1. Virus Inoculación y purificación de las VLP plantas de Nicotiana benthamiana <ol><li> Producir recubiertas T7-ARN transcritos de Potato virus X (PVX)-basados ​​en plásmidos vectores que transportan MRFV de tipo salvaje (wt) y las proteínas Cys-mutado capa (CP) 12 genes, utilizando T7-mMessage mMachine de Ambion Kit. </li><li> Para cada reacción de transcripción T7, inocular dos hojas completamente expandidas de N. benthamiana con 10 reacciones mu l y se incuban las plantas durante 10 días en invernadero, a 60% de humedad durante 16 h con luz (25.000-30.000 lux) a 25 ° C y 8 horas de oscuridad a 20 ° C. </li><li> Harves camisetas N. benthamiana infectadas por el virus hojas 10 días después de la inoculación (dpi), pese el tejido de la planta (50 gramos), y meterlo en hielo. </li><li> Homogeneizar el material vegetal en una mezcladora en presencia de 100 ml de una solución enfriada de 0,5 M de citrato sódico, pH 5,5 (uso 2 ml de tampón por gramo de tejido de la planta). </li><li> Filtrar homogeneizado a través de 3 capas de cheeseclanto y clarificar por centrifugación a 27.000 xg durante 30 min. Transferir el sobrenadante a un cilindro graduado fría, medir el volumen y, a continuación, transferir a un matraz estéril fría. Descartar el sedimento. </li><li> Procesar el sobrenadante mediante la adición de 10% de polietilenglicol 8.000 (PEG 8.000), se agita durante 15 min a 4 ° C, y se incuba durante 45 min a 4 ° C para precipitar las partículas parecidas a virus-(VLPs)-PEG matriz. </li><li> Centrifugar la muestra a 27.000 xg durante 20 min a 4 ° C y retirar el sobrenadante. </li><li> Procesar el precipitado mediante la adición de 8 ml de 0,05 M tampón citrato de Na, 5,5 que contiene 2% de Triton X-100 pH. Remolino tubos de la centrífuga bien para liberar el sedimento. Transferir el sedimento y tampón a un vaso de precipitados estéril enfriada. Enjuague los tubos inmediatamente con 2 ml del mismo tampón y añadir al vaso de precipitados. Se agita la suspensión a 4 ° C hasta que los gránulos se disolvieron (generalmente 1 hr). </li><li> Clarificar por centrifugación durante 5 min a 27.000 xg, desechar el sedimento, y proceso el sobrenadante por centrifugación a 140.000 xg durante 2 horas a 4 ° C. </li><li> Resuspender el precipitado en 1 ml de 0,05 M de citrato sódico, pH 5,5 y colocar la suspensión en la parte superior de un gradiente de sacarosa al 10-40% hecha en 0,05 M de citrato sódico, pH 5,5. </li><li> Centrifugar el gradiente durante 3 horas a 140.000 xg a 4 ° C. </li><li> Brillar una luz sobre la parte superior del tubo de centrífuga y recoger la banda de dispersión de luz superior a 4 cm de la parte superior del tubo, se diluye con 0,05 M de citrato sódico, pH 5,5, y se centrifuga durante 3 horas a 140.000 xg a 4 ° C. </li><li> Resuspender el precipitado en 0,5 ml de agua estéril y se almacena a 4 ° C durante hasta seis meses. </li><li> Cuantificar la concentración de las VLP realizando un ensayo de proteínas de Bradford. El rendimiento de las VLP es de entre 1 y 3 mg / g de tejido de la planta. </li></ol> 2. Fluoresceína-5-maleimida-etiquetado Reacciones de VLPs <ol><li> Preparar una solución 1 mM de fluoresceína-5-maleimida (427,37 g / mol) en 50 mM de sodio Phosphate tampón, pH 7,0, 1 mM EDTA. Mezclar utilizando el vórtice y verificar la completa disolución. Evite que el tubo de la luz utilizando una lámina de aluminio. </li><li> Añadir fluoresceína-5-maleimida para VLPs producidas en la etapa 1 y mezclar. Usar un exceso molar de 25-veces de fluoresceína-5-maleimida para la cantidad molar de sulfhidrilos. Generalmente, usando 30 l de VLPs en la concentración de 1 g / l y 60 l de la solución de fluoresceína-5-maleimida produce resultados aceptables. </li><li> Se incuba la reacción durante 2 horas a temperatura ambiente o durante la noche a 4 ° C. </li><li> Terminar la reacción mediante la adición de ditiotreitol (DTT) a una concentración final de 50 mM. </li><li> Eliminar sin reaccionar fluoresceína utilizando una columna de centrifugación de desalado (Thermo Scientific Inc, Rockford IL) y centrifugación durante 2 min a 1.500 xg, a temperatura ambiente. </li><li> Para análisis SDS-PAGE, añadir 10 l de 2 x SDS-PAGE de tampón de muestra de Laemmli a 10 l de cada reacción. La tienda de la proteína marcada protegido de la luz a 4 º C hasta por un mes ode un solo uso en alícuotas a -20 ° C hasta 3 meses. </li></ol> 3. Etiquetado de Reacción y Determinación de Constitución Biotina <ol><li> Utilice la desalación columnas de centrifugado (Thermo Scientific Inc., Rockford IL) para llevar a cabo un intercambio de tampón de agua a salina tamponada con fosfato (PBS), pH 7,0 de las VLPs producidas en el paso 1. </li><li> Preparar una solución madre 20 mM de maleimida-PEG2-biotina mediante la adición de 190 l de PBS en n-Weigh maleimida-PEG2-biotina y mezclar pipeteando arriba y abajo. </li><li> Añadir maleimida-PEG2-biotina a las VLPs y mezclar. Usar un exceso molar de 10 veces de reactivo de soluciones VLPs ≤ 2 mg / ml. </li><li> Se incuba la reacción a temperatura ambiente durante 4 h. </li><li> Eliminar no reaccionado maleimida-PEG2-biotina usando una columna de centrifugación de desalado (Thermo Scientific Inc, Rockford IL) y centrifugación durante 2 min a 1.500 xg, a temperatura ambiente. </li><li> Determine los moles de biotina por moles de VLP utilizando el Kit de cuantificación Pierce Biotina (Thermo Scientific) y foguientes instrucciones del fabricante. </li></ol> 4. La reticulación de Cys 2-VLP y el péptido F <ol><li> VLPs que resultó en residuos de cisteína disponibles para la reticulación en el paso 2 (Cys 2-VLPs) se utilizó en la etapa siguiente. Preparar una nueva reticulación solución madre (28,63 mM) disolviendo 10 mg de NHS-peg4-maleimida reticulante en 680 l de sulfóxido de dimetilo. </li><li> Disolver un 17 (F)-amino ácido péptido largo (LLPNMOKDKEACAKAPL) en 50 l de tampón de conjugación (1 x PBS, pH 7,2, 1 mM EDTA) en una concentración de 0,1 mM. </li><li> Añadir 4 l de agente de reticulación a 50 l de péptido disuelto (proteína-NH2) en 1 mM de concentración final (que es un exceso molar de 10-veces para 0,1 mM de solución de péptido). </li><li> Se incuba la reacción durante 2 horas a 4 ° C. </li><li> Purificar reticulado péptido usando una columna de desalación (Thermo Scientific Inc., Rockford IL) equilibrada con tampón de conjugación (1 x PBS, pH 7,2, 1 mM EDTA) mediante centrifugación a 1.500 xgdurante 1 min a temperatura ambiente. </li><li> Configurar una mezcla de reacción de la combinación de VLPs (proteína-SH) y reticulado péptido (proteína-NH2) en una relación molar determinada por el número de tioles y aminas activadas implicadas en la reacción y la diferencia de peso molecular entre VLPs y péptido. </li><li> Incubar la mezcla de reacción a temperatura ambiente durante 2 horas a 4 ° C. </li><li> Para el análisis de transferencia Western, mezclar el producto resultante conjugado 1:1 con tampón de Laemmli, hervir durante 10 min, y proceder con SDS-PAGE en un molde pre-10-20% Tris-Glicina gel (Invitrogen), seguido de electrotransferencia sobre una membrana de nitrocelulosa (Invitrogen). Bloquear las membranas con 1 x PBS, pH 7,2 que contiene 0,05% de Tween-20 (PBS-Tween) y 5% de polvo de leche descremada y la sonda con el péptido de anticuerpos específicos seguidos por la fosfatasa apropiada anticuerpo secundario marcado. </li></ol>

<ol>
	<li>Barnhill, H., Reuther, R., Ferguson, P. L., Dreher, T. W., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnip+yellow+mosaic+virus+as+a+chemoaddressable+bionanoparticle.">Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.</a> Bioconj. Chem. 18, 852-859 (2007).</li><li>Chapman, S., Kavanagh, T., Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+vector+for+gene+expression+in+plants.">Potato virus X as a vector for gene expression in plants.</a> Plant J. 2, 549-557 (1992).</li><li>Chen, C., Kwak, E. S., Stein, B., Kao, C. C., Dragnea, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+gold+particles+in+viral+capsids.">Packaging of gold particles in viral capsids.</a> J. Nanosci. Nanotechnol. 5, 2029-2033 (2005).</li><li>Fowler, C. E., Shenton, W., Stubbs, G., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+liquid+crystals+as+templates+for+the+interior+design+of+silica+mesophases+and+nanoparticles.">Tobacco mosaic virus liquid crystals as templates for the interior design of silica mesophases and nanoparticles.</a> Advanced Materials. 13, 1266-1269 (2001).</li><li>Gazit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biomolecular+templates+for+the+fabrication+of+metal+nanowires.">Use of biomolecular templates for the fabrication of metal nanowires.</a> FEBS. J. 274, 317-322 (2007).</li><li>Gillitzer, E., Wilts, D., Young, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+a+viral+cage+for+multivalent+presentation.">Chemical modification of a viral cage for multivalent presentation.</a> Chem. Commun. , 2390-2391 (2002).</li><li>Hammond, R. W., Hammond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+capsid+proteins+assemble+into+virus-like+particles+in+Escherichia+coli.">Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.</a> Virus Res. 147, 208-215 (2010).</li><li>Hermamson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (1991).</li><li>Kaiser, C. R., Flenniken, M. L., Gillitzer, E., Harmsen, A. L., Harmsen, A. G., Jutila, M. A., Douglas, T., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+studies+of+protein+cage+nanoparticles+demonstrate+broad+tissue+distribution+and+rapid+clearance+in+vivo.">Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.</a> Int. J. Nanomed. 2, 715-733 (2007).</li><li>Knez, M., Bittner, A. M., Boes, F., Wege, C., Jeske, H., Maisse, E., Kern, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotemplate+synthesis+of+3-nm+nickel+and+cobalt+nanowires.">Biotemplate synthesis of 3-nm nickel and cobalt nanowires.</a> Nano Lett. 3, 1079-1082 (2003).</li><li>Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+CuCl2+concentration+on+the+aggregation+and+mineralization+of+Tobacco+mosaic+virus+biotemplate.">Effect of CuCl2 concentration on the aggregation and mineralization of Tobacco mosaic virus biotemplate.</a> J. Colloid. Interface. Sci. 297, 554-560 (2006).</li><li>Lvov, Y., Haas, H., Decher, G., Mohwald, H., Mikhailov, A., Mtchedlishvily, B., Morgunova, E., Vainshtein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+deposition+of+alternate+layers+of+polyelectrolytes+and+a+charged+virus.">Successive deposition of alternate layers of polyelectrolytes and a charged virus.</a> Langmuir. 10, 4232-4236 (1994).</li><li>Natilla, A., Hammond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+virus-like+particles+expressed+in+tobacco+plants:+a+new+platform+for+cysteine+selective+bioconjugation+peptide+display.">Maize rayado fino virus virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display.</a> J. Virol. Methods. 178, 209-215 (2011).</li><li>Rae, C. S., Khor, I. W., Wang, Q., Destito, G., Gonzalez, M. J., Singh, P., Thomas, D. M., Estrada, M. N., Powell, E., Finn, M. G., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+trafficking+of+plant+virus+nanoparticles+in+mice+via+the+oral+route.">Systemic trafficking of plant virus nanoparticles in mice via the oral route.</a> Virology. 343, 2224-2235 (2005).</li><li>Raja, K. S., Wang, Q., Gonzalez, M. J., Manchester, M., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+virus-polymer+materials.+Synthesis+and+properties+of+PEG-decorated+Cowpea+mosaic+virus.">Hybrid virus-polymer materials. Synthesis and properties of PEG-decorated Cowpea mosaic virus.</a> Biomacromolecules. 4, 472-476 (2003).</li><li>Royston, E., Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silica-coated+Tobacco+mosaic+virus.">Characterization of silica-coated Tobacco mosaic virus.</a> J. Colloid Interface Sci. 298, 706-712 (2006).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+in+the+Tobacco+mosaic+virus.">Dual-surface modification in the Tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Young, M., Willits, D., Uchida, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+as+biotemplates+for+materials+and+their+use+in+nanotechnology.">Plant viruses as biotemplates for materials and their use in nanotechnology.</a> Annu. Rev. Phytopathol. 46, 361-384 (2008).</li></ol>

La mención de los nombres comerciales de los productos comerciales de la publicación es únicamente con el propósito de proporcionar información específica y no implica recomendación ni aprobación por el Departamento de Agricultura de EE.UU..

El método presentado aquí permite un análisis muy sensible y rápida de cisteínas reactivas presentes en la superficie de VLPs producidos por plantas así como en otros complejos de proteínas. Las maleimidas son tiol reactivos específicos, que reaccionan con sulfhidrilo libres que contienen moléculas para formar enlaces tioéter estables. Este método se basa en la especificidad de las maleimidas para reaccionar con grupos sulfhidrilo que no participan en las interacciones con otros aminoácidos. Preservar la est...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> El número de catálogo </td><td> Comentarios </td></tr><tr><td> Thinwall, Ultra-Clear Tubos </td><td> Beckman </td><td> 344059 </td><td></td></tr><tr><td> mMessage mMachine T7 Kit </td><td> Tecnologías de la Vida </td><td> AM1344M </td><td></td></tr><tr><td> Fluoresceína-5-maleimida </td><td> Thermo Scientific Life Technologies </td><td> 46130 F150 </td><td> 46130 está fuera de orden con sustituto F150 </td></tr><tr><td> Pierce Biotina Kit cuantificación </td><td> Thermo Scientific </td><td> 28005 </td><td></td></tr><tr><td> EZ-Link maleimida PEG2-Biotina, No-Weigh Formato </td><td> Thermo Scientific </td><td> 21901 </td><td></td></tr><tr><td> SM (PEG) n Reticuladores </td><td> Thermo Scientific </td><td> 22107 </td><td></td></tr><tr><td> 10-20% de Tris-Glicina gel </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Tampón de Laemmli </td><td> Bio-Rad </td><td> 1610737 </td><td></td></tr><tr><td> Tris Glicina SDS funcionamiento de amortiguación </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> Tris Glicina Transfer Buffer </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> Membrana de nitrocelulosa Sandwich Papel de Filtro </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> Fosfatasa Etiquetada anticuerpo purificado por afinidad para IgG de conejo </td><td> Kirkegaard y Perry Laboratories </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP sustrato de fosfatasa </td><td> Kirkegaard y Perry Laboratories </td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

Imitando y explotar las propiedades del virus y las características físico-químicas y físicas es una promesa de aportar soluciones a algunos de los desafíos más apremiantes del mundo. La enorme gama y los tipos de virus, junto con sus propiedades intrigantes potencialmente dar un sinfín de oportunidades para las aplicaciones en las tecnologías basadas en el virus. Los virus tienen la capacidad de auto-ensamblarse en partículas con forma discreta y tamaño, la especificidad de la simetría, la polivalencia, y propiedades estables bajo un amplio rango de condiciones de temperatura y pH. No es sorprendente que, con una gama tan notable de las propiedades, los virus se proponen para uso en biomateriales 9, las vacunas 14, 15, materiales electrónicos, herramientas, químicos y electrónicos molecular contenedores 4, 5, 10, 11, 16, 18, ​​12. A fin de utilizar los virus en la nanotecnología, deben ser modificados a partir de sus formas naturales para impartir nuevas funciones. Este desafiante proceso se puede realizar a través de varios mecanismos incluyendo la modificación genética del genoma viral y químicamente unir moléculas extrañas o deseada a los grupos reactivos de partículas de virus 8. La capacidad de modificar un virus depende principalmente de las propiedades físico-químicas y físicas del virus. Además, las modificaciones genéticas o físico-químico necesario llevar a cabo sin afectar negativamente a la estructura del virus nativo y la función virus. Rayado fino del maíz de virus (MRFV) proteínas de la capa auto-ensamblan en Escherichia coli productoras de VLPs estables y vacío que están estabilizadas por proteína-proteína y las interacciones que se pueden utilizar en aplicaciones basados ​​en virus tecnologías 8. VLPs producidas en plantas de tabaco se examinaron como un andamio sobre el que puede ser una variedad de péptidos covalentemente muestra 13. Aquí, se describen los pasos para 1) determinar cuál de las cisteínas accesible al disolvente en una cápside de virus están disponibles para la modificacióncatiónico, y 2) Bioconjugate péptidos a las cápsidas modificadas. Mediante el uso de los residuos de aminoácidos nativos o mutacionalmente inserta-ácidos y las tecnologías estándar de acoplamiento, una amplia variedad de materiales han sido presentados en la superficie de virus de plantas tales como, virus del mosaico del bromo 3, virus del moteado del clavel 12, caupí virus del moteado clorótico 6, del mosaico del tabaco 17 virus, virus mosaico amarillo del nabo 1, y MRFV 13.

La expresión transitoria de mutantes MRFV capa de proteínas (CP) genes en N. benthamiana plantas en un vector PVX VLPs basadas productoras se describe en la figura 1. La modificación capa MRFV gen de la proteína se amplifica por PCR y se coloca entonces bajo el control transcripcional del promotor subgenómico de CP duplicada en un vector basado en PVX, pP2C2S 2, (un regalo de D. Baulcombe, Laboratorios Sainsbury, Norwich, Inglaterra). En vitro la transcripción del ARN ...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Un método para analizar la accesibilidad al disolvente del grupo tiol de residuos de cisteína de<em&gt; Maize rayado fino virus</em&gt; (MRFV)-partículas similares a virus (VLPs), seguido por un péptido reacción de reticulación se describe. El método tiene la ventaja de la disponibilidad de varios grupos químicos en la superficie de las VLP que pueden ser dianas para reacciones específicas.

El análisis de la accesibilidad al disolvente de los residuos de cisteína en<em&gt; Maize rayado fino virus</em&gt; Partículas similares al virus producidas en<em&gt; Nicotiana benthamiana</em&gt; Plantas y reticulación de péptidos a VLPs

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K vistas.  Agricultural Research Service, United States Department of Agriculture. Un método para analizar la accesibilidad al disolvente del grupo tiol de residuos de cisteína de Maize rayado fino virus (MRFV)-partículas similares a virus (VLPs), seguido por un péptido reacción de reticulación se describe. El método tiene la ventaja de la disponibilidad de varios grupos químicos en la superficie de las VLP que pueden ser dianas para reacciones específicas.

Video: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Aislamiento y Análisis del Genoma de viriones individuales utilizando &quot;Genómica solo virus &#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Investigación

Inmunología e Infección

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análisis del daño epitelial Producido por<em&gt; Entamoeba histolytica</em&gt; Infección

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modelado El ciclo de vida de Ebola Virus Bajo Nivel de Bioseguridad 2 Condiciones Con-Virus como partículas que contienen Tetracistronic minigenomas

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Desarrollo de derivados de células madre Las células T reguladoras específicas de antígeno contra la autoinmunidad

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

El uso de la técnica de superposición Soft-agar para la detección de compuestos inhibidores en bacterias producidas

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

La combinación de análisis de la DNA en una extracción de crudo del virión con el análisis de ARN de hojas infectadas para descubrir nuevos genomas de Virus

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Establecimiento de la infección Viral y análisis de la interacción huésped-Virus en Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

Medición de Cytometric del flujo de producción de ROS en los macrófagos en respuesta a Cross-linking FcγR

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...