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 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 (premio no. DE-FG02-91ER20021) y por el Departamento de Energía de EE.UU. (DOE) de los Grandes Lagos Centro de Investigación de Bioenergía (DOE BER Oficina de Ciencia-DE-FC02 07ER64494).

1. El aislamiento de la pared celular <ol><li> moler aproximadamente el 60-70mg de aire o congelar material vegetal seco con 5.5 mm bolas de acero inoxidable en un tubo de 2 ml Sarstedt tapón de rosca con un retschmill (1 min, 25 Hz). Una alternativa, el uso de una molienda de alto rendimiento y dispensación iWall robot llamado se describe en la Parte I 3. </li><li> eliminar las bolas de acero antes de continuar con el procedimiento celda de aislamiento de la pared El protocolo detallado de la preparación del material de la pared celular se muestra en la Parte I 3. Para completar aquí los pasos escritos del protocolo. </li><li> añadir 1,5 ml de etanol al 70% acuoso, 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 recoger el sobrenadante </li><li> resuspender el pellet en 500 l de acetona </li><li> evaporar 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 vuelva a suspender el precipitado en 1,5 ml de 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> enfriar la suspensión en el hielo </li><li> añadir los siguientes agentes de la pastilla: 35 l de 0,01% Sodiumazide (NaN 3), 35 l de amilasa (50 μg/1mL H 2 O, a partir de especies de Bacillus, SIGMA); (; SIGMA 18,7 unidades de acidopullulyticus bacilo) 17 pululanasa l . 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> centrifugación (10.000 rpm, 10 min) y eliminar el sobrenadante que contiene almidón solubilizado </li><li> lavar el resto de pellet tres veces mediante la adición de 1,5 ml de agua, agitación, centriguation y decantación del agua de lavado. </li><li> resuspender el pellet en 500 l de acetona </li><li> evaporar 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. Matriz de composición de polisacáridos Este método es esencialmente una modificación del método publicado por Albersheim 1. <ol><li> Para determinar la composición de monosacáridos del material de la pared pesan 2 mg de material de la pared celular en 2 ml tubos starstedt ya sea a mano o en forma highthroughput con el iWall, un robot robots de molienda y de pesaje. </li><li> Añadir 20 l de una solución de inositol (5mg/ml) como estándar interno. Para una muestra de 2 mg de la pared celular se recomienda añadir 100 ug. </li><li> enjuagar las paredes del tubo con 250 ul 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> Para la hidrólisis ácido débil añadir 250 ul de ácido 2M trifluoroacético (TFA) para cada muestra. Añadir TFA cuidadosamente para asegurar que no es el material salpicó a las paredes del tubo. </li><li> tapa y se incuba durante 90 minutos a 121 ° C en un calentador. </li><li> enfriar los bloques de calentamiento y las muestras en hielo. </li><li> centrifugar los tubos a 10.000 rpm durante 10 min. </li><li> transferencia de 100 ul del sobrenadante ácido que contiene el polisacárido matriz derivados de monosacáridos a una tapa de vidrio viales tornillo con cuidado de no perturbar el material precipitado. Los pellets se pueden utilizar para el ensayo de celulosa cristalina a continuación (véase el punto 3.) </li><li> evaporar el TFA en el tubo de vidrio con una corriente suave de aire en un dispositivo de evaporación. </li><li> Añadir 300 l 2-propanol, vórtice y se evapora a 25 ° C (de repetición para un total de tres veces) </li><li> El primer paso del procedimiento de acetato de derivación alditol es llevar a cabo una reducción de los monosacáridos a su alditoles correspondiente. Para ello se añaden 200 l de una solución de borohidruro de sodio a cada muestra seca. Prepare una solución fresca cada vez que el uso de 10 mg de borohidruro de sodio por 1 ml de hidróxido de amonio 1M. </li><li> dejar vial de vidrio a temperatura ambiente durante 1,5 horas </li><li> neutralizar la solución mediante la adición de 150 ul de ácido acético glacial </li><li> vortex y se evapora a 25 ° C. </li><li> añadir 250 vórtice l de ácido acético / metanol (1:9, v / v), y se evapora a 25 ˚ C </li><li> añadir 250 l de metanol y se evapora bajo una corriente de aire (de repetición para un total de tres veces) </li><li> Para la acetilación de la alditoles, se añaden 50 l de anhídrido acético y 50 l de piridINE, vortex e incubar durante 20 minutos a 121 ° C en un calentador. </li><li> muestras frescas en el bloque con hielo, mientras que esperar a disminuir temperatura a la temperatura ambiente aproximadamente. </li><li> se evaporan los reactivos bajo una suave corriente de aire a temperatura ambiente. Tenga cuidado: acetatos alditol son muy volátiles. </li><li> añadir 200 l de tolueno y se evapora en el aire (x3) </li><li> En los pasos finales de la acetatos alditol se extraen. En primer lugar, añadir 500 ul de acetato de etilo y agitar ligeramente. </li><li> añadir 2 ml de agua, tubos de tapón y mezclar. </li><li> tubos de centrífuga a 2.000 rpm durante 5 minutos para obtener claras capas separadas (acetato de etilo en la parte superior, el agua en la parte inferior) </li><li> pipeta 50 ul de la capa de acetato de etilo en GC / MS viales con insertos. </li><li> diluida por la adición de 100 ul de acetona a la GC-vial y la tapa. El volumen de muestra y el volumen de dilución puede ser ajustado para evitar la sobrecarga del GC / MS, si la concentración de la muestra es demasiado alto. El GC-vial se puede almacenar a 4 ° C, si el análisis de GC / MS no proceder de inmediato </li><li> Las muestras se inyectan en un GC que está equipado con un MS cuadrupolo, pero un detector de ionización de llama es también adecuado. A Supelco SP-2380 (30 mm x 0,25 mm x 0,25 m de espesor película) columna se utiliza con un retraso de 4 minutos disolvente y un caudal de 1.5ml/min. Muestras inyectadas son sometidos a la siguiente programa de temperaturas: retención inicial a 160 ° C durante 2 minutos, a 20 ° C / min rampa a 200 ° C y mantener durante 5 min, a 20 ° C / min rampa a 245 ° C y mantener 12 min, pico a 270 ° C y mantener durante 5 minutos antes de refrescarse a la temperatura inicial de 160 ° C 2.26) picos se identifican con los perfiles de masa y / o tiempos de retención de las normas.. Monosacáridos se cuantifican sobre la base de las curvas de calibración. </li></ol> 3. Contenido de celulosa cristalina Este método es esencialmente descrita por Updegraf 8. Hay una serie de materiales de partida para este procedimiento: elementos aislados de la pared celular (ver 1) o material de la pared que ya ha sido tratado con 2M TFA (véase el punto 2.8) o bien el resto de pellets inmediatamente después del tratamiento con ácido (véase el punto 2.8) o TFA un pellet, que ha sido lavada con 2-propanol y se secan. <ol><li> Añadir a la pastilla de AGT en el tornillo de tubo tapado de vidrio de 1 ml de reactivo Updegraff (Ácido acético: ácido nítrico, agua, 08:01:02 v / v). </li><li> Tapar el tubo con fuerza, vórtice, y el calor en un calentador a 100 ° C durante 30 min. Como resultado de este tratamiento sólo celulosa cristalina sigue siendo insoluble en el sedimento. </li><li> Enfriar las muestras en el bloque de hielo a temperatura ambiente o frías </li><li> las muestras se centrifugan a 10.000 rpm durante 15 minutos </li><li> Desechar el sobrenadante asegurando que la pastilla no se altera y no material de la pastilla se quita. Para ello dejar aprox. 150 ul de sobrenadante en el tubo. </li><li> añadir 1,5 ml de agua, agitar, centrifugar y desechar el sobrenadante como se indicó anteriormente </li><li> repetir el lavado procedimiento 3 veces más con 1,5 ml de acetona </li><li> Aire seco en pelets muy suavemente con el aire, o deje secar durante la noche en el banco </li><li> el pellet (celulosa cristalina) está completamente hidrolizada en glucosa por lo que se llama una hidrólisis Saeman. Para ello añadir 175 l de ácido sulfúrico al 72% del tubo Sarstedt </li><li> incubar a temperatura ambiente durante 30 minutos, vortex e incubar durante otros 15 minutos </li><li> añadir 825 l de agua y agitar </li><li> centrifugar las muestras a 10.000 rpm durante 5 min. Puede haber un poco de material marrón insoluble, la lignina, que queda en el tubo. </li><li> El contenido de glucosa en el sobrenadante se analizó mediante el ensayo colorimétrico antrona. Este ensayo se realiza en una placa de 96 pocillos de microtitulación de poliestireno. </li><li> Para la curva estándar utiliza un stock de glucosa 1mg/ml (almacenado a 0 ° C) y crear duplicados 0, 2, 4, 6, 8 y 10 ug normas pipeteando 0, 2, 4, 6, 8 y 10 ul en el pozo correspondiente por separado. Llene cada ul y hasta 100 con el agua. </li><li> Añadir 10 l de cada sobrenadante de la muestra y 90 ml de agua en celdas separadas, pero en la placa de microtitulación que el estándar. </li><li> añadir 200 l de reactivo de antrona recién preparada (antrona disuelve en ácido sulfúrico concentrado, con 2 mg de antrona / ml de ácido sulfúrico) </li><li> de calor de placas durante 30 minutos a 80 ° C en un horno (esparcidor de calor de aluminio). Muestras de glucosa que contienen a su vez de amarillo a azul-verde. </li><li> dejar enfriar la placa de la temperatura ambiente y agitar enérgicamente. </li><li> leer la absorción de la placa de 625 mm con un lector de placas microtiter. </li><li> Glucosa (y por lo tanto el contenido de celulosa cristalina) se calcula sobre la base de la absorción en comparación con la curva estándar establecido en la misma placa. </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. La matriz de polysaccharide composición se destaca por un cromatograma de ejemplo la identificación de los azúcares presentes típicos de las paredes celulares vegetales, fucosa, ramnosa, xilosa, arabinosa, galactosa, manosa y glucosa (y el inositol estándar interno). El principal componente de hemicelulosa de álamo es xilano como lo demuestra el contenido de alta xilosa. Sin embargo, la abundancia de estos azúcares pueden variar dependiendo de la materia prima used4. El nivel de glucosa en este análisis se deriva de la xiloglucano hemicelulosa y celulosa amorfa. Debido al análisis de los datos pueden ser presentados como% en moles o ug / mg material de la pared (o el peso en seco). El contenido de celulosa cristalina se explica por sí, se puede esperar que los valores de entre el 20-50% del peso seco de la pared. Con base en los resultados presentados aquí y en I3 parte de la composición lignocelulósica de madera de álamo es de 21% de lignina, hemicelulosa del 30% y el 41% de celulosa cristalina. El resto sería cenizas. <img src="/files/ftp_upload/1837/1837fig1.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. Composición de la matriz de polisacáridos se establece después de tratar el material de la pared con un ácido débil (2M TFA), derivación de los monosacáridos solubilizada resultante de sus acetatos alditol, y el análisis por GC-MS. El residuo del tratamiento ácido débil se lava con el reactivo Updegraff llamados dejando sólo la celulosa insoluble crystlline atrás. La celulosa se disuelve por el ácido sulfúrico y se cuantifica mediante un ensayo colorimétrico para determinar el contenido de glucosa. Al mismo tiempo, el contenido y la composición de la lignina se puede determinar como se describe en la Parte I 3. <img src="/files/ftp_upload/1837/1837fig2.jpg" alt="Figura 2" /> Figura 2:. Análisis exhaustivo lignocelulósicos de la madera de álamo, Astillas de madera de álamo (Populus tremoloides) fueron sometidos al protocolo descrito. Arriba a la izquierda: la composición de polisacáridos Matrix; Fuc fucosa, ramnosa Rha, Ara arabinosa, xilosa XYL; hombre manosa, galactosa Gal; Glc glucosa, inositol estándar interno.

<ol>
	<li>Albersheim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+analysis+of+sugars+in+plant+cell+wall+polysaccharides+by+gas-liquid+chromatography.">A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography.</a> Carbohydr. Res. 5, 340-340 (1967).</li><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> Annu Rev Plant Biol. 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+I:+Lignin.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part I: Lignin.</a> Journal of Visualized Experiments. , (2010).</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>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,+characterisation+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endotransglycosylases.">Discovery, characterisation and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endotransglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li><li>UPDEGRAF, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEMIMICRO+DETERMINATION+OF+CELLULOSE+IN+BIOLOGICAL+MATERIALS.">SEMIMICRO DETERMINATION OF CELLULOSE IN BIOLOGICAL MATERIALS.</a> Anal. Biochem. 32 (3), 420-420 (1969).</li></ol>

Los métodos descritos permiten una rápida evaluación cuantitativa de la composición de la biomasa de plantas lignocelulósicas. El método permite la determinación de la composición de dichos materiales incluyendo la composición de azúcares de los polisacáridos de la matriz es decir, la hemicelulosa, el contenido de celulosa cristalina. El rendimiento de los distintos métodos de análisis por persona varía. Utilizando los protocolos descritos aquí, 20 muestras se pueden procesar para las composiciones de polisacáridos de la matriz y 30 para el contenido de celulosa cristalina. 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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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Trifluoroacetic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
<tr>
<td>myo-Inositol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5125</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Borohydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>213462</td>
<td>&nbsp;</td>
<tr>
<td>Pyridine</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3348-01</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>320102</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>7890A GC/5975C MSD</td>
<td>&nbsp;</td>
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<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>Retsch Mill</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>TissueLyser II</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>
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comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates

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; 2</sup&gt; Y otros productos tales como materiales biocompuesto<sup&gt; 6</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, y la lignina de polifenoles<sup&gt; 5</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 una contribución importante a la fuerza y ​​la integridad estructural de toda la planta. A pesar de su rigidez necesaria, la pared celular es una entidad muy dinámica, que es metabólicamente activa y juega un papel crucial en numerosas actividades de las células, como crecimiento de las plantas y la diferenciación<sup&gt; 5</sup&gt;. 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 (Figura 1). El método comienza con la preparación de material de desalmidonadas pared celular. La lignocelulosa resultantes se dividen para determinar su composición de monosacáridos de la hemicelulosa y la matriz de otros polysaccharides1, y su contenido de celulosa cristalina<sup&gt; 7</sup&gt;. El protocolo para el análisis de los componentes de la lignina en la biomasa lignocelulósica se discute en la Parte I<sup&gt; 3</sup&gt;.

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

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 los hidratos de carbono de pared derivados.

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

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 ...

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

Unit 1

Plant Cell Structure and Organization

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29.9K 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 los hidratos de carbono de pared derivados.

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

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 ...

Investigación

Biología

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

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

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 characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we 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. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

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 II: purification and culture of human islets) using a modified automated method.

De páncreas humano de aislamiento Islet: Parte II: Purificación y Cultura de los islotes

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

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 I: Lignin

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 transportation1 and other products such as biocomposite materials7. 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 (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. 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 depolymerization 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. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

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

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 the polyphenolic lignin.

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

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 ...