Este projecto foi apoiado pela concessão 1R03MH08127-01 e 7R03MH08127-02 do NIH para Q. Li, e pelos fundos IMPACTO do Departamento de Medicina da UAB para Q. Li. O apoio do Fundo Molette e da Universidade de Auburn é apreciado. Agradecemos também as assistências técnicas de Ms. Pulin Che eo Sr. Volodymyr Musiienko durante o curso do trabalho.

1. Células, vírus e a compostos antivirais <ol><li> Manter células BSR, de um derivado de rim de hamster bebé (BHK), células 10, em meio de Eagle modificado por Dulbecco (DMEM) contendo 5% de soro fetal de vitelo (FCS), 100U/ml penicilina e 100 ug / ml de estreptomicina. </li><li> Para todos os três ensaios, células da placa em DMEM com 1% de FCS, 100U/ml penicilina e 100 ug / ml de estreptomicina, como anteriormente 5 optimizado. Este meio é referido como meio de ensaio para os três ensaios. </li><li> Incubar todas as células na incubadora a 37 ° C, com 5% de CO 2 e 80-95% de humidade. </li><li> Placa-purificar e propagar o tipo 10 BTV (BTV-10), tal como descrito anteriormente 8. Diluir BTV-10 em meio de ensaio para cada ensaios designados. </li><li> Dissolve-se todos os compostos de ensaio em DMSO para formar uma unidade com a concentração de 10 mM. Guarde os estoques a -20 ° C. </li><li> Dilui-se os compostos a concentrações desejadas, utilizando meio de ensaio para o designadod ensaios 2. </li></ol> 2. Ensaio baseado no CPE utilizando o kit CTG <ol><li> Células BSR semente em um de 384 poços de microplacas (preto, formato de 16 x 24), através de MicroFlo selecione distribuidor. A densidade de sementeira é de 5.000 células / poço, e o volume de sementeira é de 20 uL para análise de eficácia antiviral. </li><li> Incubar as células por 2-3 horas até obter células aderente à placa completamente. </li><li> Adicionar composto antiviral com uma concentração final de 10 uM a cada poço. Misturá-lo completamente. </li><li> Diluir a BTV título desejado e adicionar 5 mL de BTV com denotado MOI para cada poço. Para o controle bem, adicione 5 mL de meio de ensaio. Incubar as células infectadas durante 72 horas a 37 ° C, com 5% de CO 2 e 80-95% de humidade. </li><li> Aos 72 hpi, degelo e equilibrar tampão CTG CTG e o substrato liofilizado até à temperatura ambiente antes de usar. Reconstituir a solução homogénea reagente CTG misturando o liofilizado enzima / substrato e o reagente de tampão de acordo com a poinstruções e do fabricante. </li><li> Equilibrar as placas de ensaio a temperatura ambiente durante 15 min. </li><li> Adicionar um volume igual (25 ul) de reagentes de CTG para cada poço de um distribuidor. Após incubação durante 15 minutos à temperatura ambiente na escuridão, medir os sinais de luminescência utilizando um leitor multi-modo, com um tempo de integração de 0,1 seg. </li></ol> 3. Ensaio de dose-resposta <ol><li> As células BSR semente em uma de 384 poços de microplacas (preto; formato de 16 x 24), através de um dispensador, com uma densidade de sementeira de 5000 células / cavidade em um volume de 20 ul de semeadura para ensaio de eficácia antiviral e 25 uL para o ensaio de citotoxicidade, respectivamente . </li><li> Incubar as células durante 2-3 horas a 37 ° C, com 5% de CO 2 e 80-95% de humidade até que as células estão bem ligados à placa. </li><li> Processo de ensaio de um quarto da área da placa de 384 poços, para cada composto (equivalente a uma placa de 96 poços), como mostrado na Tabela 1. Alocar oito repetições para cada concentraçãonum único ensaio. Atribuir a primeira coluna como o controlo positivo sem adição de composto e vírus, e a última coluna (12 ª) como controlo negativo, adicionando apenas o vírus sem compostos. </li><li> Dilui-se os compostos para 50 uM em meio de ensaio e adicionado à coluna 2 nd a 20 ul / poço para o ensaio de eficácia antiviral. Misturar o composto cinco vezes usando uma pipeta de 8 canais semi-automático para uma concentração de 25 uM. </li><li> Usando uma pipeta semi-automática de 8 canais, transferir 20 ul de mistura em 2 ª coluna para a coluna seguinte (3 º), misturar bem para formar uma concentração de 12,5 | iM. Repetir este processo por meio da transferência de 20 uL de mistura de 3 a coluna para a coluna seguinte (4 mil) para formar uma outra diluição de duas vezes com uma concentração de 6,25 uM. Repita esta diluição em série de duas vezes até a 11 ª coluna. Aspirar e descartar 20 ul da mistura em 11 ª coluna após a adição e a mistura da amostraound. </li><li> Adicionar BTV-10, com base na MOI de 0,01, para cada poço da coluna 2 para a coluna 11 com um volume de 5 ul / poço. Após a adição do vírus, a concentração de composto final em cada coluna devem ser: 20 uM em coluna de 2, 10 ^ M na coluna 3, e continuou com uma diluição de duas vezes para baixo para a coluna 11 com uma concentração final de 0,04 uM. </li><li> Adicionar 5 mL de meio para a coluna como uma célula única de controlo (positivo) e 5 ul / poço de VFC à coluna 12, tal como infecção por vírus da febre catarral apenas controle (negativo). </li><li> Dilui-se os compostos para uma concentração inicial de 200 pM para o ensaio de citotoxicidade. Da mesma forma, adicionar 25 ul / poço de composto para a coluna 2 e 5x misturado com pipeta semi-automática de 8 canais para uma concentração de 100 uM. Não adicione BTV. </li><li> Realizar a diluição série de duas vezes por aspiração 25 mL da coluna 2 para a coluna vizinha 3, e continuou até a última coluna (12 º). Na coluna 12, depois de mistura, aspirado e descartar 25 ul de mixture. A concentração final na coluna 2 deve ser de 100 ^ M e a 12 ª coluna deve estar a 0,2 uM. A coluna 1 é o único controlo celular. </li><li> Para ambos os ensaios de citotoxicidade e eficácia antiviral, incubar as placas a 37 ° C, com 5% de CO 2 e 80-95% de humidade durante 72 horas pós-tratamento. Medir a viabilidade celular utilizando o kit CTG como descrito acima (protocolo passos 2,5-2,7). </li></ol> 4. Time-of-Adição (TOA) Assay <ol><li> Células BSR Semente da coluna 1-24 em 384 poços de microplacas (preto, formato de 16 x 24), através de um distribuidor em 5.000 células / poço e que o volume de semeadura é de 15 mL / poço. </li><li> Para cada composto, utilizam todos os vinte e quatro colunas com oito réplicas para cada ponto de tempo no meio da placa de 384 poços (Tabela 2). Atribuir coluna 1 como células só controle adicionando 10 ul / poço de meio de ensaio. Mark coluna 24, tal como infecção por vírus da febre catarral apenas controle (controle negativo) pela adição de 5 ul / poço de ensaio meio de umd 5 ul / poço de vírus a uma MOI de 0,01 e um volume final de 25 ul / poço. </li><li> Selecione as colunas de números pares da coluna 2-22 como antiviral coluna avaliação da eficácia em diferentes horas após a infecção (hpi). Nessas colunas, infectar as células com 5 mL / poço BTV no MOI de 0,01. No hpi diferente, também adicionar 5 uL / ​​poço composto diluído a cada poço para formar um volume final de 25 ul / poço. Para o denotado -2 e -1 hpi adicionar composto de células BSR antes da infecção BTV. Para 0 hpi, adicione o composto e BTV para a cultura ao mesmo tempo. </li><li> Em paralelo, designam as colunas com números ímpares de coluna de 3-23 como composto apenas controla, de que o composto foi adicionado em diferentes pontos de tempo como designado (-2, -1, 0, 1, 2, 4, 8, 16, 24, 48 hpi). Nestas colunas, adicionar 5 mL de meio / poço de ensaio e 5 pL / cavidade diluída composto para proporcionar um volume final de 25 ul / poço. </li><li> Após o tratamento, as células incubar a 37 ° C, 5% de CO 2, com 80-95% de humidade. </li><li> Determinar a viabilidade celular às 72 hpi usando o kit de CellTiter-Glo, tal como descrito anteriormente (protocolo de passos de 2,5-2,7). </li></ol> 5. Análise de Dados <ol><li> Processar todos os dados utilizando software apropriado primeira in-house com base nos sinais luminescentes obtidos através do leitor multi-modo. Determinar o valor médio (média), o desvio padrão (STDEV) de cada tratamento, bem como as variações de coeficientes (CV), que requer um valor não superior a 10%. </li><li> Transfira os dados processados ​​a partir do software acima para uma ferramenta de software bioestático e gráfico. Levar a cabo a análise de regressão não-linear para determinar os valores de EC 50 e CC 50. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Para a identificação inicial de visitas antivirais, um dos passos chave para a descoberta da droga anti-viral e de desenvolvimento consiste em desenvolver ensaios robustos, que inclui a selecção de um marcador quantificável, o desenvolvimento de um protocolo simples, obter sinais suficientes e menos do que 10% CV. A maioria das telas bioquímicos ou à base de células são projetados para fornecer um ponto de partida química baseada na mais robusta, ensaio simples e barato, devido à reprodutibilidade requerida n...

BTV é um protótipo de cadeia dupla de RNA vírus do gênero Orbivirus, família Reoviridae. BTV é uma das doenças mais importantes da pecuária nacional, incluindo ovelhas, cabras, gado e outros animais domésticos, com a perda de 3.000 milhões dólares americanos / ano em todo o mundo 1,2. O exótico sorotipo BTV é um importante patógeno animal enumerados nas &quot;USDA alta Consequence Pecuária Patógenos&quot;. Recentemente, o re-emergente de BTV causou um grande surto da doença em bovinos e ovinos em vários países em todo o norte da Europa 3,4. Como resultado do seu significado económico e como um sistema modelo, BTV tem sido o objecto de estudos moleculares, genéticos e estruturais extensas, e várias vacinas têm sido desenvolvidos. No entanto, devido à falta de ensaios adequados para a descoberta da droga anti-viral, não existem medicamentos antivirais disponíveis contra BTV. Em recente pesquisa de alto rendimento campanha (HTS), utilizando o sistema BTV como modelo, nós developed, otimizado e validado um ensaio baseado em CPE para identificar potenciais antivirais de amplo espectro contra arbovírus 5. Ensaio baseado CPE é um ensaio bem conhecido que tem sido utilizado na descoberta de drogas anti-viral contra um número de vírus que induzida rápida e observável CPE / apoptose 5-7. No nosso sistema, infecção pós BTV, CPE é evidente em células de vertebrados, incluindo HeLa, BSR, e HEK 293T 8. CPE induzida por vírus da febre catarral pode ser monitorizada e quantificado usando vários métodos de detecção de viabilidade celular, incluindo o kit de reagente de viabilidade celular CellTiter Glo (kit CTG) 9. Este kit determina o número de células viáveis ​​em cultura com base na quantificação de ATP celular apresentado, o que sinaliza a presença de células vivas, metabolicamente activas. Sob condições otimizadas, o ensaio baseado em CPE aqui apresentado mostrou sua viabilidade com o protocolo de um passo &quot;misturar e medir&quot;, e flexibilidade com sinais luminescentes estáveis. Enquanto isso, os compostos tóxicos reducing viabilidade celular irá ser excluída no presente ensaio baseia-CPE. O ensaio baseado CPE mostrou a sua robustez e fiabilidade para a descoberta da droga anti-viral contra o vírus da febre catarral, e tem sido utilizada para rastrear o NIH Molecular Bibliotecas de Moléculas Pequenas Repository (MLSMR), que conduz à identificação de seis novos aglomerado de potencial composto principal antiviral (s ) 5. Quando um composto antiviral potencial foi identificado usando o ensaio baseado em CPE, que terá de ser submetida ao ensaio de dose-resposta de dez concentração para determinar o intervalo de eficácia antiviral e citotoxicidade 2. A eficácia antiviral, representada como a concentração inibidora 50% (IC50) ou a concentração eficaz 50% (CE 50), é a concentração de uma droga que inibe a CPE induzida por vírus a meio caminho entre a linha de base e no máximo. A citotoxicidade dos antivirais, ou seja, a concentração de 50% de citotoxicidade (CC 50), é a concentraçãode um fármaco induzir 50% de citotoxicidade entre a linha de base e no máximo. O índice selectivo (SI), denotada como 50% de Si (SI 50) é calculado a partir de CC50 / IC 50, que determina a especificidade do anti-viral contra a CPE induzida por vírus. A IC50 (ou EC50), CC 50 e SI 50 valores são medidas importantes para determinar se um composto antiviral é potente e selectivo para o desenvolvimento de drogas. Quando um antiviral mostrou nenhuma toxicidade manifesta in vitro, no entanto, impedido de vírus induzido CPE e o ciclo de vida viral produtiva, é importante caracterizar a sua MOA 2. Iniciamos tal caracterização através da realização de ensaio ToA para determinar a possível etapa (s) do ciclo de vida viral que é afetada pelo antiviral. Geralmente, composto antiviral foram adicionados às células em diferentes épocas pré-ou pós-infecção de vírus. Se os antivirais foram adicionados às células infectadas pôr ao seu alvo step durante o curso da infecção, ela iria resultar em menor actividade, quando comparada com a que foi adicionada antes do passo. Assim, o estudo ToA é crítico para a determinação da eficácia anti-viral de um composto, e o seu alvo potencial, quer no ciclo de vida viral ou a máquina hospedeira envolvidas no ciclo de vida viral. Para todos os três ensaios, a viabilidade celular foi determinada utilizando o kit CTG seguindo as instruções do fabricante 5. Este sistema de detecção emite sinais de luminescência adequadas que poderiam ser analisadas por diversos softwares em casa. Cada ensaio foi validado e executada, pelo menos, em triplicado, com oito réplicas. Para todos os dados obtidos, três parâmetros, incluindo o valor médio (AVE), o desvio padrão (STDEV), e variação de co-eficiente (CV) foram analisados ​​para determinar a robustez do ensaio. Uma vez que a robustez do ensaio foi determinado, os dados irão ser ainda analisada e representada usando vários biostatics e graficaferramentas c 2.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>1134218</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>1000185</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gbico</td>
			<td>1049769</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>CellTiter-Glo (CTG) kit</td>
			<td>Promega</td>
			<td>TB288</td>
			<td>For cell viability measurement</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>S25309B</td>
			<td>Diluted from 95%</td>
		</tr>
		<tr>
			<td>Antiviral huashilcompounds</td>
			<td>NIH MLSMR and de novo synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTV-10</td>
			<td>ATCC</td>
			<td>VR-187</td>
			<td></td>
		</tr>
		<tr>
			<td>BSR cell</td>
			<td>Developed in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy-II multi-mode microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td>For luminescent signal reading</td>
		</tr>
		<tr>
			<td>MicroFlo select dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td>Adding cells, virus, and reagents</td>
		</tr>
		<tr>
			<td>384-well flat-bottom microplate</td>
			<td>CORNING</td>
			<td>28908031</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gen. 5 software</td>
			<td>BioTek</td>
			<td></td>
			<td>For analysis of reading outputs from Synergy-II multi-mode microplate reader</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>Version 5</td>
			<td>For biostatic analysis and plot</td>
		</tr>
	</tbody>
</table>

assays for the identification of novel antivirals against bluetongue virus

Para identificar potenciais antivirais contra BTV, temos desenvolvido, otimizado e validado três ensaios aqui apresentados. O ensaio baseado CPE foi o primeiro ensaio foi desenvolvido para avaliar se um composto mostrou qualquer eficácia antiviral e foram utilizados para rastrear o composto biblioteca grande. Enquanto isso, a citotoxicidade dos antivirais podem também ser avaliadas utilizando o ensaio baseado em CPE. O ensaio dose-resposta foi concebido para determinar a gama de eficácia para o antiviral seleccionado, isto é, 50% da concentração inibitória (IC50), ou a concentração eficaz (EC 50), bem como a sua gama de citotoxicidade (CC 50). O ensaio ToA foi utilizado para o estudo inicial Moa para determinar o mecanismo subjacente dos novos antivirais durante BTV ciclo de vida viral ou o possível efeito sobre acolhimento maquinaria celular. Estes ensaios são essenciais para a avaliação da eficácia antiviral no sistema de cultura de células, e têm sido usados ​​para nossas pesquisas recentes levamção para a identificação de uma série de novos medicamentos antivirais contra BTV.

1. Eficácia antiviral de composto O ensaio de ECP de base celular foi desenvolvido, optimizado e validado in vitro utilizando o kit à base de CTG luminescente para identificar novos medicamentos antivirais contra BTV como descrito anteriormente de 2,5. O ensaio de resposta de dez dose foi empregada para reflectir a eficácia antiviral e citotoxicidade de um composto de chumbo identificados medindo o número de células metabolicamente viáveis ​​em cultura com base na ...

Watch this Scientific Journal Video about Assays for the Identification of Novel Antivirals against Bluetongue Virus at JoVE.com

Assays for the Identification of Novel Antivirals against Bluetongue Virus

Três ensaios, incluindo o efeito citopático (CPE) baseado em ensaio, o ensaio de dose-resposta e (TOA) ensaio de Tempo-de-adição ter sido desenvolvido, optimizado e validado e utilizado para identificar novos medicamentos antivirais contra o vírus da febre catarral (VFC), bem como para determinar o possível mecanismo de ação-de-(MOA) para antivirais recentemente identificados.

Ensaios para a identificação de novos antivirais contra a febre catarral ovina vírus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

assays-for-identification-novel-antivirals-against-bluetongue

Research

JoVE Journal

Immunology and Infection

13.9K visualizações.  University of Alabama at Birmingham.  Três ensaios, incluindo o efeito citopático (CPE) baseado em ensaio, o ensaio de dose-resposta e (TOA) ensaio de Tempo-de-adição ter sido desenvolvido, optimizado e validado e utilizado para identificar novos medicamentos antivirais contra o vírus da febre catarral (VFC), bem como para determinar o possível mecanismo de ação-de-(MOA) para antivirais recentemente identificados. 

Vídeo: Assays for the Identification of Novel Antivirals against Bluetongue Virus

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Concentração Determinação Viral Através de placa Ensaios: Usando tradicionais e novos Sistemas de sobreposição

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Imunologia e Infecção

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Imunofluorescência para monitorar a Incorporação celular de lactoferrina humana e sua Associated atividade antiviral contra o vírus da hepatite C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

No início virais Ensaios de entrada para a identificação e avaliação de compostos antivirais

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Qualitativos e quantitativos Os ensaios para a detecção e caracterização de antimicrobianos Proteína

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Desenvolvimento e Identificação de uma subpopulação Novel dos fagócitos Human Giant neutrófilos derivado<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Ensaios de caracterização de romance endoteliais reguladores envolvidos na resposta inflamatória de triagem

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extração rápida, segura e simples Manual cabeceira de ácidos nucleicos para a detecção de vírus em amostras de sangue total

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...