We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Trabajo Animal realizado en el Centro Nacional de Salud de la fauna de USGS fue realizado de acuerdo con la Oficina de NIH de directrices Laboratorio Animal Welfare y bajo cuidado de los animales institucional y el empleo protocolo # EP080716. Los tejidos de animales cazadores-cosechada fueron regalos de la Wisconsin o Michigan Departamentos de Recursos Naturales. 1. Preparación de la solución NOTA: Las soluciones que se indican a continuación son necesarios para la preparación del sustrato de ensayo CER en la Sección 2 a continuación. Preparar cada una de estas soluciones de soluciones de concentración de valores más altos; concentraciones recomendadas para las soluciones madre se dan entre paréntesis después de cada nombre químico respectivo incluido. Preparar todas las soluciones frescas sobre la preparación del sustrato y se almacena a 4 ° C hasta su uso. <ol><li> Para tampón de lisis, preparar una solución de la siguiente en 10 mM Tris, pH 7,5 (Tris 1 M, pH 7,5 solución valores recomendados): 100 mM scloruro de odio (NaCl, 1 M de solución madre), ácido etilendiaminotetraacético 10 mM (EDTA, EDTA 500 mM, pH 8,0 solución stock), 0,5% NP-40 (solución madre 10%), 0,5% de desoxicolato (DOC, solución madre 10% ). </li><li> Para tampón de conversión, preparar una solución de sulfato de 0,05% de dodecil de sodio (SDS, 10% de solución de stock) y 0,5% de Triton X-100 (solución al 10% de stock) en 1 salina tamponada con fosfato (PBS, 10, pH 7,4 solución madre). </li><li> Para soluciones caotrópicas, preparar dos 3 M soluciones de clorhidrato de guanidina (GdnHCl, 8 M de valores) en 1x PBS (solución 10x). Ajustar el pH de una de las soluciones a 3,5 ± 0,05 con ácido clorhídrico concentrado (HCl). Comprobar que el pH de la segunda solución se mantiene a un pH de 7,4 ± 0,05 y ajustar usando HCl concentrado o hidróxido de sodio (NaOH) si es necesario. </li></ol> 2. Prepare pares CER ensayo de sustrato <ol><li> Obtener el tejido cerebral sano, no-TSE-infectado de especies de interés y preparar un volumen de peso-por-10%(W / v) de homogeneizado en tampón de lisis utilizando cualquiera de los siguientes métodos: Dounce, molino de bolas, o de homogeneización mortero y mano de mortero. Además refinar resultante homogeneizados sometiéndolos a homogeneización jeringa y aguja, el uso de agujas de calibre creciente hasta que el substrato puede ser aspirado y expulsado con facilidad a través de una aguja de la jeringa 27 G. <ol><li> Para sustratos preparados a partir de roedores y otros pequeños mamíferos, homogeneizar todo el cerebro (s); para sustratos preparados a partir de los mamíferos más grandes, use OBEX (tronco cerebral) para preparar los homogeneizados de tejido. Al seleccionar la cantidad de homogeneizado de cerebro, prepare no más de 50% de la capacidad de la centrífuga utilizada para la precipitación de proteínas en el paso 2.6. </li><li> Si la calidad del tejido cerebral adquirido es cuestionable, evaluar los niveles de PrP C por SDS-PAGE e inmunoblot 16 17 antes de sustrato preparación. </li></ol></li><li> Divida homogeneizado de cerebro en dos volúmenes iguales en tubos cónicos de plástico etiquetadas separadas. Añadir GdnHCl (3 M soluciones en 1 PBS) a pH 3,5 o bien 7,4 a cada tubo en una proporción de 1: 1 volumen de homogeneizado de cerebro. </li><li> Compruebe los valores de pH de las dos soluciones. Ajustar el pH de la pH 3,5 sustrato a pH ± 0,05 usando HCl concentrado. Asegúrese de que el pH de la otra sustrato se mantiene a un pH de 7,4 ± 0,05 y ajustar el pH si es necesario con HCl o NaOH según sea necesario. Evitar sobrepasar valores de pH mediante la adición juiciosa de ácido o base. </li><li> Girar soluciones en un mezclador extremo sobre extremo a temperatura ambiente (RT) durante 5 h. </li><li> Precipitado de proteína mediante la adición de cuatro volúmenes de metanol a las soluciones de sustrato en cada tubo cónico, vórtice para mezclar bien, y se incuba a -20 ° C durante 16-18 h. </li><li> Las muestras de sedimentos por centrifugación a 13000 × g durante 30 min a 4 ° C. Decantar con cuidado y desechar el sobrenadante y dejar que el metanol se evapore de los pellets. Utilice bastoncillos de algodón para ayudar en la absorción de metanol aferrado a la parte interior del tubo. No permita bolitas a la sobre-dry, y una vez metanol ya no es visible, continúe con el siguiente paso. </li><li> Bolitas de proteína Resuspender en tampón de conversión. Utilizar una cantidad de tampón de conversión igual a la cantidad de material de partida homogeneizado de cerebro dispensado en cada tubo en el paso 2.2. NOTA: Es fundamental que el búfer de conversión utilizado para volver a suspender gránulos de proteína tanto de pH 3,5 y los preparativos de sustrato 7,4 tratados con la solución está definida en el paso 1.2 anterior (que contiene bajos niveles de detergentes y un pH fisiológico). </li><li> Soluciones brevemente con ultrasonidos sustrato en un aparato de ultrasonidos cuphorn (10 seg en ~ 30% de la potencia máxima), alícuota en 0,5 a 1,0 ml volúmenes de 1,5 ml tubos de microcentrífuga etiquetados. Realizar una inmunotransferencia de control de calidad (paso 2.9) en una parte alícuota de cada sustrato. Guarde otras alícuotas a -80 ° C hasta el momento de realizar el ensayo CER (Sección 4). </li><li> Realizar el control de calidad en los pares de sustrato CER antes de su uso (Figura 2). Estos pasos aseguran que los sustratos CER provide óptimos resultados en los estudios de conversión. <ol><li> Asegurar cantidades comparables de PrP C en los dos sustratos. Comparar los niveles de PrP en el 10-25 l de cada sustrato por SDS-PAGE e inmunotransferencia 16 17. Si los niveles de proteína en el pH 7,4 y 3,5 son sustratos dentro de ~ 10% uno del otro, los pares de sustrato son apropiados para su uso en estudios de CER. NOTA: Los inmunoblots PrP no deben mostrar evidencia de una extensa degradación PrP C. La inmunorreactividad de PrP debe ser principalmente&gt; 20 kDa. Bandas de menos de esta masa molecular pueden ser productos de degradación. Patrones de bandas deben ser más o menos equivalente entre pares de sustrato. </li><li> A medida que la PrP C en ambos sustratos debe ser sensible PK, tratar 10-25 l de cada sustrato con PK a una concentración final de 100 mg / ml y digerir las muestras a 1000 rpm a 37 ° C durante 1 hora en el termo-agitador. Evaluar cualquier señal PrP restante por SDS-PAGE 16 y 17 de inmunotransferencia.Sustratos con PrP res no son apropiados para su uso en estudios de CER. </li></ol></li></ol> 3. Preparar Agente TSE Semillas <ol><li> Obtener el tejido cerebral infectado por el TSE a partir de especies de interés y preparar un 10% (w / v) de homogeneizado en 1X PBS pH 7,4 usando los métodos enumerados en el paso 2.1. NOTA: Las semillas también podrían producirse a partir de otros tejidos infectados por el TSE fuera del sistema nervioso central, sin embargo, la eficiencia de la conversión de sustratos derivado del cerebro por semillas derivadas de tejidos periféricos infectadas por el TSE aún no se ha evaluado experimentalmente en la literatura publicada. </li><li> Alícuota resultante homogeneizado (s) en 100 a 300 volúmenes mu l en 0,5 ml tubos de microcentrífuga y se almacena a -80 ° C hasta el momento de realizar el ensayo CER. </li></ol> Ensayo 4. CER NOTA: El ensayo CER implica la adición de una cantidad relativamente pequeña de PrP TSE (siempre en la &quot;semilla&quot;) de una especieen un exceso relativo de PrP C (proporcionado en el cerebro no infectada &quot;sustrato&quot;) de otra especie que ha sido parcialmente desnaturalizadas, ya sea en pH 3,5 o 7,4. Tras un período de incubación prolongada agitación, la conversión basado en plantillas de PrP C en PrP TSE en cada reacción se evalúa la señal densitométrico de PrP res que queda después de la digestión PK y detección inmunoblot. La comparación de los niveles de PrP res en los sustratos previamente desnaturalizado a pH 3,5 vs. 7,4 proporciona una medida de la barrera de especies. Una visión general experimental de este procedimiento de ensayo se proporciona en la Figura 1. <ol><li> Lentamente se descongele pares de sustrato CER no infectadas (ensayo requiere volúmenes iguales de sustratos previamente desnaturalizado a ambos pH 3.5 y 7.4) y soluciones de semillas TSE-infectados por su inclusión en la parte superior de un lecho de hielo (aproximadamente 1 hr tiempo de descongelación). </li><li> Una vez descongelado completamente, aspirado y luego expulsar a cada solución de sustrato a través de una27 G aguja de la jeringa varias veces para volver a homogeneizar. Tratar brevemente con ultrasonidos cada solución semilla infectada con TSE durante 10 segundos a ~ 30% de la potencia máxima. Mantenga todas las soluciones en hielo mientras se prepara reacciones de conversión. </li><li> Label 5 tubos individuales bajos vinculantes, de paredes finas de PCR por agente de la EET están probando. </li><li> Preparar reacciones de conversión en estos tubos mediante la adición de 5 l de solución de semilla infectada TSE-10% a 95 l de (a) sustrato CER preparados a pH 7,4 y (b) CER sustrato preparado a pH 3,5. <ol><li> Para cada muestra experimental, preparar reacciones paralelas en pares sustrato CER previamente desnaturalizado tanto a pH 3,5 y 7,4. </li><li> Optimizar proporciones de semillas agente de la EET sustrato cerebral no infectada a través de la determinación empírica. Semilla común: relaciones de sustrato empleado mantener un volumen total de reacción de 100 l y l incluyen 1:99, 2:98 y 5:95 l l. Para la mayoría de aplicaciones, la semilla 5:95 l: relación de volumen de sustrato es apropiado y es lo que se presenta en ºes el protocolo. </li></ol></li><li> Preparar tres muestras de control por agente EET se ensayaron como sigue: (a) añadir 5 l de solución de semilla infectada TSE-10% a tampón de conversión de 95 l como control para la entrada de PrP res; añadir tampón de conversión de 5 l a 95 l de sustrato preparado en (b) pH 3,5 y (c) pH 7,4 como controles para la conversión no específica, no moldeado. </li><li> En resumen vórtice cada muestra en su tubo de PCR cerrado para mezclar semillas y sustrato bien. Siga con un pulso momentáneo en una baja velocidad de banco mini-centrífuga para eliminar cualquier volumen de la mezcla de reacción atrapado en el tubo de la tapa PCR. </li><li> Cargar las muestras en tubos de PCR individualmente etiquetados en un banco superior termo-agitador equipado para aceptar tubos de PCR. Agitar las muestras a 1.000 rpm a 37 ° C durante 24 horas. </li><li> Después de agitación, añadir sarcosilo a una concentración final de 2% (w / v) y PK a una concentración final de 100 mg / ml. Muestras Digest en 1000 rpm a 37 ° C durante 1 hora en el termo-agitador. NOTA: La adición de sarcosilo a muestras ayudas post-conversión en PK digestión de PrP C. Señales de falsos positivos en muestras no cabezas de serie (Paso 4.5) son virtualmente eliminados por adición de sarcosilo. </li><li> Añadir tampón de muestra de SDS-PAGE, las muestras de calor a 95 ° C durante 5 minutos y resolver restantes PrP res en cada muestra por SDS-PAGE seguido de inmunotransferencia 16 17. NOTA: Después de la adición de tampón de muestra y calefacción, muestras podrá inmediatamente procesada o muestras se pueden almacenar a -20 ° C o -80 ° C antes de la inmunotransferencia. Todos los datos aquí presentados fueron generados usando los sistemas de gel y transferencia Novex NuPAGE. Las muestras individuales del ensayo de CER se trataron con tampón de muestra y el agente reductor de acuerdo a las instrucciones del fabricante. Las muestras se calentaron a continuación a 95 ° C durante 5 min y 30 l de cada muestra se cargó en un 12% de bis-tris geles prefabricados. </li><li> Calcular el ratio de eficiencia de conversión (CER) como una medidade barrera de las especies. Medir los niveles de PrP res por densitometría 17 y dividen la densidad total de la muestra de pH 7,4 por la del pH 3.5 muestra. Multiplicar por 100 para producir un porcentaje. </li><li> Recopilar datos de réplicas experimentales independientes para generar una media CER ± desviación estándar para una especie con un agente de la EET dado. </li></ol>

<ol>
	<li>Colby, D. W., Prions Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harb Perspect Biol. 3 (1), a006833 (2011).</li><li>Hill, A. F., Collinge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+strains+and+species+barriers.">Prion strains and species barriers.</a> Contrib Microbiol. 11, 33-49 (2004).</li><li>Scott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+hamster+prion+protein+produce+species-specific+scrapie+infectivity+and+amyloid+plaques.">Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.</a> Cell. 59 (5), 847-857 (1989).</li><li>Prusiner, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenetic+Studies+Implicate+Interactions+between+Homologous+Prp+Isoforms+in+Scrapie+Prion+Replication.">Transgenetic Studies Implicate Interactions between Homologous Prp Isoforms in Scrapie Prion Replication.</a> Cell. 63, 673-686 (1990).</li><li>Telling, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+Propagation+in+Mice+Expressing+Human+and+Chimeric+Prp+Transgenes+Implicates+the+Interaction+of+Cellular+Prp+with+Another+Protein.">Prion Propagation in Mice Expressing Human and Chimeric Prp Transgenes Implicates the Interaction of Cellular Prp with Another Protein.</a> Cell. 83, 79-90 (1995).</li><li>Hill, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+prion+strain+causes+vCJD+and+BSE.">The same prion strain causes vCJD and BSE.</a> Nature. 389 (6650), 448-450 (1997).</li><li>Torres, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+modulating+the+prion+species+barrier+and+its+passage+consequences.">Elements modulating the prion species barrier and its passage consequences.</a> PLoS One. 9 (3), e89722 (2014).</li><li>Groschup, M. H., Buschmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+prion+diseases.">Rodent models for prion diseases.</a> Vet Res. 39 (4), 32 (2008).</li><li>Korth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abbreviated+incubation+times+for+human+prions+in+mice+expressing+a+chimeric+mouse-human+prion+protein+transgene.">Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.</a> Proc Natl Acad Sci U S A. 100 (8), 4784-4789 (2003).</li><li>Kocisko, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+specificity+in+the+cell-free+conversion+of+prion+protein+to+protease-resistant+forms:+a+model+for+the+scrapie+species+barrier.">Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.</a> Proc Natl Acad Sci U S A. 92 (9), 3923-3927 (1995).</li><li>Raymond, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+molecular+barrier+limiting+susceptibility+of+humans,+cattle+and+sheep+to+chronic+wasting+disease.">Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.</a> EMBO J. 19 (7), 4425-4430 (2000).</li><li>Fernandez-Borges, N., de Castro, J., Castilla, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+the+transmission+barrier.">In vitro studies of the transmission barrier.</a> Prion. 3 (4), 220-223 (2009).</li><li>Zou, W. Q., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidic+pH+and+detergents+enhance+in+vitro+conversion+of+human+brain+PrPC+to+a+PrPSc-like+form.">Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form.</a> J Biol Chem. 277 (46), 43942-43947 (2002).</li><li>Li, L., Coulthart, M. B., Balachandran, A., Chakrabartty, A., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+barriers+for+chronic+wasting+disease+by+in+vitro+conversion+of+prion+protein.">Species barriers for chronic wasting disease by in vitro conversion of prion protein.</a> Biochem Biophys Res Commun. 364 (4), 796-800 (2007).</li><li>Morawski, A. R., Carlson, C. M., Chang, H., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+prion+protein+conversion+suggests+risk+of+bighorn+sheep+(Ovis+canadensis)+to+transmissible+spongiform+encephalopathies.">In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies.</a> BMC Vet Res. 9, 157 (2013).</li><li>Chandler, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encephalopathy+in+mice+produced+by+inoculation+with+scrapie+brain+material.">Encephalopathy in mice produced by inoculation with scrapie brain material.</a> Lancet. 1 (7191), 1378-1379 (1961).</li><li>Browning, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prions+from+mule+deer+and+elk+with+chronic+wasting+disease+to+transgenic+mice+expressing+cervid+PrP.">Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.</a> J Virol. 78 (23), 13345-13350 (2004).</li><li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+sheep+and+goats+with+transmissible+mink+encephalopathy+virus.">Experimental infection of sheep and goats with transmissible mink encephalopathy virus.</a> Can J Vet Res. 51 (1), 135-144 (1987).</li><li>Rubenstein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+surveillance+and+antigen+conformation+determines+humoral+immune+response+to+the+prion+protein+immunogen.">Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.</a> J Neurovirol. 5 (4), 401-413 (1999).</li><li>Castilla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+species+barrier+by+PrP(Sc)+replication+in+vitro+generates+unique+infectious+prions.">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134 (5), 757-768 (2008).</li><li>Kubista, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27, 95-125 (2006).</li><li>Atarashi, R., Sano, K., Satoh, K., Nishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quaking-induced+conversion:+a+highly+sensitive+assay+for+prion+detection.">Real-time quaking-induced conversion: a highly sensitive assay for prion detection.</a> Prion. 5 (3), 150-153 (2011).</li><li>Ladner-Keay, C. L., Griffith, B. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaking+Alone+Induces+De+Novo+Conversion+of+Recombinant+Prion+Proteins+to+beta-Sheet+Rich+Oligomers+and+Fibrils.">Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to beta-Sheet Rich Oligomers and Fibrils.</a> PLoS One. 9 (6), e98753 (2014).</li><li>Mathiason, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+domestic+cats+to+chronic+wasting+disease.">Susceptibility of domestic cats to chronic wasting disease.</a> J Virol. 87 (4), 1947-1956 (2013).</li><li>Seelig, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

Para completar con éxito de este protocolo, se debe prestar atención a los niveles de PrP C en el tejido cerebral infectado utilizado para la preparación del sustrato (paso 2.1.2) y la semilla al sustrato ratio para reacciones de conversión (paso 4.4.2). En nuestra experiencia, los cerebros pueden ser extraídos para su uso como sustrato CER luego de un período sustancial post-mortem, siempre que la PrP C está presente por inmunotransferencia (Figura 4). De hecho, algunos aut...

Encefalopatías espongiformes transmisibles (EET, enfermedades de priones) son un grupo de enfermedades neurodegenerativas fatales con periodos de incubación prolongados que afectan a una variedad de animales y seres humanos. El agente etiológico putativo de las EET se compone de un isómero mal plegada de la proteína priónica host (PrP) que es capaz de auto-propagación por conversión basado en plantillas de la forma celular normal de PrP (PrP C) en una, asociada a la enfermedad infecciosa forma (PrP TSE) que se acumula en los tejidos del sistema nervioso central del huésped infectado 1. Priones mamíferos infecciosas en general transmiten desde el host-a-host de una manera específica de la especie, lo que ha dado lugar al concepto de la &quot;barrera de las especies TSE&quot; limitar los eventos de transmisión entre especies 2. Los determinantes biológicos de la barrera de especies prión no se comprenden bien. Secuencia de aminoácidos similitud entre la infecciosa PrP TSE y el anfitrión PrP C cUna influencia fuertemente si la conversión tiene lugar 3-5, pero sigue siendo insuficiente para explicar todos los eventos de transmisión de priones observados in vivo 6,7. Por lo tanto, la caracterización de las barreras de especies de EET se ha basado en gran medida en estudios de desafío de los animales: la exposición de los animales no tratados previamente de una especie dada a los priones de otro y midiendo el tiempo de incubación resultante de la aparición de la enfermedad y la tasa de ataque como indicadores de la eficacia de la transmisión. Los ratones que expresan PrP C a partir de especies heterólogas también se utilizan para este tipo de estudio 8. Los costos asociados a la producción de ratones transgénicos y bioensayos priónicas prolongadas, así como las consideraciones éticas del uso de los animales, son obstáculos para la investigación experimental de la EET barreras entre especies. Evaluación de la barrera de la especie humana a las EET se basa en ratones modificados para expresar humana PrP C. Estos ratones requieren largos períodos de incubación a sucumbir a las EET o modificaciones a T humanosél molécula PrP C humana de inicio rápido de la enfermedad 9. Los períodos de incubación de las EET no humana en estos ratones pueden extenderse más allá de la vida útil normal de ratón haciendo la interpretación de los resultados negativos desafiantes. Los primates no humanos también han sido utilizados como indicadores para el estudio de las barreras entre especies humanas, pero estos estudios están llenos de los mismos desafíos que otros tipos de experimentación con animales y primates no humanos pueden no recapitular precisamente la enfermedad a medida que avanza en el huésped humano. Bioensayos animales siguen siendo el método &quot;estándar de oro&quot; para medir la susceptibilidad de una especie a dicha enfermedad, pero los obstáculos, los costos y la ética de estos estudios en animales vivos han obligado investigación sobre alternativas. Un número de ensayos in vitro, basados ​​en la evaluación de la conversión de PrP C anfitrión a una proteinasa K (PK) estado resistentes (PrPres) cuando se sembró por PrP TSE, se han desarrollado y utilizado para investigar TSE spECIES barreras 10-12. Ejemplos de ensayos in vitro incluyen ensayos libres de células de conversión, la proteína misfolding amplificación cíclica (EPCP), y el ratio de eficiencia de conversión (CER) ensayo de 10-14. Aunque ninguno de estos ensayos tienen en cuenta los factores periféricos que intervienen en las barreras de especies después de la infección natural, todo puede ser útil para identificar hosts potencialmente susceptibles para las EET. Aquí presentamos el protocolo para el ensayo CER, en el que dos desnaturalizados sustratos PrP C derivadas de homogeneizados cerebrales normales se utilizan en las principales reacciones de conversión del prión de banco (Figura 1) 13. PrP C en el sustrato desnaturalizado a pH 7,4 sólo se puede convertir en PrPres por PrP TSE se sembraron en la reacción en ausencia de una barrera de las especies 14. En contraste, la desnaturalización de PrP C en el otro sustrato a pH 3,5 permite que se convierte en PrP res después de la incubacióncon PrP TSE de cualquier especie y sirve como un control para la conversión. La relación de conversión de PrP C a PrP res en pH 7.4 sustrato con relación a la del sustrato pH 3.5 proporciona una medida de la barrera de especies. Hemos encontrado que el ensayo CER predice conoce barreras entre especies de ratones de laboratorio a diferentes EET y han utilizado el ensayo en los esfuerzos para predecir la barrera de las especies de numerosas especies de mamíferos, entre ellos el borrego cimarrón, a la caquexia crónica (CWD) y otras EET 14, 15. Los investigadores interesados ​​en una herramienta que permite la detección rápida de la EET barreras entre especies o la evaluación de la conversión de PrP C -a-PrP res encontrarán esta metodología útil.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

El uso exitoso del ensayo CER es dependiente en gran medida de la calidad de los pares de sustrato utilizado en reacciones de conversión. Por esta razón, siguiendo los procedimientos para preparar pares sustrato CER, un inmunoblot de control de calidad debe llevarse a cabo (Figura 2). Para ambos sustratos, ensayo de 10-25 l por inmunotransferencia. PrP C debe ser fácilmente detectable en cada sustrato y los niveles de PrP C debe ser aproximadamente la misma entre los dos. Inmuno...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

La evaluación de la encefalopatía espongiforme transmisible Especies Barreras con un<em&gt; In Vitro</em&gt; Prion Análisis de Conversión de proteínas

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K vistas.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Video: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Investigación

Medicina

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

Aislamiento de oligómeros de PrP soluble e insoluble en el cerebro humano normal

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulación de páncreas Neuroplasticidad:<em&gt; In Vitro</em&gt; Dual-neurona Plasticidad Ensayo

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizando adeno-asociado virus como una herramienta para estudiar las barreras de retina en la enfermedad

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Un modelo del cochinillo de Neonatología encefalopatía hipóxico-isquémica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelado Encefalopatía del Prematuro El uso prenatal hipoxia-isquemia con lipopolisacárido intraamniótica en ratas

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

La especificidad de la evaluación de Medicamentos contra el cáncer<em&gt; In Vitro</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como<em&gt; In Vitro</em&gt; Herramienta de detección en la investigación de la audición

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...