Este trabajo fue apoyado por el Fondo Científico de Austria (FMF, P20619 y P22988 para BZ).

Microondas preparación de muestras asistida <ol><li> Antes del inicio de la preparación de muestras es necesario programar el procesador automático de tejidos de microondas para la microscopia electrónica (Leica EM AMW, Leica Microsystems, Viena, Austria) con los siguientes protocolos (Tabla 1) para la preparación de muestras asistida por microondas para TEM: <img src="/files/ftp_upload/2950/2950table1.jpg" alt="Tabla 1" /> Tabla 1. Protocolo para la preparación de la muestra mediante la irradiación de microondas. Las distintas columnas muestran (de izquierda a derecha): <ol><li> Número Vial (nr Vial.) Representa el orden en que se carga el vial en el carrusel del procesador. </li><li> Paso del proceso actual. </li><li> Reactivos en los viales. </li><li> Duración de la etapa actual. </li><li> La temperatura máxima que se alcanza en el vial antes de la irradiación de microondas se apaga. </li><li> Establecimiento de la irradiación de microondas: aumento continuo = bruscos de temperatura, manteniendo la temperatura; Pendiente = aumento de la temperatura suave, la temperatura final alcanzada al final; pulsada = aumento rápido de la temperatura, la energía apagado hasta que la temperatura bajó 5 ° C, el poder reanudó alcance la temperatura . </li><li> La potencia máxima de la irradiación de microondas. </li></ol></li><li> Recién preparar las soluciones para las diversas fases descritas en el protocolo de preparación de la muestra (por resina epoxi Agar 100 ver 1.7), llenarlos en los viales designados de acuerdo con el protocolo programado (Tabla 1), la carga de los viales en el carrusel, a continuación, insertar el carrusel en el procesador de tejidos de microondas, y, finalmente, la carga de la primera copa en la cámara de mono-modo. </li><li> Cortar secciones pequeñas de las hojas (1mm 2) de Nicotiana tabacum infectadas con el Virus del mosaico del tabaco (TMV), con una hoja de afeitar en una placa de cera de modelar en una caída de 3% de glutaraldehído (Agar Scientific Ltd., Stansted, Inglaterra) en 60 mM Sørensen tampón fosfato (pH 7,2) a temperatura ambiente. </li><li> La transferencia de las secciones con pinzas finas de inmediato en las cestas designadas con una abertura de malla de aproximadamente 200μm. Pila de las canastas en la parte superior de cada uno y los inserta en la cámara de mono-modo. Se debe tener cuidado de que las muestras se cubren constantemente con solución fijadora durante la carga y el apilamiento de las canastas para que no se sequen. </li><li> Inicie el microondas previamente programada la asistencia de protocolo de preparación de muestras para la fijación, deshidratación e infiltración. </li><li> Mientras que la preparación de muestras se realiza automáticamente por el procesador de tejido microondas continuar con tinción negativa con el material vegetal restante como se describe en la sección 3 (tinción negativa). </li><li> Recién preparar Agar 100 resina epoxi mediante la mezcla de los componentes que se exponen: Agar llenar 24g 100, 16 g de anhídrido succínico dodecenil, y 10 g de anhídrido metil nadic (para todos los componentes ver Agar Scientific Ltd., Stansted, Inglaterra) en un vaso de plástico de calor a 40 ° C y mezclar bien. Añadir 1,2 g de bencil dimetilamina y mezclar bien. Rellene Agar 100 resina epoxi en la polimerización designado se forma justo antes de que el protocolo de preparación de la muestra llega a su fin (por ejemplo, durante el paso 22 en la tabla 1). </li><li> Después de que el protocolo está terminado (después del paso 22 en la tabla 1) la liberación de las cestas apiladas que contienen las muestras se infiltraron desde la cámara de mono-modo en el último frasco de carrusel. Retire el carrusel desde el dispositivo de microondas, desapilar las cestas y cargarlos usando pinzas finas en las formas de polimerización designado. Se debe tener cuidado de que las muestras están siempre cubiertos de resina epoxi Agar 100 durante desapilado y la carga para que no se sequen. </li><li> Pila de las formas de polimerización en la parte superior de cada uno. Se debe tener cuidado de que las muestras están siempre cubiertos de resina epoxi Agar 100 durante el apilado y carga de manera que no se sequen. </li><li> Eliminar los viales utilizados anteriormente en el carrusel del procesador de tejidos de microondas, que carga con las cestas apiladas e insertar en el carrusel en el procesador de tejidos de microondas. </li><li> Iniciar el protocolo de polimerización previamente programada (tabla 1). </li><li> Mientras que la polimerización se lleva a cabo automáticamente por el procesador de tejidos microondas, examinar las redes de tinción negativa con un microscopio electrónico de transmisión [por ejemplo, Philips CM10 TEM, FEI (anteriormente Philips), Eindhoven, Países Bajos] y llevar a cabo el análisis de imágenes como se describe en la sección 3 y 4 ( tinción negativa y análisis de imágenes). </li><li> Después de que el protocolo está terminado eliminar las formas de polimerización de la cámara de mono-modo, desapilar las formas de polimerización y eliminar los bloques polimerizados que contienen las muestras. Ahora están listos para ser seccionados con un microtomo. </li></ol> 2. Corte y seccionamiento <ol><li> Introduzca uno o más bloques en los titulares de la muestra por separado para el corte ultra fino con la muestra en quedarse encima de 1 cm del soporte. </li><li>Recorte el bloque con un condensador de ajuste muestra para TEM (por ejemplo, Leica Reichert Ultratrim, Leica Microsystems), de modo que una cara del bloque de máx. 1 mm de longitud y 200μm de espesor que contiene material de la hoja tanto como sea posible se logra (tamaño de bloque cara podría ser necesario ajustar el tamaño de la cuchilla de diamante). </li><li> Sección del bloque con un ultramicrótomo (por ejemplo, Reichert Leica Ultracut S, Leica Microsystems) mediante el uso de un cuchillo de diamante en un ángulo de cuchilla de 45 º (por ejemplo, Diatome Ultra 45, Gröpl, Tulln, Austria). Espesor de la sección debe ser ajustado a alrededor de 70 a 90 nm y la velocidad de corte debe estar alrededor de 1 mm / s. Recoger varias secciones con una formvar (Agar Scientific Ltd.) recubierto de cobre o níquel 200 rejilla de malla cuadrada. </li><li> Después de la tinción de las secciones de la parrilla con citrato de plomo (Agar Scientific Ltd., citrato de plomo 1,1 g disueltos en 42 ml de agua doblemente destilada y 8 ml de NaOH 1N) durante 5 minutos en una placa de petri parcialmente lleno con NaOH para crear un CO 2 sin medio ambiente y durante 15 minutos con acetato de uranilo al 1% (Agar Scientific Ltd.) disuelto en agua destilada a temperatura ambiente. Lave las rejillas con agua destilada durante 1 minuto entre cada paso después de la tinción. Deje secar al aire de las rejillas en una celda de la malla. </li><li> Examinar las secciones con un microscopio electrónico de transmisión (por ejemplo, Philips CM10 TEM, FEI, Eindhoven, Holanda). </li></ol> 3. Tinción negativa <ol><li> Cosecha de aproximadamente 100 mg de material de las hojas infectadas con TMV y preparar la savia bruta por homogeneizar el material por 2 minutos con una hoja de afeitar en un portaobjetos en 100μl de 60 mm Søfrensen (pH 7,2). </li><li> Transferencia de 20μl del homogeneizado resultante en el primer pozo de un portaobjetos de microscopio recubierto de teflón, con 4 o más pozos (como alternativa también es posible transferir el homogeneizado en un trozo de parafina). </li><li> Coloque una rejilla Formvar recubierto en la parte superior de la homogeneizado con el formvar (Agar Scientific Ltd.) cara revestida mirando hacia la baja y se incuba durante 5 minutos. </li><li> Lave la red 2 veces durante 2 minutos cada uno mediante la colocación de la rejilla en la parte superior de dos gotas de 200μl Sørensen 60 mM (pH 7,2). </li><li> Incubar la red durante 1 minuto con una solución recién preparada de ácido fosfotúngstico al 2% (Agar Scientific Ltd.) en una solución 60 mM Sørensen (pH 6,5). </li><li> Retire la rejilla y deje que se seque al aire en una celda de la malla. </li><li> Examinar la red con un microscopio electrónico de transmisión (por ejemplo, Philips CM10 TEM, FEI, Eindhoven, Holanda). Tomar por lo menos 10 imágenes elegidas al azar de viriones con tinción negativa (por lo menos 10 o más viriones debe ser visible en cada imagen) con el microscopio electrónico de transmisión con un aumento de 21000X primaria o superior. Se debe tener cuidado de que todas las imágenes tienen el mismo aumento. </li></ol> 4. De análisis de imagen <ol><li> Medir la longitud y la anchura de al menos 100 elegidas al azar partículas del virus solo en las micrografías tomadas de las muestras con tinción negativa mediante el uso de cualquier análisis de imágenes de software [por ejemplo, células D (Olympus, la Vida y la Ciencia de los Materiales Europe GmbH, Hamburgo, Alemania) con el herramienta de análisis de partículas o Optimas 6.5.1 (Media Cybernetics Inc., de Bethesda, Maryland, EE.UU.)]. </li><li> Calcular medias y desviaciones estándar para lograr una longitud y anchura media de las partículas del virus visualizado por los métodos de tinción negativa y TEM. </li><li> Compare las características de longitud y ultraestructurales (obtenido en 2.5) con un tamaño de los virus y las alteraciones ultraestructurales inducidas por virus de las enfermedades conocidas en la literatura con el fin de identificar claramente las enfermedades causadas por virus. </li></ol> 5. Los resultados representativos: Después de la preparación de muestras asistida por microondas típico TMV inducida por alteraciones ultraestructurales, tales como grandes áreas que contienen viriones alineados en forma paralela se puede observar con el microscopio electrónico de transmisión en el citosol de las células infectadas por Nicotiana tabacum (Fig. 1A). Además, en la savia bruta de TMV hojas infectadas partículas de TMV se pudo observar como flexuoso, en forma de barra estructuras después de la tinción negativa (Fig. 1B). Análisis de imágenes de 100 partículas de virus reveló un tamaño promedio de TMV de 280 nm de longitud y 17nm de ancho (Fig. 2). La ultraestructura de las células infectadas con TMV y el tamaño de los viriones observada en este estudio se encontró que de acuerdo con TMV inducida por las propiedades ultraestructurales en el tabaco y el rango de tamaño de partículas de TMV previamente reportados en la literatura 9-15. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Figura 1" /> Figura 1. Micrografías electrónicas de transmisión de las células de la hoja de TMV-infectados y los viriones. A) La imagen muestra la ultraestructura de las células infectadas con TMV mesófilo de la hoja de Nicotiana tabacum después de la preparación de muestras asistida por microondas de la planta. Tenga en cuenta la gran superficie de los viriones paralelos alineados que se acumuló en el citosol.C = cloroplastos con almidón (St), M = mitocondria, N = núcleo, V = vacuola, Bar = 2μm. B) La imagen muestra los viriones que fueron detectados por tinción negativa en la savia de las hojas infectadas. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Figura 2" /> Figura 2. Tamaño relativo de los viriones. Distribución relativa de la longitud y la anchura de TMV-partículas después de la tinción negativa de la savia de las hojas infectadas, ya que apareció en el microscopio electrónico. Los valores medios (media longitud / anchura media ± desviación estándar) se calcula a partir de 100 viriones.

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No hay conflictos de interés declarado.

Asistida por microondas de preparación de muestras de plantas para TEM se ha demostrado que la oferta de datos ultraestructurales rápido y fiable a las pocas horas 3,4,16. La preservación fina estructural de las membranas de los organelos y logra con el método utilizado en este estudio fue similar a las muestras convencionales y cryofixed 5,15 con la ventaja de una reducción masiva en la fijación de la muestra y la incrustación de tiempo de 3 días o más a cerca de 2 horas. Esto representa el protocolo de muestreo más rápido de preparación para TEM actualmente disponibles en la literatura. El método descrito en este estudio combinado asistida por microondas de preparación de muestras de plantas para TEM con los métodos de tinción negativa, que permite una identificación clara y rápida de la TMV inducida por alteraciones ultraestructurales y el agente viral en sí. TMV alteraciones inducidas ultraestructurales podría ser investigado después de la poda, corte y post-tinción en aproximadamente 4 horas después del inicio de la fijación en el microscopio electrónico de transmisión. Usando el modo totalmente automático la preparación de muestras liberó a los investigadores para llevar a cabo la tinción negativa en el ínterin con el fin de determinar el tamaño y el ancho del agente viral. Por lo tanto, podemos concluir que este método permite un diagnóstico claro y rápido de las enfermedades de virus de la planta en alrededor de medio día, que es de gran importancia para su futuro uso en experimentos científicos en la agricultura y la planta de fitopatología. Como este método también podría utilizarse para el diagnóstico rápido de enfermedades de los animales y humanos que tiene un gran potencial para su futura aplicación en medicina y patología veterinaria.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agar 100 resin </td>
			<td>Agar Scientific Ltd.</td>
			<td>R1043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dodecenyl succinic anhydride</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1051</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methyl nadic anhydride</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzyl dimethylamine</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1210</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1260A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1213</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formvar</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1202</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">&nbsp;</td>
		</tr>
		<tr>
			<td>Leica EM AMW</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leica (Reichert) Ultratrim</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Leica (Reichert) Ultracut S</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Diatome Ultra 45</td>
			<td>Gr&ouml;pl</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Philips CM10 TEM</td>
			<td>FEI (formerly Philips)</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Cell D</td>
			<td>Olympus Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optimas 6.5.1</td>
			<td>Media Cybernetics Inc.</td>
			<td>&nbsp;</td>
			<td>Newer version is available</td>
		</tr>
	</tbody>
</table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Las investigaciones de los cambios ultraestructurales inducidas por virus son a menudo necesarios para identificar claramente las enfermedades virales en las plantas. Con la preparación de muestras convencionales de microscopía electrónica de transmisión (TEM) estas investigaciones pueden durar varios días 1,2 y por tanto no adecuada para un diagnóstico rápido de enfermedades virales de las plantas. La fijación de microondas se pueden utilizar para reducir drásticamente el tiempo de preparación de muestras para las investigaciones de TEM con similares resultados ultraestructurales observados después de la preparación de muestras convencional 3-5. Diferentes dispositivos de microondas por encargo están disponibles que pueden ser utilizados para la fijación de éxito y la inclusión de muestras biológicas para investigación TEM 5-8. En este estudio hemos demostrado sobre el virus del mosaico del tabaco (TMV) las plantas infectadas con Nicotiana tabacum que es posible diagnosticar alteraciones ultraestructurales en las hojas en la mitad de una semana a través de la preparación de muestras asistida por microondas TEM. Hemos elegido para llevar a cabo este estudio con un dispositivo de microondas disponibles en el mercado, ya que lleva a cabo la preparación de muestras casi en su totalidad de forma automática 5 a diferencia de los otros dispositivos disponibles en muchos pasos todavía tiene que realizarse de forma manual 6.8 y por lo tanto más tiempo y mucho trabajo. Como preparación de la muestra se realiza de forma totalmente automática tinción negativa de las partículas virales en la savia de los restantes TMV hojas infectadas y el siguiente examen de la ultraestructura y el tamaño se puede realizar durante la fijación e inclusión.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Este estudio describe un método que permite el diagnóstico rápido y libre de enfermedades virales en las plantas cerca de la mitad de un día mediante el uso de una combinación de preparación de muestras asistida por microondas para la planta de microscopía electrónica de transmisión y los métodos de tinción negativa.

Asistida por microondas diagnóstico rápido de enfermedades de virus de plantas por microscopía electrónica de transmisión

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K vistas.  University of Graz. Este estudio describe un método que permite el diagnóstico rápido y libre de enfermedades virales en las plantas cerca de la mitad de un día mediante el uso de una combinación de preparación de muestras asistida por microondas para la planta de microscopía electrónica de transmisión y los métodos de tinción negativa.

Video: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

El diagnóstico rápido del virus de la influenza aviar en aves silvestres: El uso de un portátil de RT-PCR y reactivos liofilizados en el campo

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Investigación

Inmunología e Infección

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detección del virus infeccioso de campo recogidos por los mosquitos del ensayo cultivo celular Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Estructura del VIH-1 Asambleas cápside por crio-microscopía electrónica y reconstrucción iterativa helicoidal del espacio real

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

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

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

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

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

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Microscopía Electrónica de Alta Resolución de la<em&gt; Helicobacter pylori</em&gt; Cag secreción tipo IV Sistema Pili Producido en condiciones variables de Hierro disponibilidad

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

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

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

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

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

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

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologías para el estudio<em&gt; B. subtilis</em&gt; Las biopelículas como modelo para la caracterización de Pequeño Biofilm inhibidores de moléculas

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Los protocolos para la investigación de la distribución original de los tejidos, modo de transmisión, y el efecto sobre la aptitud de un anfitrión Densovirus en el gusano de la cápsula del algodón

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extracción rápido, seguro y sencillo Manual cabecera de ácido nucleico para la detección del Virus en muestras de sangre entera

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

Sinergia antimicrobiana prueba asistida por la impresora de inyección de tinta matriz tablero automatizado y el método Manual de tiempo de matar

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...