Agradecemos a CAPES-Brasil, Brasil-CNPq, FAP/DF para apoio financeiro. Nós estamos gratos ao Dr. Hugo Costa Paes para a revisão do manuscrito.

1. soluções e meios de comunicação
<ol><li>Prepare 2 X Roswell Park Memorial Institute (RPMI) 1640 suporte, tampão fosfato salino (PBS), caldo de Sabouraud dextrose e ágar de Sabouraud dextrose conforme tabela 1.</li>
</ol>2. condições de crescimento do inóculo fúngicas
<ol><li>Armazene todas as estirpes fúngicas como estoques congelados em glicerol de 35% a-80 ° C, até que seja necessário.</li>
	<li>Execute as seguintes etapas antes de cada experimento.<ol>
<li>Por cepas de Candida albicans :<ol>
<li>Descongelar um frasco de estoque e transferir 200 µ l a 10 mL de caldo de Sabouraud dextrose em um tubo de polipropileno estéril 50 mL com tampa e cultura durante a noite a 30 ° C, com agitação (200 rpm). Lembre-se de inclinar o tubo e deixar a tampa um pouco aberta para melhor aeração da cultura.</li>
		</ol>
</li>
		<li>Para cepas de Cryptococcus neoformans :<ol>
<li>Raspe a superfície congelada de um frasco de estoque. As células em placas de ágar Sabouraud dextrose da placa e incubar durante 48 h a 30 ° C. Depois visível crescimento de colônias isoladas, manter as placas no frigorífico (4 ° C) selado com película de parafina para até 15 dias.</li>
			<li>Recolher uma colônia isolada de médio porte da placa usando um palito estéril ou ansa inoculando estéril e inocular 10 mL de caldo de Sabouraud dextrose em um tubo cônico estéril 50 mL. Incube durante aproximadamente 24 horas a 30 ° C, sob agitação (200 rpm).</li>
			<li>Não exceda a incubação de 24 h. Lembre-se de inclinar o tubo e deixar a tampa um pouco aberta para melhor aeração. É sempre bom padronizar a fase de crescimento das células antes de cada ensaio, já que este é um fator importante que afecta a resistência antimicrobiana. Nota: Ambos os fungos foram cultivados a 30 ° C, de rápida propagação em nossos experimentos, mas a temperatura pode ser alterada para refletir os objetivos do estudo, o tratamento por exemplo clínico (37 ° C). Mais importante, este tempo de incubação inicial deve ser previamente estabelecido para cada isolado fúngico e mantido ao longo de todos os testes para garantir a reprodutibilidade.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Após o crescimento de fungos, colete as células por centrifugação dos tubos cónicos a 1.200 x g durante 5 min à temperatura ambiente. Desprezar o sobrenadante e adicionar 10 mL de PBS.</li>
	<li>Ressuspender as células e centrifugar novamente a 1.200 x g durante 5 min à temperatura ambiente.</li>
	<li>Repeti a lavagem PBS e centrifugação mais duas vezes. Após a terceira lavagem, Ressuspender as células em 5 mL (de acordo com o tamanho da pelota) de 2 X RPMI-1640 médio.</li>
	<li>Prepare-se 1 mL de uma diluição de 1: 100 ou 1:1,000 em um tubo de microcentrifugadora (dependendo da turvação de suspensão de células).</li>
	<li>Alíquota 10 µ l desta diluição, coloque-o em uma câmara de hemocytometer e conte o número total de células nos quadrantes quatro canto sob o microscópio. Calcular a concentração pela fórmula: (total de número de celular/4) x diluição fator x 104 (constante de diluição de câmara).<ol>
<li>Considere uma viabilidade de 100% se condições de viabilidade contagens e crescimento têm sido sistematicamente correlacionadas para o isolado a ser estudado. Nota: A padronização e controle de qualidade das contagens de viabilidade podem ser feitos por trás-chapeamento de acordo com o Comité Europeu de antifúngicos método de concentração inibitória mínima (CIM) teste de susceptibilidade aos antimicrobianos (EUCAST) para leveduras10 .</li>
		<li>Se a correlação de crescimento/viabilidade não foi estabelecida para o isolado sob estudo, medir a viabilidade de fungosa pela contagem de células vivas em um hemocytometer com o auxílio de corantes que cor seletivamente as células mortas, como Floxina B11, trypan azul 12, ou Janus verde13. Use as células fúngicas para este protocolo somente se a viabilidade da população neste momento é de 90% ou acima. Nota: Corantes mortos/vivos podem não funcionar bem com o fungo de escolha. Por favor, teste-os antes de usar. Por exemplo, o corante azul de Tripan não funciona bem com o c. neoformans.</li>
	</ol>
</li>
	<li>Depois disso, preparar as suspensões celulares em 2 X RPMI-1640 médias (2 X ajustado inóculo em meio RPMI-1640). Para placas de 96 poços, considere um volume de 5 mL por cada placa.<ol>
<li>Para todas as cepas de c. albicans , prepare uma suspensão de célula estoque de 4 x 103 células/mL. Esta concentração é 2x da concentração celular final em cada poço (2 x 103 células/mL).</li>
		<li>Para cepas de c. neoformans , prepare uma estoque cultura de células de 2 x 104 células/mL. Esta concentração é duas vezes a concentração celular final em cada poço (1 x 104 células/mL).</li>
		<li>Para outros fungos, teste com um antifúngico conhecido qual concentração será ideal para o estudo particular em relação ao tempo de incubação. Tempo de duplicação e metabolismo do fungo em consideração.</li>
	</ol>
</li>
</ol>3. peptídeos (agente desconhecido)
<ol><li>Armazenar os peptídeos liofilizados a-20 ° C e dissolvê-los em água desionizada antes de cada experimento. O tempo máximo de armazenamento, uma vez dissolvido em água vai depender da natureza de cada peptídeo.</li>
	<li>Prepare alíquotas de duas vezes a maior concentração final testados no ensaio (2 X). Idealmente, prepare um pequeno número de alíquotas com suficiente peptídeo para uma utilização evitar ciclos de congelamento e descongelamento. Nota: A escolha da concentração deve ser baseada na literatura e as propriedades do peptide. Recomenda-se iniciar as diluições de série com cerca de 100 µM do peptide e, em seguida, diminuir ou aumentar este intervalo de concentração dependendo dos resultados obtidos.</li>
</ol>4. referência antifúngicos (controles positivos)
<ol><li>Para aqueles diluído em água: preparar uma solução X 2 de maior concentração da análise (ver secção 5: ensaio antifúngicos para as etapas de diluição).<ol>
<li>Por cepas de c. albicans , prepare uma solução de 128 µ g/mL de fluconazol ou 128 µ g/mL de caspofungina. A concentração de placa para ambos será 64 µ g/mL.</li>
		<li>Por cepas de c. neoformans , prepare uma solução de 32 µ g/mL de anfotericina B (solução hidrossolúvel). A concentração de placa será 16 µ g/mL. Nota: Normalmente anfotericina B é diluído em dimetilsulfóxido (DMSO), desde que este antifúngicos é pouco solúvel em água. No entanto, existem preparações hidrossolúveis anfotericina B disponível comercialmente.</li>
	</ol>
</li>
	<li>Para antifúngicos diluídos em um solvente orgânico: preparar uma 100 X solução estoque em DMSO e, em seguida, diluir a 10 X na água para uso. Nota: Assim, no poço, a concentração final de DMSO não exceda 1%. Um poço onde o fungo irá crescer em meios contendo 1% DMSO como controle é necessário, dado que alguns fungos não toleram bem esta concentração do solvente. Lembre-se que o DMSO é fotossensível, então cubra o prato com papel alumínio ou coloque-o em uma câmara escura para a duração do período de incubação.</li>
</ol>5. antifúngico ensaio
Nota: Os ensaios In vitro antifungais são executados com base no teste de susceptibilidade de caldo como de orientações Clinical and Laboratory Standards Institute (CLSI) M27-A3 com algumas modificações.
<ol><li>Preparar uma diluição serial dupla de cada peptídeo e antifúngico em microplacas de poliestireno de 96 poços para um volume final de 50 µ l de controle.<ol>
<li>Usando uma pipeta Adicione 100 µ l do peptide/antifúngico na concentração X 2 da mais alta desejada concentração final nas colunas 1-3, em linha A.</li>
		<li>Usando uma pipeta multicanal, adicionar 50 µ l de água estéril em outros poços, nas linhas B-H.</li>
		<li>Remover 50 µ l de poços com a maior concentração (linha A), transferir para o próximo bem da próxima concentração (linha B) e homogeneizar.</li>
		<li>Repita os passos acima até o poço com a menor concentração e descartar 50 µ l desta bem (linha H). Deixe colunas 11 e 12 (linhas A, B, C) com água apenas para controle em branco ou controle negativo/crescimento.</li>
		<li>Adicionar 50 µ l do 2 X inóculo ajustado em meio RPMI-1640 a cada poço (colunas 1-3 plus coluna 11 (controle negativo/crescimento)); a concentração final para c. albicans será 2 x 103 células/mL e a concentração final para cepas de c. neoformans será 104 células/mL.</li>
		<li>Prepare-se controles em branco (50 µ l água + 50 µ l 2 X RPMI-1640 meio sem células) e controle negativo/crescimento (50 µ l água + 50 µ l ajustado inóculo 2 X RPMI-1640 médio sem antifúngico) e carga na placa bem como descrito acima.</li>
	</ol>
</li>
	<li>Repita o procedimento para cada composto a ser testado e o controle de antifúngico escolhido (colunas 4-10). Nota: As diluições de série podem ser feitas verticalmente como o experimento abaixo, ou horizontalmente na placa (ou seja, se o número de diluições de determinado composto/extracto que precisam ser testados é de oito ou mais).<ol>
<li>Se os compostos, referência drogas, ou qualquer dos seus componentes são fotossensíveis, realizar o ensaio em luz reduzida, cobrir as placas com papel alumínio ou coloque em uma câmara escura, durante a incubação do.</li>
	</ol>
</li>
	<li>Considere as seguintes recomendações opcionais.<ol>
<li>Selo da placa com uma placa de tampa transparente que permite a troca gasosa, como este ajuda a reduzir a evaporação.</li>
		<li>Coloque a placa em uma câmara úmida; Isso também ajudará a reduzir a evaporação.</li>
	</ol>
</li>
	<li>Incube as placas a 37 ° C por 24 h ou 48h para todas as cepas de c. albicans e por 48 h com 200 rpm agitação por c. neoformans. O volume final inferior (100 µ l) garante que não há repercussões ocorre. Nota: Nenhuma diferença significativa foi observada entre as leituras de MIC em 24 h e 48 h para cepas de c. albicans . No entanto, leituras em 48 h são mais fáceis de Visualizar.</li>
	<li>Observe todos os poços, com o auxílio de um microscópio óptico invertido, antes do período de incubação e depois verificar as alterações na morfologia, bem como para verificar se há sinais de contaminação. Nota: As leituras podem ser feitas visualmente e fotografadas no final do experimento. Antes de cada leitura, homogeneizar ligeiramente a placa. As leituras também podem ser realizadas medindo seu OD em 600 nm, se agrupa de célula ou filamentação não são observados.</li>
	<li>Realizar os experimentos que pelo menos três vezes em datas distintas.</li>
</ol>

<ol>
	<li>Armstrong-James, D., Meintjes, G., Brown, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neglected+epidemic:+fungal+infections+in+HIV/AIDS.">A neglected epidemic: fungal infections in HIV/AIDS.</a> Trends Microbiol. 22 (3), 120-127 (2014).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">Immunity to fungal infections.</a> Nat Rev Immunol. 11 (4), 275-288 (2011).</li><li>Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+drug+resistance:+mechanisms,+epidemiology,+and+consequences+for+treatment.">Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment.</a> Am J Med. 125 (1 Suppl), S3-S13 (2012).</li><li>Hancock, R. E., Diamond, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cationic+antimicrobial+peptides+in+innate+host+defences.">The role of cationic antimicrobial peptides in innate host defences.</a> Trends Microbiol. 8 (9), 402-410 (2000).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snake+cathelicidin+from+Bungarus+fasciatus+is+a+potent+peptide+antibiotics.">Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics.</a> PLoS One. 3 (9), e3217 (2008).</li><li>Du, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AaeAP1+and+AaeAP2:+novel+antimicrobial+peptides+from+the+venom+of+the+scorpion,+Androctonus+aeneas:+structural+characterisation,+molecular+cloning+of+biosynthetic+precursor-encoding+cDNAs+and+engineering+of+analogues+with+enhanced+antimicrobial+and+anticancer+activities.">AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.</a> Toxins (Basel). 7 (2), 219-237 (2015).</li><li>Benincasa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+activity+of+five+cathelicidin+peptides+against+clinically+isolated+yeasts.">Fungicidal activity of five cathelicidin peptides against clinically isolated yeasts.</a> J Antimicrob Chemother. 58 (5), 950-959 (2006).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reference Method for Broth Dilution Antifungal Susceptibiliy Testing of Yeasts; Approved Standard -Third Edition. CLSI document M27-A3. , (2008).</li><li>Guilhelmelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Scorpion+Venom-Derived+Antifungal+Peptides+against+Planktonic+Cells+of+Candida+spp.+and+Cryptococcus+neoformans+and+Candida+albicans+Biofilms.">Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.</a> Front Microbiol. 7, 1844 (2016).</li><li>Roongruangsree, U. T., Kjerulf-Jensen, C., Olson, L. W., Lange, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+Tests+for+Thick+Walled+Fungal+Spores+(ex:+Oospores+of+Peronospora+manshurica).">Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica).</a> Journal of Phytopathology. 123 (3), 244-252 (1988).</li><li>Boedijn, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+as+stain+for+fungi.">Trypan blue as stain for fungi.</a> Stain Technol. 31 (3), 115-116 (1956).</li><li>Goihman-Yahr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71 (2), 73-83 (1980).</li><li>Tati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histatin+5-spermidine+conjugates+have+enhanced+fungicidal+activity+and+efficacy+as+a+topical+therapeutic+for+oral+candidiasis.">Histatin 5-spermidine conjugates have enhanced fungicidal activity and efficacy as a topical therapeutic for oral candidiasis.</a> Antimicrob Agents Chemother. 58 (2), 756-766 (2014).</li><li>Petrou, M. A., Shanson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Cryptococcus+neoformans+by+the+NCCLS+microdilution+and+Etest+methods+using+five+defined+media.">Susceptibility of Cryptococcus neoformans by the NCCLS microdilution and Etest methods using five defined media.</a> J Antimicrob Chemother. 46 (5), 815-818 (2000).</li><li>Zaragoza, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+analysis+of+variables+for+standardization+of+antifungal+susceptibility+testing+of+nonfermentative+yeasts.">Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts.</a> Antimicrob Agents Chemother. 55 (4), 1563-1570 (2011).</li><li>Rodriguez-Tudela, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shaking+on+antifungal+susceptibility+testing+of+Cryptococcus+neoformans:+a+comparison+of+the+NCCLS+standard+M27A+medium,+buffered+yeast+nitrogen+base,+and+RPMI-2%+glucose.">Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose.</a> Antimicrob Agents Chemother. 44 (2), 400-404 (2000).</li><li>Beggs, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase+in+relation+to+ketoconazole+and+miconazole+susceptibilities+of+Candida+albicans.">Growth phase in relation to ketoconazole and miconazole susceptibilities of Candida albicans.</a> Antimicrob Agents Chemother. 25 (3), 316-318 (1984).</li><li>Alcouloumre, M. S., Ghannoum, M. A., Ibrahim, A. S., Selsted, M. E., Edwards, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+properties+of+defensin+NP-1+and+activity+against+Cryptococcus+neoformans+in+vitro.">Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.</a> Antimicrob Agents Chemother. 37 (12), 2628-2632 (1993).</li></ol>

Os autores não têm nada para divulgar.

Como testes podem analisar a potencial atividade antifúngica de um alvo composto usando pequenas quantidades do composto e ao mesmo tempo testá-lo em uma gama de concentrações. Nesse sentido, o presente protocolo é recomendado como um primeiro passo na triagem para potenciais novos compostos antifúngicos. O protocolo aqui apresentado é baseado no protocolo M27-A3, inicialmente projetado para auxiliar na seleção da terapia antifúngica em clínicas e pode ser adaptado a uma variedade de novos compostos antifúngi...

Infecções fúngicas tornaram-se uma preocupação médica importante nas últimas décadas, tendo aumentado consideravelmente principalmente devido a um aumento do número de indivíduos imunocomprometidos, tais como aqueles submetidos a tratamento de câncer e aqueles que vivem com HIV/AIDS ou transplante de órgãos,1,2. No entanto, um conjunto muito limitado de drogas antifúngicas disponíveis e o número crescente de relatórios sobre resistência fúngica-lhes contribuam para os grandes problemas em relação a terapêutica de micoses sistêmicas3.
Uma potencial fonte de novos compostos antifúngicos são peptídeos antimicrobianos (AMPs), pequenos peptídeos catiônicos produzidos por muitos organismos como parte de sua resposta imune inata para infecção4. No entanto, o método de triagem para testar estes compostos contra patógenos fúngicos não é padronizado. Diferentes procedimentos têm sido utilizados para avaliar a atividade antifúngica de AMPs, às vezes para o mesmo modelo microorganismo5,6,7. Essas diferenças e a falta de detalhes em alguns protocolos complicam comparações entre compostos e dificulta a reprodutibilidade.
Uma forma de padronizar os testes de novos candidatos a fármacos é seguir diretrizes usadas para definir a susceptibilidade em ambientes clínicos, tais como o clínico e orientações Laboratory Standards Institute (CLSI) M27-A3. No entanto, estes testes de sensibilidade antifúngica são demasiado restritivas e não leva em variação de consideração no metabolismo em toda a espécie, como eles foram criados apenas para alguns agentes selecionados. Por exemplo, eles não levam em conta as necessidades metabólicas de leveduras não-fermentação.
Este protocolo permite a avaliação da atividade dos compostos antifúngicos em potencial e é implementado aqui para a procura de peptídeos antifúngicos. Ele se baseia o teste de sensibilidade como caldo de orientações CLSI M27-A3 com modificações que otimizam a exibição de novos compostos8,9. Estas alterações permitem o uso de pequenas quantidades de composto, variações de temperatura ou inóculo inicial e de diferentes meios de comunicação para o crescimento ideal do pré-teste, enquanto padronizar os resultados com o uso de antifúngicos de referência como controles. Esse método, com o uso de placas de cultura multi bem, torna possível para um grande número de compostos de tela de forma rápida e confiável.
Devido à sua flexibilidade inerente, este protocolo pode ser usado com diferentes classes químicas de compostos e contra outros microorganismos, com algumas adaptações.

<table>
	<tbody>
		<tr>
			<td>Media and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td>
			<td>Thermo Fisher</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propane sulfonic acid (MOPS) (o que a gente usa tem um s&#243;dio, completa o nome dele please)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use to buffer 2X RPMI medium</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Din&#226;mica</td>
			<td>1528-1</td>
			<td>137 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>J.T.Baker</td>
			<td>3040-01</td>
			<td>2.7 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>V000129</td>
			<td>10 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>60230</td>
			<td>2 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud dextrose broth</td>
			<td>BD</td>
			<td>238230</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud Dextrose Agar</td>
			<td>BD</td>
			<td>210950</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>V000123</td>
			<td>35% for (solu&#231;&#227;o de estoque? Criopreserva&#231;&#227;o?)</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td>Para dilui&#231;&#227;o das drogas na dilui&#231;&#227;o seriada</td>
		</tr>
		<tr>
			<td>Antifungal drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluconazole</td>
			<td>Sigma-Aldrich</td>
			<td>F8929</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspofungin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1160</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dish petri</td>
			<td>J.Prolab</td>
			<td>0304-5</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Solution Reservoir</td>
			<td>KASVI</td>
			<td>K30-208</td>
			<td>Use to pippet the solutions using the multichannel pippet</td>
		</tr>
		<tr>
			<td>Equipment and other materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Nikon</td>
			<td>E200MV</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugue</td>
			<td>Thermo Fisher</td>
			<td>MegaFuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ethik Technology</td>
			<td>403-3D</td>
			<td>Set to 37&#176; C</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E25</td>
			<td>Set to 37&#176; C, 200 RPM</td>
		</tr>
		<tr>
			<td>Cell counting chamber, Neubauer</td>
			<td>BOECO Germany</td>
			<td>BOE 13</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>HTL</td>
			<td>5123</td>
			<td></td>
		</tr>
	</tbody>
</table>

broth microdilution in vitro screening: an easy and fast method to detect new antifungal compounds

Infecções fúngicas tornaram-se uma importante condição médica nas últimas décadas, mas o número de drogas antifúngicas disponíveis é limitado. Nesse cenário, a busca de novos medicamentos antifúngicos é necessária. O relatado aqui detalha um método péptidos de tela para suas propriedades antifúngicas. Ele se baseia o teste de sensibilidade como caldo de orientações Clinical and Laboratory Standards Institute (CLSI) M27-A3 com modificações de acordo com a pesquisa de peptídeos antimicrobianos como potenciais novos antifúngicos. Este protocolo descreve um ensaio funcional para avaliar a atividade dos compostos antifúngicos e pode ser facilmente modificado para se adequar a qualquer classe particular de moléculas sob investigação. Desde que os ensaios são realizados em placas de 96 poços, usando volumes pequenos, uma triagem em larga escala pode ser concluída em um curto espaço de tempo, especialmente se realizado em um ambiente de automação. Este procedimento ilustra como um protocolo clínico padronizado e ajustável pode ajudar a busca de trabalho-banco de novas moléculas para melhorar a terapia de doenças fúngicas.

O MIC é definido como a menor concentração de compostos antimicrobianos que inibe completamente o crescimento fúngico visível no final do período de incubação. Desde que o objectivo do presente protocolo é ter um método rápido de antifúngicos potencial de tela, qualquer bem com clara mídia similar aos poços em branco é considerado um resultado positivo, Considerando que qualquer bem com turbidez análoga dos poços de controle negativo/crescimento considerados negativos. No...

Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Um fácil e adaptável como caldo para a seleção de extratos e compostos antifúngicos.

Como caldo em Vitro rastreio: um método fácil e rápido para detectar novos compostos antifúngicos

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

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Research

JoVE Journal

Biology

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

18.7K visualizações.  University of Brasília. Um fácil e adaptável como caldo para a seleção de extratos e compostos antifúngicos.

Vídeo: Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

Usando uma pinça Laser Para Manipulação neurônios isolados In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Biologia

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Protein Assay Overlay membrana: um protocolo para testar Interação entre proteínas solúveis e insolúveis In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Fluxos de medição de cálcio rápido no Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

Construção de uma compilação de fácil Facility Zebrafish Acessível e

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Um cromossomo Dropping Ar seco rápido Método de Preparação Adequado para FISH em plantas

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Dirigido Método Evolução<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Biblioteca Criação e Triagem

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Um método fácil para a planta polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Um método de inoculação fácil e flexível para avaliar com precisão os fenótipos de infecção por oídio de Arabidopsis e outras plantas

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Como caldo em Vitro rastreio: um método fácil e rápido para detectar novos compostos antifúngicos

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