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 pelo Departamento de Energia dos EUA (DOE) dos Grandes Lagos Centro de Pesquisa em Bioenergia (DOE BER Escritório de Ciência DE-FC02-07ER64494) e pelas Ciências Química, Geociências e Biociências Divisão do Escritório de ciências básicas da energia, o Office of Science , Departamento de Energia dos EUA (sem prêmio. DE-FG02-91ER20021).

1. Isolamento da parede celular <ol><li> Moer cerca de 60-70mg de ar ou liofilizados material vegetal com 5,5 milímetros esferas de aço inoxidável em um tubo de 2 ml Sarstedt tampa de rosca usando um iWall, um robô de moagem e distribuição (30 s). Uma alternativa não-robótica procedimento de baixo rendimento utilizando um moinho de bolas (retschmill) é apresentada na Parte II 2. </li><li> Adicionar 1,5 ml de 70% de etanol aquoso para o material do solo dispensado, e vortex completamente. </li><li> Centrifugar a 10.000 rpm por 10 min para sedimentar o resíduo de álcool insolúveis. </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> Centrifugar a 10.000 rpm por 10 min e Aspirar ou decantar o sobrenadante. </li><li> Ressuspender pellet em 500 mL 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 ressuspender o sedimento em 1,5 ml de sódio 0,1 M tampão acetato pH 5,0. </li><li> Cap os 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> Adicionar os seguintes agentes para o pellet: 35 mL de 0,01% de azida de sódio (NaN3), 35 mL de amilase (50 mcg / mL H 2 O, a partir de espécies de Bacillus, Sigma); 17 pululanase mL (17,8 unidades de acidopullulyticus Bacillus; 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> Lave o pellet restantes três vezes pela adição de 1,5 ml de água, vortex, centrifugação, decantação e da água de lavagem. </li><li> Ressuspender pellet em 500 mL de acetona. </li><li> Evaporar o solvente com uma corrente 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. Teor de lignina Este método é baseado em um método relatado por Fukushima e Hatfield 3. <ol><li> Pesar 1-1,5 mg de material preparado da parede celular (ver 1) para balão volumétrico de 2 ml deixando um tubo vazio para um em branco. </li><li> lavar paredes do tubo com 250 mL 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> Delicadamente adicionar 100 ml de solução de brometo de acetila acabado de fazer (25% v / v brometo de acetila em ácido acético glacial) ao longo das paredes do tubo para evitar respingos. </li><li> Cap balão e aquecer a 50 ° C por 2 horas </li><li> Calor por uma hora adicional com vórtex a cada 15 minutos. </li><li> Fresco no gelo à temperatura ambiente. </li><li> Adicionar 400 mL de hidróxido de sódio 2M e 70 mL de recém-preparados 0,5 M cloridrato de hidroxilamina. Vortex frascos volumétricos. </li><li> Encher o balão volumétrico exatamente à 2,0 ml marca com ácido acético glacial, tampa e inverter várias vezes para misturar. </li><li> Pipeta de 200 mL da solução para um UV 96 placa específica bem e lido em um leitor de ELISA em 280nm. </li><li> Determinar a porcentagem de lignina solúvel em brometo de acetila (ABSL%), utilizando um coeficiente adequado (Poplar = 18,21; Gramas = 17,75; Arabidopsis = 15,69) com a seguinte fórmula: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figura 1" /> Multiplicação de ABSL%, com 10 resultados na unidade de parede ug / mg de células Ele ajuda a fazer pelo menos 3 placa lê a média da absorbância (abs) desde partículas pode causar uma ligeira variação nos valores de absorbância. Nota: 0,539 centímetro representa a extensão de caminho, mas dependendo da placa esta pode ter de ser determinado. </li></ol> 3. Composição lignina Este método é adotado a partir de um método recente publicado por Robinson e Mansfield 5. <ol><li> Transferir aproximadamente 2 mg de material de parede celular (ver 1.) Em um tubo de vidro tampado para thioacidolysis parafuso. </li><li> preparar cuidadosamente a 2,5% de boro trifluoreto dietil etherate (BF 3), 10% etanotiol solução (EtSH). Você deve usar um balão cheio de gás nitrogênio para deslocar o volume perdido na garrafa dioxano com nitrogênio. Dioxano é muito perigoso, não tome amostras ou equipamento fora do capô. Volumes necessários para a preparação da solução por amostra: 175 mL dioxano, 20 EtSH mL, 5 mL BF 3. </li><li> Adicionar 200 mL de EtSH, BF 3, solução de dioxano para cada amostra. </li><li> Headspace Purge frasco com gás nitrogênio e tampa imediatamente. </li><li> Aquecer a 100 ° C por 4 horas com suave a cada hora de mistura. </li><li> Reação final de resfriamento em gelo por 5 minutos. </li><li> Adicionar 150 mL de bicarbonato de sódio 0.4M, vortex </li><li> Para o clean-up adicionar 1 ml de água e 0,5 ml de acetato de etila vórtice, e deixe fases distintas (acetato de etila na água superior, na parte inferior). </li><li> Transferência de 150 mL da camada de acetato de etila em um tubo de 2 ml Sarstedt. Certifique-se de falta de água é transferida. </li><li> Evaporar o solvente por um concentrador de ar. </li><li> Adicionar 200 mL de acetona e evaporar (repetir para um total de duas vezes remover o excesso de água). </li><li> Para a derivatização TMS adicionar 500 mL de acetato de etila, 20 l de piridina, e 100 l de N, O-bis (trimetilsilil) acetamida a cada tubo. </li><li> incubar por 2 horas a 25 ° C. </li><li> Transferência de 100 ul da reação em um frasco de GC / MS e adicione 100 ml de acetona. </li><li> Analisar as amostras por GC equipado com um espectrômetro de massa quadrupolar ou detector de ionização de chama. Uma coluna HP-Agilent 5MS está instalado (30 mm X 0,25 mm X 0,25 espessura de filme). O gradiente de temperatura a seguir é usado com um atraso de 30 min solvente e um caudal de 1,1 ml / min: segure inicial a 130 ° C por 3 min, uma rampa de 3 ° C / min até a 250 ° C e segure por 1 min; permitir equilíbrio com a temperatura inicial de 130 ° C. </li><li> Picos são identificados por tempos de retenção relativos usando tetracosano padrão interno (opcional) ou por íons de massa característica do espectro de 299 m / z, 269 m / z, e 239 monômeros m / z de S, G e H, respectivamente (ver fig. 2). A composição dos componentes da lignina é quantificado definindo a área do pico total para 100% </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. Um exemplo de cromatograma da separação de lignina-componentes após thioacidolysis e TMS derivatização é mostrado. Claramente, a abundância relativa de siringil (S), guaiacil (G), e p-hidroxifenol (H) unidades pode ser determinada. O conteúdo de lignina solúvel em brometo de acetila é auto-explicativo, pode-se esperar valores entre 20-50% do peso seco da parede. Deve-se notar que o brometo de acetila não solubilizar todos os presentes de lignina na parede, e que o grau de solubilização pode variar dependendo do material. No entanto, este método é relativamente fácil de realizar e rápida e dá uma excelente aproximação do teor de lignina em um material lignocelulósico. <img src="/files/ftp_upload/1745/1745fig1.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. Material da parede é tratado com brometo de acetila ea lignina solubilizada quantificados por espectroscopia de UV. Para a determinação da composição de lignina, material de parede é submetido a thioacidolysis. Os compostos fenólicos solubilizados sofrer derivatização TMS e pode então ser separados e quantificados por GC-MS análise. Da composição do polissacarídeo da matriz e protocolo de conteúdo cristalino da celulose é discutido na Parte II 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figura 2" /> Figura 2:. Fichas análise abrangente lignocelulósicos de madeira de álamo madeira de álamo (Populus tremoloides), foram submetidos aos protocolos descritos. Ligin composição; H p-hidroxifenil; G guaiacil; unidades S siringil.

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

Os métodos descritos permitem uma rápida avaliação quantitativa do teor de lignina e composição da biomassa vegetal lignocelulósica. Usando o robô iWall cerca de 350 amostras podem ser moídos e dispensados ​​por dia. O rendimento dos vários métodos analíticos por pessoa varia. Utilizando os protocolos descritos aqui, 30 amostras podem ser processadas para teor de lignina, e 15 para a composição de lignina por dia. 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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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hydroxylamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>255580</td>
<td>&nbsp;</td>
<tr>
<td>Acetyl Bromide</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>135968</td>
<td>&nbsp;</td>
<tr>
<td>Ethanethiol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E3708</td>
<td>&nbsp;</td>
<tr>
<td>Borontrifluoride diethyl etherate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15719</td>
<td>&nbsp;</td>
<tr>
<td>N,O,-Bis(trimethylsilyl) acetimide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15241</td>
<td>&nbsp;</td>
<tr>
<td>Dioxane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>296309</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6890 GC/5975B MSD</td>
<td>(lignin composition)</td>
</tr>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

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; 1</sup&gt; E outros produtos, como materiais biocomposite<sup&gt; 7</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 diversas (polissacarídeos da matriz, a lignina e polifenol<sup&gt; 6</sup&gt; E, assim, às vezes chamado de lignocelulose. No entanto, as paredes celulares das plantas evoluíram para ser recalcitrantes à degradação como paredes oferecem resistência à tração para as células e as plantas inteiras, afastar os patógenos, e permitir que a água para ser transportado em toda a planta, no caso de árvores de até mais de 100 m acima nível do solo. 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 uma média a high-throughput análise. Nesta primeira parte vamos nos concentrar na análise da lignina de polifenóis (Figura 1). O método começa com a preparação do material da parede celular destarched. A lignocelulose resultantes são, então, dividir para determinar o seu teor de lignina por solubilização acetylbromide<sup&gt; 3</sup&gt;, E da composição de lignina sua em termos de sua siringil, unidades guaiacil e p-hidroxifenil<sup&gt; 5</sup&gt;. O protocolo para analisar os hidratos de carbono na biomassa lignocelulósica incluindo conteúdo de celulose e composição do polissacarídeo matriz é discutido na Parte II<sup&gt; 2</sup&gt;.

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

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

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 da lignina polifenólicos.

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

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

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

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K 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 da lignina polifenólicos.

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

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

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

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

Isolamento Islet humana Pancreatic: Parte I: Digestão e Coleta de tecido pancreático

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

Biologia

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 ...

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

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 II: Carbohydrates

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

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

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

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte II: Os hidratos de carbono

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

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

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

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

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 ...