS ha una sensibilità elevata per la tubercolosi e MDRTB, è un metodo di cultura relativamente rapido, fornisce contemporaneamente test di sensibilità farmaco per MDRTB, ed è accessibile a risorse limitate a poco meno di 3 dollari per i test per la TBC e MDRTB.

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

L&#39;osservazione microscopica-droga suscettibilità (MODS) è un test a basso costo, low-tech strumento di alte prestazioni di rilevamento della tubercolosi (TB) e la tubercolosi multi-resistente (MDRTB). Questo video descrive il liquido MODS mezzi metodo cultura.

Il metodo MODS per la diagnosi di tubercolosi e di tubercolosi multiresistente

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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Research

JoVE Journal

Biology

18.9K Views.  The Warren Alpert Medical School of Brown University. L'osservazione microscopica-droga suscettibilità (MODS) è un test a basso costo, low-tech strumento di alte prestazioni di rilevamento della tubercolosi (TB) e la tubercolosi multi-resistente (MDRTB). Questo video descrive il liquido MODS mezzi metodo cultura.

Video: 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.

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

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

Ricerca

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.

Espressione, solubilizzazione detergente, e purificazione di un trasportatore di membrana, la proteina multiresistente MexB Resistenza

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.

Il metodo di coltura degli embrioni roditori intero utilizzando il Rotator tipo di sistema cultura Bottiglia

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).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di 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.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica Bicelle

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.

Un metodo semplice per l&#39;imaging Arabidopsis Foglie Utilizzando Perfluorodecalin come Media Imaging infiltrativa

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.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

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.

Un metodo per lo screening e validazione di mutazioni resistenti contro 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 ...

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.

Metodo per la valutazione degli effetti di un intervallo di lunghezze d&#39;onda e intensità di / vicino-infrarosso di terapia della luce rossa sullo stress ossidativo<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.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

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