Somos gratos a Matthew Robert Weatherhead para excelente serviço técnico e John Ralph, da Universidade de Wisconsin para conselhos valiosos, discussões e amostra de madeira de álamo. Este trabalho foi financiado pelas Ciências Química, Geociências e Biociências Divisão do Escritório de ciências básicas da energia, Secretaria de Ciência, Departamento de Energia dos EUA (prêmio não. DE-FG02-91ER20021) e pelo Departamento de Energia dos EUA (DOE) dos Grandes Lagos Bioenergia Research Center (DOE BER Escritório de Ciência DE-FC02-07ER64494).

1. Isolamento da parede celular <ol><li> moer cerca de 60-70mg de ar ou congelar material vegetal seco com 5,5 milímetros esferas de aço inoxidável em um tubo de tampa de 2ml Sarstedt parafuso usando um retschmill (1 min, 25 Hz). Uma alternativa, o uso de uma moagem de alto rendimento e distribuição iWall robô chamado é descrito na Parte I 3. </li><li> remover as bolas de aço antes de continuar com o procedimento de isolamento da parede celular O protocolo detalhado da preparação do material da parede celular é apresentado na parte I 3. Para completar os passos aqui escritos do protocolo. </li><li> adicionar 1,5 ml de 70% de etanol aquoso, e completamente vortex </li><li> centrifugação a 10.000 rpm por 10 min para o resíduo de álcool pellet insolúvel </li><li> Aspirar ou decantar o sobrenadante </li><li> adicionar 1,5 ml de solução de clorofórmio / metanol (1:1 v / v) ao resíduo do tubo e agitar cuidadosamente para ressuspender o sedimento </li><li> centrifugação a 10.000 rpm por 10 min e Aspirar ou decantar o sobrenadante </li><li> ressuspender pellet em 500 ul de acetona </li><li> evaporar o solvente com um fluxo de ar a 35 ° C até secar Se necessário amostras secas podem ser armazenadas em temperatura ambiente até ulterior processamento. </li><li> Para iniciar a remoção do amido da amostra re-suspender o sedimento em 1,5 ml de 0,1 M sodiumacetate tampão pH 5.0. </li><li> tampa dos tubos Sarstedt e calor durante 20 min. a 80 ° C em um bloco de aquecimento. </li><li> legal da suspensão sobre o gelo </li><li> adicione os seguintes agentes para o pellet: 35 mL de Azida de sódio 0,01% (NaN 3), 35 mL de amilase (50 μg/1mL H 2 O, a partir de espécies de Bacillus, SIGMA); 17 pululanase mL (18,7 unidades de acidopullulyticus bacilo; SIGMA) . Tampe o tubo e vortex completamente. </li><li> A suspensão é incubado durante a noite a 37 ° C no shaker. Orientar os tubos na horizontal assessores melhorou mistura. </li><li> suspensão de calor a 100 ° C por 10 min em um bloco de aquecimento para terminar a digestão. </li><li> centrifugação (10.000 rpm, 10 min) e descarte de amido contendo sobrenadante solubilizados </li><li> lavar o pellet restantes três vezes pela adição de 1,5 ml de água, vórtex, centriguation, e decantação da água de lavagem. </li><li> ressuspender pellet em 500 ul de acetona </li><li> evaporar o solvente com um fluxo de ar a 35 ° C até secar. Pode ser necessário também para quebrar o material no tubo com uma espátula para secar melhor. O material seco apresenta parede celular isolada (lignocelulose). Se necessário amostras secas podem ser armazenadas em temperatura ambiente até ulterior processamento. </li></ol> 2. Composição da matriz de polissacarídeo Este método é essencialmente uma modificação do método publicado pela Albersheim 1. <ol><li> Para determinar a composição monossacarídica do material da parede pesam 2 mg do material da parede celular em tubos de 2ml starstedt à mão ou em uma forma highthroughput usando o iWall, um robô robótico de moagem e pesagem. </li><li> Adicionar 20 uL de uma solução de Inositol (5mg/ml) como padrão interno. Para uma amostra de 2 mg de parede celular recomenda-se adicionar 100 ug. </li><li> lavar paredes do tubo com 250 ul de acetona para recolher o material da parede celular na parte inferior do tubo, e evaporar a acetona muito gentil com fluxo de ar. </li><li> Para a hidrólise ácida fraca adicionar 250 ul de ácido trifluoroacético 2M (TFA) para cada amostra. Adicionar TFA cuidadosamente para garantir que nenhum material é espirrada para cima as paredes do tubo. </li><li> tampa bem e incubar por 90 min a 121 ° C em um bloco de aquecimento. </li><li> arrefecer os blocos de aquecimento e amostras no gelo. </li><li> centrifugar os tubos a 10.000 rpm por 10 min. </li><li> transferir 100 ul do sobrenadante ácido contendo o polissacarídeo derivada da matriz de monossacarídeo a um vidro frascos com tampa de rosca tomando cuidado para não perturbar o material da pelota. O pellet pode ser usado para o ensaio de celulose cristalina abaixo (ver 3). </li><li> evaporar a TFA no tubo de vidro sob um fluxo suave de ar em um dispositivo de evaporação. </li><li> adicionar 300 ml 2-Propanol, vortex e evaporar a 25 ° C (repetir para um total de três vezes) </li><li> O primeiro passo do procedimento alditol acetato de derivatização é executar uma redução da monossacarídeos aos seus alditols correspondente. Para este efeito, adicionar 200 mL de uma solução de borohidreto de sódio para cada amostra seca. Prepare uma nova solução de cada vez usando 10 mg de borohidreto de sódio por 1 ml de hidróxido de amônio 1M. </li><li> deixar frasco de vidro em temperatura ambiente por 1,5 horas </li><li> neutralizar a solução, adicionando 150 ul de ácido acético glacial </li><li> vortex e evaporar a 25 ° C. </li><li> adicionar 250 mL de ácido vortex / metanol (1:9, v / v), acético e evaporar a 25 ˚ C </li><li> adicionar 250 mL de metanol e evaporar sob corrente de ar (repetir para um total de três vezes) </li><li> Para a acetilação do alditols, adicione 50 ul de anidrido acético e 50 ul de Pyridine vortex e incubar por 20 min a 121 ° C em um bloco de aquecimento. </li><li> amostras fresco no bloco baixo com gelo, enquanto espera para diminuir a temperatura para cerca de temperatura ambiente. </li><li> evaporar os reagentes sob uma ligeira corrente de ar à temperatura ambiente. Tenha cuidado: acetatos alditol são altamente voláteis. </li><li> adicionar 200 mL de tolueno, e evaporar sob ar (x3) </li><li> Nas etapas finais do acetatos alditol são extraídos. Primeiro, adicionar 500 ul de acetato de etila e agite levemente. </li><li> adicionar 2 ml de água, tubos de tampa e vortex. </li><li> tubos de centrífuga a 2.000 rpm por 5 min para obter claras camadas separadas (acetato de etila em cima, água em baixo) </li><li> pipeta 50 ul da camada de acetato de etila em GC / MS frascos com inserções. </li><li> diluir adicionando 100 ul de acetona para a GC frasco e tampa. O volume de amostra e os volumes de diluição pode ser ajustado para evitar sobrecarregar o GC / MS, se a concentração da amostra é demasiado elevado. O GC frasco pode ser armazenado a 4 ° C, se a análise GC / MS não se proceder imediatamente </li><li> As amostras são injetadas em um GC que está equipado com um MS quadrupolo, mas um detector de ionização de chama também é adequado. A Supelco SP-2380 (30mm X 0,25 milímetros x 0,25 mM filme espessura) coluna é usada com um atraso 4min solvente e um fluxo de 1.5ml/min. Amostras injetadas são submetidos ao programa de temperatura seguinte: segure inicial a 160 ° C por 2 min, uma rampa de 20 ° C / min a 200 ° C e mantenha por 5 min, uma rampa de 20 ° C / min a 245 ° C e manter 12 min; pico a 270 ° C e mantenha por 5 min antes de resfriamento para a temperatura inicial de 160 ° C 2.26) Peaks são identificados por perfis de massa e / ou tempos de retenção dos padrões.. Monossacarídeos são quantificados com base em curvas padrão. </li></ol> 3. Conteúdo de celulose cristalina Este método é essencialmente descrito por Updegraf 8. Há uma série de materiais de partida para este procedimento: material de paredes celulares isoladas (ver 1) ou na parede de material que já foi tratado com TFA 2M (ver 2.8) ou o pellet restante imediatamente após o tratamento ácido (ver 2.8) ou um TFA pellet, que foi lavado com 2-propanol e secas. <ol><li> Adicionar ao pellet TFA no vidro tampado parafuso tubo 1 ml de reagente Updegraff (ácido acético: ácido nítrico: água, 08:01:02 v / v). </li><li> Tubo de tampa bem vórtice, e calor em um bloco de aquecimento a 100 ° C por 30 min. Como resultado deste tratamento apenas celulose cristalina permanece insolúvel, no pellet. </li><li> Amostras fresco no bloco com gelo para temperatura ambiente ou mais frios </li><li> amostras centrifugue a 10.000 rpm por 15 min </li><li> Elimine o sobrenadante garantir que o pellet não é perturbado e nenhum material do pellet é removido. Para este efeito, deixe aprox. 150 ul do sobrenadante do tubo. </li><li> adicionar 1,5 ml de água, agitar, centrifugar e descartar o sobrenadante como feito acima </li><li> repetir a lavagem procedimento 3 vezes mais usando 1,5 ml de acetona </li><li> Ar seco pellet muito suavemente com o ar, ou deixe secar no banco durante a noite </li><li> o pellet (celulose cristalina) está agora completamente hidrolisada em glicose pelo que é chamado de hidrólise Saeman. Para este efeito, adicionar 175 mL de ácido sulfúrico 72% para o tubo Sarstedt </li><li> incubar à temperatura ambiente por 30 min vortex e incubar por mais 15 min </li><li> adicionar 825 mL de água e vortex </li><li> centrifugar amostras a 10.000 rpm por 5 min. Pode haver algum material marrom insolúvel, lignina, permanecendo no tubo. </li><li> O teor de glicose do sobrenadante é analisada utilizando o ensaio colorimétrico antrona. Este ensaio é realizado em uma placa de microtitulação de 96 poços de poliestireno. </li><li> Para a curva padrão de usar um estoque de glicose 1mg/ml (armazenado a 0 ° C) e criar duplicado 0, 2, 4, 6, 8, e 10 ug padrões pipetando 0, 2, 4, 6, 8, e 10 ul em bem apropriado em separado. Preencher cada ul bem até 100 com água. </li><li> adicionar 10 ml de cada sobrenadante de amostra e 90 ml de água em celas separadas, mas na placa de microtitulação, mesmo que o padrão. </li><li> adicionar 200 mL de Reagente antrona preparada na hora (antrona dissolvido em ácido sulfúrico concentrado, 2 mg antrona / ml de ácido sulfúrico) </li><li> de calor de placas por 30 min a 80 ° C em um forno (dissipador de calor de alumínio). Amostras contendo glicose passar de amarelo para azul-esverdeada. </li><li> deixe a placa fria para a temperatura ambiente e agitar vigorosamente. </li><li> ler absorção de placa a 625 mm usando um leitor de placas de microtitulação. </li><li> Glicose (e, portanto, conteúdo de celulose cristalina) é calculado com base na absorbância em comparação com a curva padrão estabelecido no mesmo prato. </li></ol> 4. Resultados representante Um exemplo de uma análise de parede é apresentado na Figura 2. Neste caso, álamo-tronco (madeira) foi analisada por vários procedimentos descritos na seção de protocolo. A matriz pocomposição lysaccharide é destacado por um cromatograma exemplo identificando o açúcar presente típico na parede celular das plantas, fucose, ramnose, xilose, arabinose, galactose, manose e glicose (eo inositol padrão interno). O principal componente hemicelulósico de álamo é xilana como demonstrado pelo conteúdo de xilose de alta. No entanto, a abundância destes açúcares pode variar dependendo do used4 matéria-prima. A glicose nesta análise é derivado do xiloglucano hemicelulose e celulose amorfa. Devido à análise dos dados pode ser apresentado como mol% ou ug / mg material da parede (ou peso seco). O conteúdo de celulose cristalina é auto-explicativo, pode-se esperar valores entre 20-50% do peso seco da parede. Com base nos resultados aqui apresentados e em I3 parte da composição lignocelulósica de álamo madeira é 21% de lignina, hemicelulose 30%, e 41% de celulose cristalina. O restante seria cinzas. <img src="/files/ftp_upload/1837/1837fig1.jpg" alt="Figura 1" /> Figura 1:. Muros Visão geral de análise lignocelulósicos Cell (lignocelulose) são isolados a partir de material vegetal bruto seco. O material da parede é então ponderado em alíquotas e subdividida para os ensaios diversos. Composição da matriz de polissacarídeo é estabelecido após o tratamento do material da parede por um ácido fraco (TFA 2M), derivatizing os monossacarídeos resultantes solubilizados a seus acetatos alditol, e análise por GC-MS. O resíduo do tratamento ácido fraco é lavado com o reagente Updegraff chamados deixando apenas de celulose insolúvel crystlline trás. A celulose é solubilizado pelo ácido sulfúrico e quantificados por um teste colorimétrico determinar o teor de glicose. Em paralelo, o conteúdo ea composição da lignina pode ser determinado conforme descrito na Parte I 3. <img src="/files/ftp_upload/1837/1837fig2.jpg" alt="Figura 2" /> Figura 2:. Fichas análise abrangente lignocelulósicos de madeira de álamo madeira de álamo (Populus tremoloides) foram submetidos ao protocolo descrito. Superior esquerdo: composição do polissacarídeo Matrix; Fuc fucose, ramnose Rha; Ara arabinose; XYL xilose; Man manose; Gal galactose; glicose Glc; padrão interno inositol.

<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>

Os métodos descritos permitem uma rápida avaliação quantitativa da composição da biomassa vegetal lignocelulósica. O método permite a determinação da composição de tais materiais, incluindo a composição de açúcar da matriz polissacarídeos ou seja, a hemicelulose, o conteúdo de celulose cristalina. O rendimento dos vários métodos analíticos por pessoa varia. Utilizando os protocolos descritos aqui, 20 amostras podem ser processadas para composições de matriz de polissacarídeo e 30 para o índice de celulose cristalina. Devido à natureza quantitativa das culturas de matérias-primas de dados ideal, variedade ou genótipos pode ser avaliada em termos de sua aptidão para a produção de biocombustível.

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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>
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<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>
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comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates

A necessidade de renováveis, neutra de carbono, e matérias-primas sustentáveis ​​para a indústria e sociedade tornou-se uma das questões mais prementes para o século 21. Este reacendeu o interesse no uso de produtos vegetais como matérias-primas industriais para a produção de combustíveis líquidos para transporte<sup&gt; 2</sup&gt; E outros produtos, como materiais biocomposite<sup&gt; 6</sup&gt;. Biomassa vegetal continua sendo uma das maiores reservas inexploradas do planeta<sup&gt; 4</sup&gt;. É principalmente composta de paredes celulares, que são compostos de polímeros ricos em energia, incluindo a celulose, hemicelulose vários, ea lignina polifenol<sup&gt; 5</sup&gt; E, assim, às vezes chamado de lignocelulose. No entanto, as paredes celulares das plantas evoluíram para ser recalcitrantes à degradação como paredes contribuir amplamente para a força e integridade estrutural de toda a planta. Apesar de sua rigidez necessária, a parede celular é uma entidade altamente dinâmica, que é metabolicamente ativa e desempenha um papel crucial em atividades celulares numerosos tais como o crescimento da planta e diferenciação<sup&gt; 5</sup&gt;. Devido às várias funções de paredes, há uma imensa diversidade estrutural entre as paredes de diferentes espécies de plantas e tipos de células dentro de uma única planta<sup&gt; 4</sup&gt;. Assim, dependendo do que as espécies cultivadas, a variedade de culturas, ou tecido de planta é usada para uma biorefinaria, as etapas de processamento para a despolimerização de produtos químicos / enzimática processos e posterior fermentação dos açúcares diferentes para biocombustíveis líquidos precisam ser ajustados e otimizados. Este fato reforça a necessidade de uma caracterização completa de matérias-primas de biomassa vegetal. Aqui nós descrevemos uma metodologia abrangente de análise que permite a determinação da composição da lignocelulose e é passível de um meio de análise de alto rendimento (Figura 1). O método começa com a preparação do material da parede celular destarched. A lignocelulose resultantes são, então, dividir para determinar sua composição monossacarídica das hemiceluloses e outra matriz polysaccharides1, e seu conteúdo de celulose cristalina<sup&gt; 7</sup&gt;. O protocolo para a análise dos componentes de lignina na biomassa lignocelulósica é discutido na Parte I<sup&gt; 3</sup&gt;.

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

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

Biomassa vegetal é um recurso de carbono neutro principais renováveis, que poderiam ser usados ​​para a produção de biocombustíveis. Biomassa vegetal consiste principalmente das paredes das células, um material estruturalmente complexo composto chamado lignocelulose. Aqui nós descrevemos um protocolo para uma análise abrangente do conteúdo e composição dos carboidratos da parede derivadas.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte II: Os 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 visualizações.  Michigan State University (MSU). Biomassa vegetal é um recurso de carbono neutro principais renováveis, que poderiam ser usados ​​para a produção de biocombustíveis. Biomassa vegetal consiste principalmente das paredes das células, um material estruturalmente complexo composto chamado lignocelulose. Aqui nós descrevemos um protocolo para uma análise abrangente do conteúdo e composição dos carboidratos da parede derivadas.

Vídeo: 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.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: Parte 1

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

Biologia

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.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: 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.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: 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.

Isolamento Islet humana Pancreatic: Parte II: Purificação e Cultura de Ilhotas Humanos

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.

Separação de células de fluorescência Activated de Protoplastos Usina

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.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte I: A 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.

Label-free In situ Imaging de lignificação na parede celular das plantas

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.

Profiling glicana de Polímeros da planta da parede celular usando 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.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

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.

Um método eficiente para Quantitativa, Análise de células Único de cromatina Modificação e Arquitetura Nuclear em Todo-mount óvulos em<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 ...