Estamos muy agradecidos a Junko Wada por su excelente asistencia técnica. Estamos agradecidos con el profesor Akio Nomoto de la amabilidad de ofrecer un vector de expresión de baculovirus para el PVR-IgG2a. Este estudio fue apoyado en parte por subvenciones-en-Ayudas a la Promoción de la Erradicación de la Poliomielitis y la Investigación Emergentes y Re-emergentes enfermedades infecciosas del Ministerio de Salud, Trabajo y Bienestar Social.

1. Preparación de medio de mantenimiento (MM) y el crecimiento medio (GM) Para la preparación de MM y GM, consulte un manual de laboratorio Polio de la OMS 1. Dulbecco modificado de Eagle (DMEM) suplementado con 2% de suero fetal bovino (FCS) y el 10% de FCS podrían ser utilizados como sustitutos de la MM y GM, respectivamente. 2. Preparación de anticuerpos anti-PV <ol><li> Reconstituir anti-PV anticuerpos (RIVM PV escribiendo antisuero) mediante la adición de agua (0,5 ml por vial) a cada vial de anticuerpos anti-PV (tipo 1, 2, ó 3). Añadir este anticuerpo soluciones reconstituida (0,5 ml cada una) a 4,5 ml de MM por separado en tubos etiquetados. Nota: Algunos lotes de anticuerpos anti-PV se proporcionan en forma reconstituida (0,5 ml por vial). Para muchos de estos anticuerpos anti-PV, la adición de agua para la reconstitución no es necesario. </li><li> Mezcla de igual volumen de reconstitución anti-PV anticuerpos. Para anti-PV1 2, 2 3, o un 3: Mezclar 1,8 ml de cada anticuerpo reconstituida (1,8 ml + 1,8 ml = 3,6 ml de cada mezcla). Para anti-PV1 +2 +3: Mezclar 1,2 ml de cada anticuerpo reconstituida (1,2 ml + 1,2 ml + 1,2 ml = 3,6 ml). Nota: las muestras PV podría contener una mezcla de diferentes serotipos de la energía fotovoltaica. Para identificar el serotipo único de la energía fotovoltaica en las muestras con mezcla de valores actuales, las mezclas de anticuerpos anti-PV (es decir, 1 +2, 2 +3, 3 +1, 1 +2 +3) es necesario. </li><li> Añadir un volumen 1 / 5 de FCS (0,72 mL) a cada anti-PV (1 +2, 2 +3, 3 +1, o 1 +2 +3) de anticuerpos (3,6 ml cada una) (final de 18,2% FCS). Nota: La adición de FCS es para la reducción de la no-específicas de aglutinación de partículas de gelatina causada por anticuerpos. </li><li> Mantenga las piscinas de anticuerpos a -20 ° C hasta su uso en el ensayo de PA. </li></ol> 3. La reconstitución de las partículas de gelatina sensibilizada <ol><li> Añadir 1,5 ml de solución tampón de reconstitución incluido en el kit a un vial de partículas de gelatina sensibilizada (siempre como polvo liofilizado). </li><li> Mezclar bien con unos golpecitos suaves </li><li> Mantener la partícula reconstituido sensibilizados a 4 ° C. De partículas de gelatina reconstituido puede estar activo al menos 2 semanas se mantiene a 4 ° C, pero debe estar preparado justo antes de la prueba. </li></ol> Nota importante: No mezcle muy diferente de las partículas de gelatina sensibilizada, porque la imagen no aglutinación de partículas de gelatina sensibilizada puede ser diferente por la gran cantidad de las partículas. Para cada experimento, un solo lote de partículas de gelatina sensibilizada debe ser utilizado. 4. PA ensayo para la identificación de PV <ol><li> Prepare una placa de micro-titulación. Para la identificación de la muestra de un virus, es necesario + 5 pozos por lo menos un control negativo (partículas sensibilizadas, sin virus y anticuerpos, así # 6 en adelante. Véase el control de virus y anticuerpos en el ejemplo). El número total de pozos es de 5 x + muestras al menos un control en el ensayo. Nota importante: Por favor, tome por lo menos un no control de virus y anticuerpos en el ensayo. Un vial de partículas sensibilizadas es aproximadamente para la identificación de 10 muestras de virus. </li><li> Añadir 12 l / pocillo de anticuerpos anti-PV o GM a la placa de micro-titulación. La disposición de la placa es el siguiente (ver también el ejemplo más abajo). En las muestras sin anticuerpos (también # 1 y 6), añadir 12 l de GM en lugar de anticuerpos anti-PV. Nota importante: Si muchas muestras de PV debe ser probado en este ensayo, pipetas multicanal o dispensador sería de gran ayuda para este paso. <table border="1"><tbody><tr><td> Bueno # 1 </td><td> Bueno n º 2 </td><td> Bueno # 3 </td><td> Bueno # 4 </td><td> Bien # 5 </td><td> Así # 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> Añadir 12 l / pocillo de muestra del virus. En una muestra sin virus (y # 6), añadir 12 l de GM en vez de la solución de virus. Nota: La muestra del virus es el sobrenadante del cultivo de las células infectadas, las células suelen L20B o células RD. Nota importante: Si muchas muestras de PV debe ser probado en este ensayo, pipetas multicanal sería de gran ayuda para este paso. <table border="1"><tbody><tr><td> Bueno # 1 </td><td> Bueno n º 2 </td><td> Bueno # 3 </td><td> Bueno # 4 </td><td> Bien # 5 </td><td> Así # 6 </td></tr><tr><td> de muestras de virus </td><td> de muestras de virus </td><td> de muestras de virus </td><td> de muestras de virus </td><td> de muestras de virus </td><td> GM </td></tr></tbody></table></li><li> Tome la solución reconstituida de partículas sensibilizadas a 4 ° C a la temperatura ambiente, y golpee suavemente para suspender por completo las partículas. Nota importante: Después de la suspensión completa de las partículas con unos golpecitos suaves, las partículas se debe agregar de inmediato a las placas antes de las partículas en suspensión precipitaciónte y la forma no homogénea solución. </li><li> Añadir 25 l / pocillo de partículas sensibles a todos los pozos. Nota importante: Después de la adición de partículas sensibilizadas, la placa debe ser inmediatamente sometidos a la placa de mezcla para empezar la reacción uniforme de las muestras. Por lo tanto, la adición de partículas sensibilizadas se debe realizar mediante el uso de dispensadores de reducir al mínimo un lapso de tiempo entre las muestras de la placa. </li><li> Coloque la placa de micro-titulación en un mezclador de la placa y la mezcla de la placa durante 30 segundos. </li><li> Coloque la placa de micro-titulación en papel blanco (para mayor comodidad de la observación visual) en una mesa a temperatura ambiente (15 ~ 30 ° C) durante 2 h. Nota importante: No mueva el plato durante y después de la reacción para la observación de los resultados y tomar fotos. La aglutinación formado no es estable y fácilmente perturbados por movimiento de las placas. </li><li> Tomar fotos de la placa. Nota importante: Vista parcial de las fotos no podía dar datos suficientes para su posterior examen y revisión de los resultados para su interpretación. Varias fotos deben ser tomadas para cubrir todos los pocillos de las placas se centra en la imagen de la aglutinación de los pozos. </li></ol> 5. Interpretación de los resultados <ol><li> Identificación de pozos positivos y negativos. Comparar la prueba de aglutinación en las muestras con la de No hay control de virus y de anticuerpos (la precipitación de partículas de gelatina, no aglutinación) (Figura 1 y 2). Imágenes de aglutinación que se distingan claramente de ningún virus y sin control de anticuerpos (no de aglutinación, el control negativo) deben ser juzgadas como positivas, y las imágenes de aglutinación que son similares a los del control negativo debe ser juzgada como negativa. Ejemplos que muestran una clara aglutinación parcial, pero sólo podía ser juzgado como positivo, pero el registro como «positiva (aglutinación parcial). </li><li> Identificación de la energía fotovoltaica. Interpretar los resultados sobre la base de los pozos de positivo y negativo con anticuerpos anti-PV anticuerpos para identificar los serotipos PV. Principio de la interpretación del resultado es similar a la de la prueba de neutralización en el sistema de cultivo celular (por ejemplo positivo para anti-P1 +2 anticuerpos indica que la muestra contiene el tipo 3 PV). </li></ol> 6. Resultados representante Resultado representativo de la PA ensayo de identificación PV se muestra en la Figura 1. Todas las cepas PV formado aglutinación de partículas de gelatina sensibilizada (No existen anticuerpos de muestras). En presencia de los correspondientes anticuerpos anti-PV, la precipitación de partículas de gelatina (no aglutinación), similar a ningún virus y sin control de anticuerpos (control negativo) fue observada (por ejemplo, PV1 (Sabin) en la presencia de anticuerpos anti-P1 +2 o anti-P3 1 anticuerpos). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Figura 1" /> Figura 1. Identificación de la energía fotovoltaica mediante el ensayo de PA. Soluciones Virus de PV1 (Sabin), PV2 (Sabin) y PV3 (Sabin) (títulos de virus de 1,2 x 10 8 CCID 50 / l 12, 8,2 x 10 7 CCID 50 / 12 l, y 3.5 x 10 7 CCID 50 / 12 l, respectivamente) fueron utilizados para la identificación mediante el ensayo de PA. No hay virus y sin control de anticuerpos mostraron la precipitación de partículas de gelatina (no de aglutinación, el control negativo). <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Figura 2" /> Figura 2. Interpretación de los pozos de positivo y negativo. Soluciones Virus de PV1 (Sabin), PV2 (Sabin) y PV3 (Sabin) (títulos de virus de 2,4 x 10 8 CCID 50 / 25 l, 1,7 x 10 8 l CCID 50 / 25, y 7.3 x 10 7 CCID 50 / 25 l, respectivamente) se diluye 1 / 20 a 1 / 320, y luego 25 l de las muestras diluidas fueron examinados por el ensayo de PA sin anti-PV anticuerpos.

<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 es un empleado del Departamento de Diseño de Nuevos Productos, Fujirebio Inc.

El único punto de este ensayo PA es la utilización de la molécula de PVR en lugar de anticuerpos anti-PV para la detección de FV 2. Esto permite una interacción específica de las partículas sensibilizadas con gelatina de PV con una afinidad uniforme para los tres serotipos de la energía fotovoltaica 3-6. Se utilizó un formulario de inmunoadhesina de PVR (PVR-IgG2a) para la sensibilización de las partículas de gelatina, ya que las formas monoméricas de PVR sólo tienen afinidades moderada al sitio ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Sensibilizados partículas de gelatina </td><td> Fujirebio Inc. </td><td></td><td> Kit de prueba </td></tr><tr><td> La reconstitución de amortiguación </td><td> Fujirebio Inc. </td><td></td><td> Kit de prueba </td></tr><tr><td> Microplacas </td><td> Fujirebio Inc. </td><td></td><td> Kit de prueba </td></tr><tr><td> Anti-PV anticuerpos </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

En la Iniciativa de Erradicación Mundial, el diagnóstico de laboratorio desempeña un papel fundamental mediante el aislamiento y la identificación de PV de las muestras de heces de parálisis flácida aguda (PFA). En la Organización Mundial de la Salud (OMS), la Red de Laboratorios Mundial de la Poliomielitis, el aislamiento y la identificación PV están llevando a cabo mediante el sistema de cultivo celular y en tiempo real RT-PCR, respectivamente. En la era posterior a la erradicación de la energía fotovoltaica, los procedimientos de identificación simple y rápida sería de gran ayuda para la rápida confirmación de casos de polio en los laboratorios nacionales. En el presente estudio, vamos a mostrar el procedimiento de ensayo de la novela de PA desarrollado para la identificación de PV. Este ensayo PA utiliza la interacción de la energía fotovoltaica receptor (PVR) y el virión molécula que es la afinidad específica y uniforme para todos los serotipos de la energía fotovoltaica. El procedimiento es sencillo (un procedimiento de paso en las placas de reacción) y rápido (los resultados se pueden obtener dentro de las 2 h de reacción), y el resultado es visualmente observado (observación de la aglutinación 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

Una partícula desarrollado recientemente la novela de aglutinación (PA) de ensayo utilizando virus molécula del receptor permite una identificación rápida y fácil de poliovirus (PV). En este artículo vamos a mostrar el procedimiento para el ensayo de PA para la identificación de PV.

Método de aglutinación de partículas para la identificación de 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 vistas.  National Institute of Infectious Diseases. Una partícula desarrollado recientemente la novela de aglutinación (PA) de ensayo utilizando virus molécula del receptor permite una identificación rápida y fácil de poliovirus (PV). En este artículo vamos a mostrar el procedimiento para el ensayo de PA para la identificación de PV.

Video: 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).

Un procedimiento de 1,5 horas para la identificación de Enterococcus Especies directamente los cultivos de sangre

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

Investigación

Inmunología e Infección

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.

La identificación basada en péptidos de motivos funcionales y sus parejas de unión

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.

Los ensayos para la identificación de nuevos antivirales contra la fiebre catarral ovina Virus

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 por delante de detección genética para la identificación de genes implicados en la resistencia no huésped


	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.

La identificación rápida de bacterias gram negativas de Cultura sangre caldo de cultivo utilizando MALDI-TOF espectrometría de masas

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.

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando 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 Serotipificación de<em&gt; Streptococcus pneumoniae</em&gt; Por partículas de 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.

El aislamiento, la identificación y purificación de células epiteliales del 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.

Ensayos pronta entrada virales para la identificación y evaluación de compuestos antivirales

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.

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

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 ...