Estamos muy agradecidos a Mateo Robert Weatherhead de excelente servicio técnico y John Ralph, de la Universidad de Wisconsin, por sus valiosos consejos, discusiones, y la muestra de madera de álamo. Este trabajo fue financiado por el Departamento de Energía de EE.UU. (DOE) de los Grandes Lagos del Centro de Investigación de Bioenergía (DOE BER Oficina de Ciencia-DE-FC02 07ER64494) y por las Ciencias Químicas, Geociencias y Ciencias Biológicas de la División de la Oficina de Ciencias Básicas de Energía, Oficina de Ciencia , EE.UU. Departamento de Energía (sin premio. DE-FG02-91ER20021).

1. El aislamiento de la pared celular <ol><li> Moler aproximadamente el 60-70mg de aire o de material liofilizado de plantas, con 5,5 mm bolas de acero inoxidable en un tubo de 2 ml tapa Sarstedt tornillo con un iWall, un robot de molienda y dosificación (30 s). Una alternativa no robótica de bajo rendimiento con un procedimiento de molienda (retschmill) se presenta en la Parte II 2. </li><li> Añadir 1,5 ml de etanol al 70% acuoso para el material molido dispensado, y agitar bien. </li><li> Centrifugar a 10.000 rpm durante 10 minutos para que sedimenten los residuos insolubles en alcohol. </li><li> Aspirar o decantar el sobrenadante. </li><li> Añadir 1,5 ml de cloroformo / metanol (1:1 v / v) al residuo y agitar el tubo a fondo para volver a suspender el pellet. </li><li> Centrifugar a 10.000 rpm durante 10 minutos y Aspirar o decantar el sobrenadante. </li><li> Resuspender el pellet en 500 l de acetona. </li><li> Se evapora el disolvente con una corriente de aire a 35 ° C hasta que se seque. Si es necesario muestras secas se pueden almacenar a temperatura ambiente hasta su posterior procesamiento. </li><li> Para iniciar la extracción del almidón de la muestra resuspender el precipitado en 1,5 ml de un 0,1 M de acetato de sodio pH 5,0 tampón. </li><li> Tape los tubos Sarstedt y el calor durante 20 minutos. a 80 ° C en un calentador. </li><li> Se enfría la suspensión en el hielo. </li><li> Añadir los siguientes agentes de la pastilla: 35 l de 0,01% de azida sódica (NaN3), 35 l de amilasa (50 mg / ml H 2 O, a partir de especies de Bacillus, Sigma), 17 pululanasa l (17,8 unidades de acidopullulyticus Bacillus; Sigma) . Tape el tubo y agitar bien. </li><li> La suspensión se incubó durante la noche a 37 ° C en la coctelera. La orientación de los tubos horizontales ayudantes mejor mezcla. </li><li> Suspensión de calor a 100 º C durante 10 min en un calentador de terminar la digestión. </li><li> Centrifugar (10.000 rpm, 10 min) y eliminar el sobrenadante que contiene almidón solubilizado. </li><li> Lave el resto de pellet tres veces mediante la adición de 1,5 ml de agua, agitación, centrifugación y decantación del agua de lavado. </li><li> Resuspender el pellet en 500 l de acetona. </li><li> Se evapora el disolvente con una corriente de aire a 35 ° C hasta que se seque. Puede ser necesario también para romper el material en el tubo con una espátula para un mejor secado. El material seco se presenta de la pared celular aislado (lignocelulosa). Si es necesario muestras secas se pueden almacenar a temperatura ambiente hasta su posterior procesamiento. </li></ol> 2. Contenido de lignina Este método se basa en un método reportado por Fukushima y Hatfield 3. <ol><li> Pesar 1 a 1,5 mg de material preparado de la pared celular (ver 1) en 2 ml matraz aforado de salir de un tubo vacío de un espacio en blanco. </li><li> enjuagar las paredes del tubo con 250 l de acetona para recoger el material de la pared celular en la parte inferior del tubo, y se evapora la acetona muy suave en el flujo de aire. </li><li> Suavemente añadir 100 ml de solución recién hecha bromuro de acetilo (25% v / v bromuro de acetilo en ácido acético glacial) a lo largo de las paredes del tubo para evitar salpicaduras. </li><li> Cap matraz aforado y se calienta a 50 ° C durante 2 horas </li><li> El calor durante una hora adicional con agitación cada 15 minutos. </li><li> Dejar enfriar en hielo a temperatura ambiente. </li><li> Añadir 400 l de hidróxido de sodio 2M y 70 l de recién preparada clorhidrato de 0,5 M de hidroxilamina. Vortex matraces aforados. </li><li> Rellene matraz aforado exactamente a la marca de 2,0 ml con ácido acético glacial, tapa e invierta varias veces para mezclar. </li><li> Pipetear 200 l de la solución en una placa de 96 UV específicos bien y leer en un lector de ELISA a 280 nm. </li><li> Determinar el porcentaje de lignina bromuro de acetilo soluble (% ABSL) mediante un coeficiente que corresponda (Álamo = 18,21; Pastos = 17,75; Arabidopsis = 15.69) con la siguiente fórmula: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figura 1" /> Multiplicación de ABSL% con 10 resultados en la unidad de la célula ug / mg pared Ayuda a hacer por lo menos tres placa se lee en un promedio de la absorbancia (ABS), ya que las partículas pueden causar una ligera variación en los valores de absorbancia. Nota: 0.539 cm representa el paso de luz, pero dependiendo de la placa de la que tenga que determinar. </li></ol> 3. Lignina Composición Este método se adoptó a partir de un método reciente publicado por Robinson y Mansfield 5. <ol><li> Transferencia de aproximadamente 2 mg de material de la pared celular (ver 1.) En un tubo de vidrio cubiertas de tornillo para tioacidolisis. </li><li> preparar cuidadosamente el 2,5% de trifluoruro de boro dietil eterato (BF 3), el 10% etanotiol (EtSH) solución. Debe utilizar un globo lleno de gas nitrógeno para desplazar el volumen perdido en la botella de dioxano con nitrógeno. El dioxano es muy peligroso, no tomar muestras o equipos fuera de la campana. Volúmenes necesarios para la preparación de la solución por muestra: 175 l dioxano, 20 EtSH l, 5 l BF 3. </li><li> Añadir 200 l de EtSH, BF 3, la solución de dioxano para cada muestra. </li><li> Purga del espacio de cabeza del vial con gas nitrógeno y la tapa inmediatamente. </li><li> El calor a 100 º C durante 4 horas con agitación suave cada hora. </li><li> Final de la reacción por enfriamiento en hielo durante 5 minutos. </li><li> Añadir 150 ml de bicarbonato de sodio 0,4 M, vortex </li><li> Para la limpieza se añade 1 ml de agua y 0,5 ml de acetato de etilo vórtice, y dejar separar las fases (acetato de etilo en la parte superior, el agua en la parte inferior). </li><li> Transferencia de 150 ml de la capa de acetato de etilo en un tubo de 2 ml Sarstedt. Asegúrese de que el agua no se transfiere. </li><li> Evaporar el solvente por un concentrador de aire. </li><li> Añadir 200 l de acetona y se evapora (repetición de un total de dos veces quitar el exceso de agua). </li><li> Para la derivación TMS añadir 500 l de acetato de etilo, 20 l de piridina, y 100 l de N, O-bis (trimetilsilil) acetamida a cada tubo. </li><li> incubar durante 2 horas a 25 ° C. </li><li> Transferencia de 100 l de la reacción en un vial de GC / MS y añadir 100 ml de acetona. </li><li> Analizar las muestras por GC equipado con un espectrómetro de masas cuadrupolo o detector de ionización de llama. Un Agilent HP-5MS columna se instala (30 mm X 0.25 mm X 0,25 m espesor de la película). El gradiente de temperatura se utiliza la siguiente con un retraso de 30 minutos disolvente y un 1,1 ml / min de caudal: retención inicial a 130 ° C durante 3 minutos, a 3 ° C / min a una rampa de 250 ° C y mantener durante 1 min, permiten el equilibrio de la temperatura inicial de 130 ° C. </li><li> Picos se identifican con tiempos de retención relativos con tetracosane patrón interno (opcional) o mediante iones característicos espectro de masas de 299 m / z, 269 m / z, y 239 monómeros m / z de S, G y H, respectivamente (ver fig. 2). La composición de los componentes de la lignina se cuantifica mediante la creación del área del pico total de 100% </li></ol> 4. Resultados representante Un ejemplo de un análisis de la pared se presenta en la Figura 2. En este caso, el álamo madre (de madera) fue analizada por los diversos procedimientos descritos en la sección de protocolo. Un cromatograma de ejemplo de la separación de la lignina, componentes después de tioacidolisis y derivación TMS-se muestra. Claramente, la abundancia relativa de syringyl-(S), guayacil (G), y p-hidroxifenol-(H) unidades se puede determinar. El contenido de lignina soluble bromuro de acetilo se explica por sí mismo, se puede esperar que los valores de entre el 20-50% del peso seco de la pared. Hay que señalar que el bromuro de acetilo no solubilizar todos los presentes la lignina en la pared, y que el grado de solubilización puede variar dependiendo del material. Sin embargo, este método es relativamente fácil de realizar y rápido y da una excelente aproximación del contenido de lignina en un material lignocelulósico. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Figura 1" /> Figura 1:. Visión general del análisis lignocelulósicos paredes celulares (lignocelulosa) están aislados de material en bruto de plantas secas. El material de la pared se pondera en alícuotas y se divide para los ensayos de varios. Material de la pared se trata con bromuro de acetilo y la lignina solubilizada cuantificado por espectroscopia UV-. Para la determinación de la composición de la lignina, el material de la pared se somete a tioacidolisis. Los compuestos fenólicos solubilizados someterse a la derivación TMS y luego pueden ser separados y cuantificados por análisis GC-MS. La composición de polisacáridos de la matriz y el protocolo de celulosa cristalina contenido se discute en la Parte II 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figura 2" /> Figura 2:. Análisis exhaustivo lignocelulósicos de la madera de álamo, Astillas de madera de álamo (Populus tremoloides) fueron sometidos a los protocolos descritos. Ligin composición; H p-hidroxifenil; G guayacil; S syringyl unidades.

<ol>
	<li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annual Review of Plant Biology. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+II:+Carbohydrates.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates.</a> J Vis Exp. , (2010).</li><li>Fukushima, R. S., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+isolation+of+lignin+for+utilization+as+a+standard+to+determine+lignin+concentration+using+the+acetyl+bromide+spectrophotometric+method.">Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.</a> J. Agric. Food Chem. 49 (7), 3133-3133 (2001).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Robinson, A. R., Mansfield, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+poplar+lignin+monomer+composition+by+a+streamlined+thioacidolysis+procedure+and+near-infrared+reflectance-based+prediction+modeling.">Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.</a> Plant J. 58 (4), 706-706 (2009).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endo-transglycosylases.">Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li></ol>

Los métodos descritos permiten una rápida evaluación cuantitativa del contenido de lignina y la composición de la biomasa de plantas lignocelulósicas. El uso del robot iWall aproximadamente 350 muestras de suelo y puede ser dispensado por día. El rendimiento de los distintos métodos de análisis por persona varía. Utilizando los protocolos descritos aquí, 30 muestras se pueden procesar por el contenido de lignina, y 15 para la composición de la lignina por día. Debido a la naturaleza cuantitativa de los cultivos de materias primas de datos óptima, variedad o genotipos pueden ser evaluados en términos de su idoneidad para la producción de biocombustibles.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hydroxylamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>255580</td>
<td>&nbsp;</td>
<tr>
<td>Acetyl Bromide</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>135968</td>
<td>&nbsp;</td>
<tr>
<td>Ethanethiol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E3708</td>
<td>&nbsp;</td>
<tr>
<td>Borontrifluoride diethyl etherate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15719</td>
<td>&nbsp;</td>
<tr>
<td>N,O,-Bis(trimethylsilyl) acetimide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15241</td>
<td>&nbsp;</td>
<tr>
<td>Dioxane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>296309</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6890 GC/5975B MSD</td>
<td>(lignin composition)</td>
</tr>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part i: lignin

La necesidad de renovables, neutral de carbono y sostenible de materias primas para la industria y la sociedad se ha convertido en uno de los problemas más acuciantes para el siglo 21. Esto ha reavivado el interés en el uso de productos vegetales como materia prima industrial para la producción de combustibles líquidos para el transporte<sup&gt; 1</sup&gt; Y otros productos tales como materiales biocompuesto<sup&gt; 7</sup&gt;. La biomasa vegetal es uno de los más grandes reservas sin explotar en el planeta<sup&gt; 4</sup&gt;. Se está compuesta mayormente por las paredes celulares que están compuestos de polímeros ricos en energía como celulosa, hemicelulosa diferentes (polisacáridos de la matriz, y la lignina de polifenoles<sup&gt; 6</sup&gt; Y por lo tanto a veces se denomina lignocelulosa. Sin embargo, las paredes celulares vegetales han evolucionado para ser recalcitrantes a la degradación de las paredes ofrecen resistencia a la tracción a las células y las plantas enteras, y evitar agentes patógenos, y permitir que el agua se transporta por toda la planta, en el caso de árboles de hasta más de 100 m por encima de a nivel del suelo. Debido a las diversas funciones de las paredes, hay una inmensa diversidad estructural dentro de las paredes de diferentes especies de plantas y tipos de células dentro de una sola planta<sup&gt; 4</sup&gt;. Por lo tanto, dependiendo de lo que las especies de cultivo, las variedades de cultivo, o tejido de la planta se utiliza para la bio-refinerías, los pasos del proceso de despolimerización por procesos químicos / enzimáticos, y posterior fermentación de los azúcares distintos a los biocombustibles líquidos deben ser ajustadas y optimizadas. Este hecho se basa la necesidad de una caracterización exhaustiva de materias primas de biomasa de las plantas. A continuación se describe una metodología completa de análisis que permite la determinación de la composición de la lignocelulosa y es susceptible a un medio de análisis de alto rendimiento. En esta primera parte nos centramos en el análisis de la lignina en polifenoles (Figura 1). El método comienza con la preparación de material de desalmidonadas pared celular. La lignocelulosa resultantes se dividen para determinar su contenido de lignina por solubilización acetylbromide<sup&gt; 3</sup&gt;, Y su lignina composición en términos de sus unidades syringyl, guayacil-y p-hidroxifenil<sup&gt; 5</sup&gt;. El protocolo para el análisis de los hidratos de carbono en la biomasa lignocelulósica incluyendo el contenido de celulosa y la composición de polisacáridos de la matriz se discute en la Parte II<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin at JoVE.com

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

La biomasa vegetal es uno de los principales de carbono-neutral recurso renovable que podría ser utilizado para la producción de biocombustibles. La biomasa vegetal se compone principalmente de las paredes celulares, un material compuesto estructuralmente complejos denomina lignocelulosa. Aquí se describe un protocolo para un análisis exhaustivo del contenido y la composición de la lignina polifenólicos.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte I: La lignina

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

comprehensive-compositional-analysis-plant-cell-walls-lignocellulosic

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Cell Biology

Biology

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K vistas.  Michigan State University (MSU). La biomasa vegetal es uno de los principales de carbono-neutral recurso renovable que podría ser utilizado para la producción de biocombustibles. La biomasa vegetal se compone principalmente de las paredes celulares, un material compuesto estructuralmente complejos denomina lignocelulosa. Aquí se describe un protocolo para un análisis exhaustivo del contenido y la composición de la lignina polifenólicos.

Video: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Aislamiento humanos islote pancreático: Parte I: La digestión y la acumulación de tejido pancreático

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Investigación

Biología

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 3

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Fluorescencia de células activadas por la clasificación de los protoplastos de la planta

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte II: Los hidratos de carbono

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Sin etiquetas In situ Imágenes de lignificación de las paredes celulares vegetales

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Perfiles de Polímeros Glycan la pared celular vegetal utilizando microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un método eficaz para Cuantitativo, Análisis de celda única de la cromatina modificación y Arquitectura Nuclear en Todo el montaje en Óvulos<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...