Nos gustaría dar las gracias a la Dra. Marie Ramos Vesper Fe para la lectura crítica del manuscrito. Este trabajo fue apoyado por el Centro Nacional para Recursos de Investigación (P20 RR0115635-6, C06 RR17417-01 y G20RR024001) y el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares (2R01 NS052609).

1. Preparación del Equipo <ol><li> Utilice un Misonix 3000 o 4000 sonicador Misonix (Farmingdale, NY) conectado a un baño de Thermo Electron Neslab EX-7 agua (Newington, NH) para mantener una temperatura constante de 37 ° C. Sonicar las muestras en 200 ul de paredes delgadas tiras de tubos de PCR con tapas abovedadas obtenidos de Thermo Scientific (Waltham, MA). </li><li> Un baño de ultrasonidos nueva marca requiere de un &quot;robo&quot; período de operación continua 9. Un robo de dos meses consiste en una ráfaga de sonicación de 40 segundos y 10 minutos ciclos de incubación con el nivel de amplitud establece en 10. Uno debería observar la erosión a lo largo de la superficie del cuerno de titanio de microplacas (Figura 1, panel B). El agua circulante se verá blanca, turbia debido a la erosión de la bocina de titanio microplaca. Cambie el agua diariamente. </li><li> La amplificación de la PrP Sc variará a través de la trompa de microplacas. La mejor eficiencia de amplificación PrP Sc se puede conseguir dentro deun radio de 5 cm desde el centro de la bocina de microplacas (Véase la Figura 1, panel C). </li><li> Una ronda de EPCP consiste en 144 ciclos. Cada ciclo consta de 5 segundos sonicación y 10 min de incubación. Esto implicará aproximadamente a 24 horas por ronda del EPCP. </li><li> La salida de potencia de la sonicación, se muestra en el panel de control del sonicador, varía con el número de tubos de PCR presentes en la rejilla del sonicador, y la temperatura del agua de circulación del baño de agua. Asegúrese de que el agua se equilibra a 37 ° C, y no llene más de 30% de soporte de tubos de la sonicador (Figura 1, panel C). Extienda los tubos de PCR a través de la trompa de microplacas. Tener al menos una fila de espacios vacíos entre las tiras de tubos PCR y no permitir más de cuatro tubos conectados entre sí por tira (Figura 1, panel C). </li><li> La fuerza de la sonicación es crítico para una amplificación prión cepa éxito. Mientras sonicación, ajustar la amplitud de tener una potencia de salidaque oscila entre 155 ~ 170 vatios. En nuestro laboratorio, el nivel de potencia se establece en seis y siete para el 3000 Misonix Misonix y 4000, respectivamente. </li><li> El uso de agua destilada en el baño de agua para evitar la acumulación de manchas de agua dura. Reemplazar esta semana el agua. Durante los ciclos de incubación y sonicación, la condensación de agua se produce en el interior de la tapa del cuerno de microplacas. Para evitar la condensación, coloque una almohadilla de calor pequeño acuario a la parte superior de la caja de la carcasa del aparato de ultrasonidos, como se muestra en la Figura 1, panel A. </li></ol> 2. Preparación de las muestras <ol><li> Todos los procedimientos con animales fueron aprobados por el Cuidado de Animales institucional Creighton University y el empleo Comisión y cumplir con la Guía para el Cuidado y Uso de Animales de Laboratorio. Antes de que cualquier recogida de tejido, el animal debe ser anestesiados y perfundidos transcardialmente con fosfato enfriada con hielo una solución salina tamponada con 5 mM de EDTA a pH 7,4 para eliminar la sangre desde el cerebro y evitar la inhibición de laEPCP reacción por la sangre 7. Recoge los tejidos animales rápidamente utilizando una técnica aséptica y tienen herramientas específicas para cada cepa de disección prión. Después de la disección, colocar el tejido en un tubo de microcentrífuga de 1,5 ml, flash que se congele en hielo seco, y almacenar a -80 ° C hasta la homogeneización. </li><li> Para preparar el sustrato EPCP (PrP C) solución, homogeneizar un cerebro de hámster infectado (ONU BH) a un 10% peso / volumen (w / v) de solución con tampón de conversión enfriado con hielo (véase la parte 3), utilizando un triturador de tejidos Tenbroeck. Mientras homogeneizar, tenga cuidado de no tener aire en el interior del molino para evitar una excesiva formación de espuma en la solución. Aclarar la solución por centrifugación a 500 xg durante 30 seg y alícuota del sobrenadante en tubos de 1,5 ml de microcentrífuga. Almacenar a -80 ° C hasta su uso posterior. </li><li> Para preparar la semilla EPCP (PrP Sc) solución, homogeneizar un material infeccioso (nodos cerebro, bazo, linfáticos, otros tejidos o el suelo contaminado) a un 10% w / v solución con wate hielo fríor y alícuota de la solución en tubos de microcentrífuga de 0,6 ml. Almacenar a -80 ° C hasta su uso posterior. </li></ol> 3. Preparación de Buffer de conversión <ol><li> Pesar 0,5093 g de Triton X-100 en un matraz aforado de 50 ml (el resultado es una concentración final de 1% de Triton X-100) (Sigma-Aldrich GmbH, Steinheim, Alemania). </li><li> Añadir una inhibidor de proteasa completo Tablet (Roche Diagnostics GmbH, Mannheim, Alemania). </li><li> Añadir 0,0931 g de EDTA (esto dará lugar a una concentración final 5 mM de EDTA) (Mallinckrodt Baker, Inc.; Phillipsburg, NJ). </li><li> Ajustar el pH a 7,4 con NaOH 1N. </li><li> Ajustar el volumen a 50 ml con fosfato de Dulbecco Buffered Saline (DPBS) (Mediatech Inc.; Manassas, VA). </li><li> Filtrar la solución a través de una membrana de 0,45 micras de nitrocelulosa con un aparato de filtración a vacío. El tampón de conversión se puede almacenar a 4 ° C durante no más de un mes. </li></ol> 4. La amplificación de las cepas de priones mediante el EPCP <ol><li>Diluir la semilla EPCP en el sustrato EPCP en una proporción de 1:20 (es decir, mezcla de 5 l de la semilla EPCP y 95 l de sustrato de la PMCA) en un tubo de PCR. Retirar y almacenar una parte alícuota de 15 l a -80 ° C para el control de unsonicated; este control unsonicated se utiliza para cuantificar la amplificación PrP Sc través de proteinasa K (PK) seguido por la digestión de Western (WB) análisis. Un control unsonicated adicional podría ser generada por la colocación de una segunda alícuota de 15 l a 37 ° C durante la duración de la reacción EPCP. Un mínimo de tres repeticiones se requiere para fines estadísticos. </li><li> Se somete el resto de la muestra (70 l) a una ronda de EPCP como se representa en la Figura 1, Panel A. Se mezcla tubos cada 5 hr para evitar la condensación de agua en la tapa abovedada del tubo de PCR. </li><li> Tubos de la muestra deben ser centrifugadas y suavemente vortex (teniendo cuidado de que la solución no va en la tapa abovedada) antes de abrir después de cualquier procedimiento que garantice Homogeneity y evitar la contaminación potencial. </li><li> Para serie EPCP (sPMCA) de corto cepas período de incubación (es decir, HY TME, HaCWD, o 263K), diluir el material sonicado en una proporción de 1:20 mediante la transferencia de 5 l de la muestra sometida a ultrasonidos desde la primera ronda en 95 l de fresco no infectada homogeneizado de cerebro (Figura 2, panel B). </li><li> Para sPMCA de largos cepas período de incubación (es decir, DY TME, 139H, 22CH, 22AH, o ME7H), diluir el material sonicado en una proporción de 1:1 por transferencia de 50 l de la muestra sometida a ultrasonidos desde la primera ronda en 50 l de fresco no infectada homogeneizado de cerebro (Figura 2, panel B). </li><li> 15 Retire los dos alícuotas de la mezcla previamente diluido sonicado (de pasos 4,4 o 4,5) y almacenar uno de ellos a -80 ° C y el otro a 37 ° C (soluciones de control para unsonicated EPCP la segunda ronda). Someter a los restos de una ronda EPCP dos. </li><li> Este procedimiento se puede repetir indefinidamente. En nuestro laboratorio, hemoshan realizado hasta quince rounds EPCP, con las propiedades de deformación permanecerán invariables. </li><li> PK digerir las muestras. Para un típico WB, mezclar 5 l de PK 80 ug / ml a 5 l de muestra (esta solución tendrá una concentración final de PK de 40 mg / ml) e incubar a 37 ° C durante una hora. Añadir 10 l de tampón de carga de gel. Hervir esta solución durante 10 min. Deje que la solución se enfríe y cargar 10 l en el gel. </li><li> Para calcular el éxito de la amplificación de realizar un análisis de WB en las muestras digeridas PK-para estimar la cantidad de PrP Sc amplificado por comparación a cualquiera, los controles unsonicated 20,21 Figura 2, panel C o a cantidades conocidas de PK-sin digerir recombinante PrP. </li></ol> 5. Evitar la contaminación cruzada Los pasos críticos tienen que ser tomadas para reducir al mínimo la contaminación cruzada y la ocurrencia de falsos positivos debido a la formación de novo de productos resistentes a la proteasa. </p&gt; <ol><li> Utilice un conjunto dedicado de herramientas de disección (es decir, fórceps, pinzas, tijeras) para la recolección de la muestra para cada cepa de prión. </li><li> Antes de homogeneización de los cerebros no infectadas para ser utilizados como un sustrato de PMCA, limpiar las pipetas con 1% de lejía. Remoje la cremallera sonicador de PCR tubo, homogeneizadores de tejidos, y bastidores de almacenamiento de tubos PCR con 1% de lejía durante 10 minutos y enjuagar con agua destilada. </li><li> Cubra el área de trabajo con un Soaker Lab fresco Versi-Dry cada vez que una nueva muestra se homogeneiza, un nuevo experimento EPCP es estar preparado, o cuando alícuotas entre rondas sPMCA. </li><li> Use guantes nuevos cada vez que nuevas muestras se manejan (especialmente cuando se transfieren alícuotas entre rondas sPMCA). </li><li> Utilice descargas cortas sonicación durante cada ciclo del EPCP. Los ciclos de sonicación 5 sec combinado con 10 minutos de incubación es suficiente para amplificar los priones sin generar de novo PrP Sc 1,20,21,23,24. Como otros laboratorios han demostrado, el aumento de latiempo de sonicación, el aumento de la amplitud de sonicación, y aumentando el número de ciclos de PMCA aumenta la probabilidad de novo de PrP Sc formación 2. </li></ol>

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No hay conflictos de interés declarado.

Desafíos de la amplificación de las proteínas priónicas infecciosas son los períodos de incubación largos y los gastos de los experimentos in vivo. La técnica EPCP es un medio rentable para amplificar los agentes infecciosos de priones. Varios laboratorios han confirmado la capacidad de PMCA para amplificar con precisión las cepas de priones in vitro 7, 9, 12, 14, 19,24. Las enfermedades por priones se pueden transmitir entre especies. Bessen y M...

<table border="1"><tbody><tr><td> Reactivo / Equipo </td><td> Fabricante </td><td> Cat. Número </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck triturador de tejidos </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 Water Bath </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> PCR de 0,2 ml tiras de tubos </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Inhibidor de Proteasa Completo </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Versi-Dry Lab Soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm calentador </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Los priones son los agentes infecciosos causantes de la encefalopatía espongiforme transmisible inevitablemente fatal (EET) en los animales y los seres humanos 9,18. La proteína prión tiene dos isoformas distintas, la no infecciosa proteína codificada por el hospedador (PrP C) y la proteína infecciosa (PrP Sc), una isoforma anormal de PrP plegado en C 8. Uno de los retos de trabajar con agentes de priones es el largo período de incubación antes de la aparición de los signos clínicos después de la inoculación de acogida 13. Este mandato tradicionalmente los estudios en animales largos y caros de bioensayo. Además, las propiedades bioquímicas y biofísicas de PrP Sc están pobremente caracterizados debido a su conformación inusual y estados de agregación. PrP Sc puede sembrar la conversión de PrP C y PrP Sc in vitro 14. El EPCP es una técnica in vitro que se Advantage de esta capacidad utilizando ciclos de sonicación y la incubación para producir grandes cantidades de PrP Sc, a un ritmo acelerado, a partir de un sistema que contiene cantidades en exceso de PrP C y pequeñas cantidades de la semilla PrP Sc 19. Esta técnica ha demostrado ser eficaz para recapitular la especificidad de especie y cepa de PrP Sc conversión de PrP C, para emular interferencia cepa de priones, y para amplificar los niveles muy bajos de PrP Sc partir de tejidos infectados, fluidos y muestras ambientales 6,7,16, 23. Este documento detalla el protocolo EPCP, incluyendo recomendaciones para minimizar la contaminación, generando resultados consistentes, y la cuantificación de los resultados. También se discuten varias aplicaciones del EPCP, incluyendo la generación y caracterización de cepas de priones infecciosos, la interferencia cepa de prión, y la detección de priones en el medio ambiente.

Misfolding proteína amplificación cíclica (PMCA) se utiliza para amplificar PrP Sc in vitro 7, 12, 14, 19, 24. El éxito de la amplificación PrP Sc se muestra por un aumento en la intensidad de la banda en las transferencias Western de la proteína priónica PK-resistente (migrando entre 19 y 30 kDa para las cepas de priones de hámster derivados) como se muestra en la Figura 3. El aumento de la intensidad de la banda después de su EPCP indica la amplificac...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Misfolding proteína amplificación cíclica (EPCP) es un ensayo in vitro para el estudio de la conversión de priones y barreras cepa y especie. También se puede utilizar como un ensayo de detección de priones.

Mal plegamiento de proteínas priones de amplificación cíclica

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K vistas.  University of Nebraska at Lincoln. Misfolding proteína amplificación cíclica (EPCP) es un ensayo in vitro para el estudio de la conversión de priones y barreras cepa y especie. También se puede utilizar como un ensayo de detección de priones.

Video: Protein Misfolding Cyclic Amplification of Prions

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedimientos para la Identificación de priones infecciosos después de pasar por el sistema digestivo de una especie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Investigación

Inmunología e Infección

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotérmico de amplificación (LAMP) Ensayos Loop mediada para la detección específica de las especies de<em&gt; Eimeria</em&gt; Que infectan pollos

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

El desarrollo de la mediación de la Loop liofilizada amplificación isotérmica reactivos para la detección de<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

En tiempo real temblando inducida por conversión de ensayo para la detección de priones CWD en materia Fecal

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Disección señalización complejos por purificación de la afinidad de complementación Bimolecular (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Análisis conductual de gran C. elegans las poblaciones utilizando un amplio campo de visión de seguimiento plataforma automatizada

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Generación, amplificación y titulación de virus sincitiales respiratorios recombinantes

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Ensayo de amplificación isotérmica mediada por bucle de transcripción inversa (RT-LAMP) para la detección específica y rápida del virus del lago Tilapia

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

Amplificación de la señal de tiramida para la tinción inmunofluorescente de la fosforilación dependiente de ZBP1 de RIPK3 y MLKL después de la infección por HSV-1 en células humanas