Somos gratos a Junko Wada por sua excelente assistência técnica. Somos gratos ao Prof Akio Nomoto por gentilmente fornecer um vetor de expressão de baculovírus para PVR-IgG2a. Este estudo foi apoiado em parte pela Grants-in-Aid para a Promoção da Erradicação da Pólio e Pesquisa Emergentes e Re-emergentes Doenças Infecciosas do Ministério da Saúde, Trabalho e Bem-Estar.

1. Preparação de meio de manutenção (MM) e de crescimento médio (GM) Para a preparação de MM e GM, consulte um manual de laboratório Poliomielite da OMS 1. Meio modificado Dulbecco Águia (DMEM) suplementado com 2% de soro fetal bovino (FCS) e 10% FCS podem ser utilizados como substitutos de MM e GM, respectivamente. 2. Preparação de anticorpos anti-PV <ol><li> Anticorpos anti-PV reconstituir (RIVM PV digitando anti-soro) por adição de água (0,5 mL por frasco) para cada frasco de anticorpo anti-PV (tipo 1, 2 ou 3). Adicionar este soluções de anticorpos reconstituída (0,5 mL cada) para 4,5 mL de MM em separado tubos rotulados. Nota: Alguns lotes de anti-PV anticorpos são fornecidos como forma reconstituída (0,5 mL por frasco). Para o lote de tais anticorpos anti-PV, além de água para a reconstituição não é necessário. </li><li> Mix volume igual de anticorpos anti-PV reconstituído. Para anti-PV1 +2, 2 +3, ou 3 +1: Misture 1,8 mL de cada anticorpo reconstituída (1,8 mL + 1,8 mL = 3,6 mL para cada mistura). Para anti-PV1 +2 +3: Misture 1,2 mL de cada anticorpo reconstituída (1,2 mL + 1,2 mL + 1,2 mL mL = 3,6). Nota: As amostras de PV poderia conter uma mistura de diferentes sorotipos do PV. Para identificar o sorotipo único do PV em amostras com mistura de PVs, piscinas de anticorpos anti-PV (isto é, 1 +2, 2 +3, 3 +1, 1 +2 +3) é necessário. </li><li> Adicione 1 / 5 do volume de FCS (0,72 mL) para cada anti-PV (1 +2, 2 +3, 3 +1, ou 1 +2 +3) de anticorpos (3,6 mL cada) (final FCS 18,2%). Nota: A adição de FCS é para a redução das não-específicas aglutinação de partículas de gelatina causada por anticorpos. </li><li> Manter o piscinas de anticorpos a -20 ° C até o uso no ensaio PA. </li></ol> 3. Reconstituição de partículas de gelatina sensibilizadas <ol><li> Adicione 1,5 mL de tampão de reconstituição incluído no kit para um frasco de partículas de gelatina sensibilizadas (fornecido como pó liofilizado). </li><li> Misture bem tocando suave </li><li> Mantenha a partícula reconstituído sensibilizados a 4 ° C. De partículas de gelatina reconstituída pode ser ativo, pelo menos, duas semanas mantido a 4 ° C, mas deve ser preparado antes do ensaio. </li></ol> Nota importante: Não misture muito diferentes de partículas de gelatina sensibilizadas, porque a imagem não de aglutinação de partículas de gelatina sensibilizadas poderia ser diferente pela grande quantidade de partículas. Para cada experimento, um único lote de partículas de gelatina sensibilizadas deve ser usado. 4. PA ensaio para identificação PV <ol><li> Prepare uma placa de micro titulação. Para a identificação de uma amostra de vírus, precisamos de 5 poços +, pelo menos, um controle negativo (partículas sensibilizadas sem vírus e de anticorpos, bem # 6 em abaixo. See No controle de vírus e anticorpos no Exemplo). Número total de poços é de 5 x + amostras controle de pelo menos um no ensaio. Nota importante: Por favor, tome pelo menos um controle Nenhum vírus e anticorpos no ensaio. Um frasco de partículas é de aproximadamente sensibilizados para a identificação de 10 amostras de vírus. </li><li> Adicione 12 mL / poço de anticorpo anti-PV ou GM para a placa micro-titulação. O arranjo do prato é a seguinte (Veja também Exemplo abaixo). Em amostras sem anticorpos (bem # 1 e 6), adicionar 12 mL da GM, em vez de anti-PV anticorpos. Nota importante: Se muitas amostras PV deve ser testado neste ensaio, pipeta multicanal ou dispensador seria útil para esta etapa. <table border="1"><tbody><tr><td> Bem # 1 </td><td> Bem # 2 </td><td> Bem # 3 </td><td> Bem # 4 </td><td> Bem # 5 </td><td> Bem # 6 </td></tr><tr><td> GM </td><td> anti-P1 +2 +3 </td><td> anti-P1 +2 </td><td> anti-P2 3 </td><td> ani P1-3 </td><td> GM </td></tr></tbody></table></li><li> Adicione 12 mL / poço de amostra de vírus. Em uma amostra sem vírus (bem # 6), adicionar 12 mL da GM, em vez de solução de vírus. Nota: a amostra do vírus é sobrenadante de cultura de células infectadas, células normalmente L20B ou células RD. Nota importante: Se muitas amostras PV deve ser testado neste ensaio, pipeta multicanal seria útil para esta etapa. <table border="1"><tbody><tr><td> Bem # 1 </td><td> Bem # 2 </td><td> Bem # 3 </td><td> Bem # 4 </td><td> Bem # 5 </td><td> Bem # 6 </td></tr><tr><td> amostra de vírus </td><td> amostra de vírus </td><td> amostra de vírus </td><td> amostra de vírus </td><td> amostra de vírus </td><td> GM </td></tr></tbody></table></li><li> Tome solução reconstituída de partículas sensibilizadas a partir de 4 ° C até a temperatura ambiente e bata suavemente completamente suspender as partículas. Nota importante: Após a suspensão completa das partículas agitando ligeiramente, as partículas devem ser adicionados imediatamente para as placas antes da precipitação de partículas suspensaste e formam solução não-homogênea. </li><li> Adicionar 25 mL / poço de partículas sensibilizadas para todos os poços. Nota importante: Após a adição de partículas sensibilizadas, a placa deve ser imediatamente submetido a placa de mistura para iniciar a reação uniforme das amostras. Portanto, a adição de partículas sensibilizadas deve ser realizada usando dispenser para minimizar um intervalo de tempo entre as amostras na placa. </li><li> Coloque a placa de micro-titulação em um misturador de prato e misture a placa por 30 segundos. </li><li> Coloque a placa de micro-titulação em papel branco (para facilidade de observação visual) em uma mesa à temperatura ambiente (15 ~ 30 ° C) por 2 h. Nota importante: Não mova a placa durante e após a reação para a observação dos resultados e tirar fotos. A aglutinação formado não é estável e facilmente perturbadas movendo as placas. </li><li> Tirar fotos do prato. Nota importante: Vista parcial das fotos não poderia dar dados suficientes para posterior exame e análise dos resultados para a interpretação. Várias fotos devem ser tomados para cobrir todos os poços das placas com foco na imagem de aglutinação dos poços. </li></ol> 5. Interpretação dos resultados <ol><li> Identificação de poços positivos e negativos. Compare a aglutinação nas amostras com o de Sem controle de vírus e de anticorpos (precipitação de partículas de gelatina, a aglutinação não) (Figura 1 e 2). Imagens de aglutinação que são claramente distinguidos das Nenhum vírus e sem controle de anticorpos (não-aglutinação, controle negativo) deve ser considerada positiva, e as imagens de aglutinação que são semelhantes ao controle negativo deve ser julgado como negativo. Exemplos que mostram aglutinação clara, mas apenas parcial pode ser considerada positiva, mas gravar como &quot;positivo (aglutinação parcial). </li><li> Identificação do PV. Interpretar os resultados com base em poços positivos e negativos com anti-PV anticorpos para identificar os sorotipos PV. Princípio da interpretação do resultado é semelhante ao do teste de neutralização em sistema de cultura de células (por exemplo, positivo para anticorpos anti-P1 2 indica que a amostra contém o tipo 3 PV). </li></ol> 6. Resultados representante Resultado representativo da PA ensaio PV identificação é mostrado na Figura 1. Todas as cepas PV formado aglutinação de partículas de gelatina sensibilizadas (Nenhum anticorpos amostras). Na presença de correspondentes anti-PV anticorpos, precipitação de partículas de gelatina (aglutinação não) semelhante ao Nenhum vírus e sem controle de anticorpos (controle negativo) foi observada (por exemplo, PV1 (Sabin), na presença de anti-P1 +2 ou anti-P3 uma anticorpos). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Figura 1" /> Figura 1. Identificação de PV por ensaio PA. Soluções Virus de PV1 (Sabin), PV2 (Sabin), e PV3 (Sabin) (títulos de vírus de 1,2 x 10 8 CCID 50 / 12 mL, 8,2 x 10 7 DICC mL 50/12, e 3,5 x 10 7 DICC 50 / 12 mL, respectivamente) foram utilizados para a identificação através do teste de PA. Nenhum vírus e sem controle de anticorpos mostraram precipitação de partículas de gelatina (não-aglutinação controle negativo). <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Figura 2" /> Figura 2. Interpretação dos poços de positivo e negativo. Soluções Virus de PV1 (Sabin), PV2 (Sabin), e PV3 (Sabin) (títulos de vírus de 2,4 x 8 CCID 10 L 50/25, 1,7 x 10 8 mL CCID 50 / 25, e 7,3 x 10 7 DICC mL 50/25, respectivamente) foram diluídos 1 / 20 a 1 / 320, e em seguida 25 mL de amostras diluídas foram examinados por ensaio PA sem anti-PV anticorpos.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

Souji Masujima é um funcionário da New Product Design Department, Fujirebio Inc.

O único ponto deste ensaio PA é a utilização da molécula de PVR em vez de anticorpos anti-PV para a detecção de PV 2. Isso permite uma interação específica das partículas sensibilizadas gelatina com PV com uma afinidade uniforme para todos os três sorotipos do PV 3-6. Nós usamos uma forma immunoadhesin de PVR (PVR-IgG2a) para a sensibilização das partículas de gelatina, porque as formas monoméricas de PVR têm apenas afinidades moderada para o único local de ligação do vírion (constante d...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Sensibilizadas-partículas de gelatina </td><td> Fujirebio Inc. </td><td></td><td> Kit de teste </td></tr><tr><td> Buffer de reconstituição </td><td> Fujirebio Inc. </td><td></td><td> Kit de teste </td></tr><tr><td> Placa de microtitulação </td><td> Fujirebio Inc. </td><td></td><td> Kit de teste </td></tr><tr><td> Anti-PV anticorpos </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

Do Global de Erradicação da Pólio, diagnóstico laboratorial desempenha um papel crítico por isolar e identificar PV a partir de amostras de fezes de paralisia flácida aguda (PFA) casos. Na Organização Mundial de Saúde (OMS) Global Polio Laboratory Network, PV isolamento e identificação estão sendo realizadas por meio do sistema de cultura celular e em tempo real de RT-PCR, respectivamente. Na era pós-erradicação do PV, procedimentos de identificação simples e rápido seria útil para rápida confirmação de casos de pólio nos laboratórios nacionais. No presente estudo, vamos mostrar o procedimento do romance de ensaio PA desenvolvidos para identificação PV. Este ensaio PA utiliza interação de PV receptor molécula (PVR) e virion que é afinidade específica e uniforme para todos os sorotipos do PV. O procedimento é simples (um procedimento passo em placas de reação) e rápido (resultados podem ser obtidos dentro de 2 h de reação), eo resultado é visualmente observados (observação de aglutinação de partículas de gelatina).

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

A recentemente desenvolvida romance de partículas de aglutinação do ensaio (PA) utilizando molécula receptora vírus permitiu uma identificação rápida e fácil de poliovírus (PV). Neste artigo, vamos mostrar o procedimento para o ensaio PA para identificação PV.

Método de aglutinação de partículas de Identificação Poliovirus

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Research

JoVE Journal

Immunology and Infection

15.2K visualizações.  National Institute of Infectious Diseases. A recentemente desenvolvida romance de partículas de aglutinação do ensaio (PA) utilizando molécula receptora vírus permitiu uma identificação rápida e fácil de poliovírus (PV). Neste artigo, vamos mostrar o procedimento para o ensaio PA para identificação PV.

Vídeo: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Um Procedimento de 1,5 horas para a Identificação de Enterococcus Espécies Diretamente de hemoculturas

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Imunologia e Infecção

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Identificação baseada em peptídeo de motivos funcionais e seus parceiros de ligação

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

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 ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS Mediada frente Triagem Genética para identificação de genes envolvidos na resistência não hospedeiras


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Rápida identificação de bactérias gram-negativas de Sangue Cultura Caldo Usando MALDI-TOF Espectrometria de Massa

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Calculando a média de Viral glicoproteína do envelope Spikes de Electron Cryotomography reconstruções com Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular sorotipagem de<em&gt; Streptococcus pneumoniae</em&gt; Por aglutinação em látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Citometria de fluxo de partículas ligadas aos Bet v 1 alérgeno no PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...