Nous sommes reconnaissants à Matthieu Robert Weatherhead pour un excellent service technique et John Ralph, Université du Wisconsin pour de précieux conseils, discussions, et l&#39;échantillon de bois de peuplier. Ce travail a été financé par le US Department of Energy (DOE) des Grands Lacs Bioenergy Research Center (DOE BER Bureau de la science-DE-FC02 07ER64494) et par les sciences chimiques, sciences de la terre et les sciences biologiques, Bureau des sciences fondamentales de l&#39;énergie, Bureau des sciences , US Department of Energy (pas de récompense. DE-FG02-91ER20021).

1. Paroi cellulaire d&#39;isolement <ol><li> Moudre grossièrement 60-70mg d&#39;air ou lyophilisés matériel végétal de 5,5 mm en acier inoxydable boules dans un tube de 2 ml Sarstedt bouchon à vis en utilisant une iWall, un robot de broyage et de distribution (30 s). Une alternative non-robotique faible débit procédure en utilisant un broyeur à billes (retschmill) est présenté dans la Partie II 2. </li><li> Ajouter 1,5 ml d&#39;éthanol aqueux à 70% de la matière broyée dispensés, et le vortex soigneusement. </li><li> Centrifuger à 10000 rpm pendant 10 min pour sédimenter le résidu insoluble dans l&#39;alcool. </li><li> Aspirer ou décanter le surnageant. </li><li> Ajouter 1,5 ml de solution de chloroforme / méthanol (1:1 v / v) au résidu et secouer le tube soigneusement de remettre en suspension le culot. </li><li> Centrifuger à 10000 rpm pendant 10 min et aspirer ou décanter le surnageant. </li><li> Resuspendre le culot dans 500 ul d&#39;acétone. </li><li> Evaporer le solvant avec un courant d&#39;air à 35 ° C jusqu&#39;à sec. Si nécessaire échantillons séchés peuvent être stockés à température ambiante jusqu&#39;au traitement ultérieur. </li><li> Pour lancer la suppression de l&#39;amidon de l&#39;échantillon Reprendre le culot dans 1,5 ml d&#39;un 0,1 M d&#39;acétate de sodium pH 5,0 tampon. </li><li> Boucher les tubes Sarstedt et de la chaleur pendant 20 min. à 80 ° C dans un bloc de chauffage. </li><li> Refroidir la suspension sur la glace. </li><li> Ajouter les agents suivants au culot: 35 pl de 0,01% d&#39;azide de sodium (NaN3), 35 ul d&#39;amylase (50 pg / mL H 2 O; à partir d&#39;espèces de Bacillus, Sigma), 17 pullulanase ul (17,8 unités provenant du Bacillus acidopullulyticus; Sigma) . Boucher le tube et le vortex soigneusement. </li><li> La suspension est incubée pendant la nuit à 37 ° C dans le shaker. Orienter les tubes horizontalement aides meilleur mélange. </li><li> Suspension de la chaleur à 100 ° C pendant 10 min dans un bloc de chauffage à mettre fin à la digestion. </li><li> Centrifugeuse (10.000 rpm, 10 min) et éliminer le surnageant contenant de l&#39;amidon solubilisé. </li><li> Laver le culot restant à trois reprises en ajoutant 1,5 ml d&#39;eau, vortex, centrifugation et décantation des eaux de lavage. </li><li> Resuspendre le culot dans 500 ul d&#39;acétone. </li><li> Evaporer le solvant avec un courant d&#39;air à 35 ° C jusqu&#39;à sec. Il peut être nécessaire aussi de briser la matière dans le tube avec une spatule pour un meilleur séchage. La matière sèche présente paroi cellulaire isolée (lignocellulosiques). Si nécessaire échantillons séchés peuvent être stockés à température ambiante jusqu&#39;au traitement ultérieur. </li></ol> 2. La teneur en lignine Cette méthode est basée sur une méthode décrite par Fukushima et Hatfield 3. <ol><li> Peser 1 à 1,5 mg de matériau de paroi cellulaire préparé (voir 1) en 2 ml fiole jaugée laissant un tube vide pour une vierge. </li><li> rincez parois du tube avec 250 pi d&#39;acétone pour recueillir le matériau du mur de cellules sur le fond du tube et évaporer l&#39;acétone très doux sous flux d&#39;air. </li><li> Ajouter délicatement 100 ul de solution de bromure d&#39;acétyle fraîchement préparés (25% v / v bromure d&#39;acétyle de l&#39;acide acétique glacial) le long des parois du tube pour éviter les éclaboussures. </li><li> Cap fiole jaugée et de la chaleur à 50 ° C pendant 2 heures </li><li> Chauffer pendant une heure supplémentaire avec les vortex toutes les 15 minutes. </li><li> Laisser refroidir sur la glace à température ambiante. </li><li> Ajouter 400 ul d&#39;hydroxyde de sodium 2M et 70 pi de chlorhydrate de 0,5 M d&#39;hydroxylamine fraîchement préparés. Vortex fioles jaugées. </li><li> Remplissez fiole jaugée exactement à la marque 2,0 ml d&#39;acide acétique glacial, capuchon et inverser plusieurs fois pour mélanger. </li><li> Pipeter 200 pl de la solution dans un UV spécifiques plaque de 96 puits et de lire dans un lecteur ELISA à 280 nm. </li><li> Déterminer le pourcentage de lignine soluble dans le bromure d&#39;acétyle (ABSL%) à l&#39;aide d&#39;un coefficient approprié (peuplier = 18,21; Graminées = 17,75; Arabidopsis = 15,69) avec la formule suivante: ABSL% Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figure 1" /> Multiplication des ABSL% avec 10 résultats dans l&#39;unité ug / mg de la paroi cellulaire Il contribue à faire au moins 3 plaque est en moyenne de l&#39;absorbance (ABS) depuis les particules peuvent causer une légère variation dans les valeurs d&#39;absorbance. Note: 0.539 cm représente la longueur de chemin, mais en fonction de la plaque présente peut-être besoin d&#39;être déterminée. </li></ol> 3. La lignine Composition Cette méthode est adoptée par une méthode récemment publiée par Robinson et Mansfield 5. <ol><li> Transférer environ 2 mg de matériau de paroi cellulaire (voir 1.) Dans un tube en verre à vis plafonnés pour thioacidolysis. </li><li> préparer soigneusement les 2,5% de trifluorure de bore éthérate diéthylique (BF 3), 10% éthanethiol (EtSH) solution. Vous devez utiliser un ballon rempli d&#39;azote gazeux pour remplacer le volume perdu dans la bouteille dioxane à l&#39;azote. Dioxane est très dangereuse, ne pas prendre des échantillons ou des équipements hors de la hotte. Les volumes nécessaires à la préparation de la solution par échantillon: 175 ul dioxane; 20 EtSH ul; 5 pl BF 3. </li><li> Ajouter 200 pl de EtSH, BF 3, solution de dioxane à chaque échantillon. </li><li> Headspace flacon purge à l&#39;azote et le bouchon immédiatement. </li><li> Chauffer à 100 ° C pendant 4 heures avec mélange en douceur à chaque heure. </li><li> Fin de réaction par refroidissement sur de la glace pendant 5 minutes. </li><li> Ajouter 150 ul de bicarbonate de sodium 0,4 M, vortex </li><li> Pour le nettoyage, ajouter 1 ml d&#39;eau et 0,5 ml d&#39;acétate, vortex éthylique et laissez phases distinctes (acétate d&#39;éthyle sur le dessus, de l&#39;eau sur le fond). </li><li> Transfert 150 pi de la couche d&#39;acétate d&#39;éthyle dans un tube de 2 ml Sarstedt. Assurez-vous que l&#39;eau n&#39;est pas transféré. </li><li> Évaporer le solvant par un concentrateur d&#39;air. </li><li> Ajouter 200 ul d&#39;acétone et évaporer (répéter pour un total de deux fois enlever l&#39;excès d&#39;eau). </li><li> Pour la dérivation TMS ajouter 500 ul d&#39;acétate d&#39;éthyle, 20 pl de la pyridine, et 100 pi de N, O-bis (triméthylsilyl) acétamide dans chaque tube. </li><li> incuber pendant 2 heures à 25 ° C. </li><li> Transférer 100 ul de la réaction dans un flacon CG / SM et ajouter 100 ul d&#39;acétone. </li><li> Analyser les échantillons par GC équipé d&#39;un spectromètre de masse quadripolaire ou un détecteur à ionisation de flamme. Un Agilent HP-5ms est installé (30 mm X 0,25 mm X 0,25 um l&#39;épaisseur du film). Le gradient de température suivante est utilisée avec un retard de 30 min de solvant et un taux de 1,1 débit ml / min: maintenez initiale à 130 ° C pendant 3 min; une 3 ° C / min à une rampe de 250 ° C et maintenir pendant 1 min; permettent équilibration de la température initiale de 130 ° C. </li><li> Les pics sont identifiés par temps de rétention relatifs à l&#39;aide tétracosane étalon interne (en option) ou par caractéristique des ions spectre de masse de 299 m / z, 269 m / z, et 239 monomères m / z pour S, G et H, respectivement (voir fig. 2). La composition de la lignine est quantifiée par la mise de la zone de crête totale de 100% </li></ol> 4. Les résultats représentatifs Un exemple d&#39;une analyse de mur est présenté dans la Figure 2. Dans ce cas, le peuplier tige (bois) a été analysée par les différentes procédures décrites dans la section du protocole. Un chromatogramme exemple de la séparation de la lignine et de composants après thioacidolysis TMS-dérivatisation est montré. De toute évidence, l&#39;abondance relative des syringyl-(S), guaiacyle-(G), et p-hydroxyphénol-(H) des unités peut être déterminé. Le contenu de la lignine soluble dans le bromure d&#39;acétyle est auto-explicatif, on peut s&#39;attendre à des valeurs comprises entre 20-50% de la muraille du poids sec. Il faut noter que le bromure d&#39;acétyle ne solubilise pas l&#39;ensemble des présents de lignine dans le mur, et que le degré de solubilisation peut varier en fonction du matériau. Cependant, cette méthode est relativement facile à réaliser et rapide et donne une excellente approximation de la teneur en lignine dans une matière lignocellulosique. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Figure 1" /> Figure 1:. Parois cellulaires Aperçu de l&#39;analyse lignocellulosique (lignocellulosiques) sont isolées de matières végétales brutes séchées. Le matériau du mur est alors pondérée en aliquots et subdivisé pour les dosages différents. Matériau du mur est traitée avec le bromure d&#39;acétyle et de la lignine solubilisée quantifiés par spectroscopie UV-. Pour la détermination de la composition de la lignine, matière du mur est soumis à thioacidolysis. Les composés phénoliques solubilisées subissent dérivation TMS et peuvent ensuite être séparés et quantifiés par analyse GC-MS. La composition de la matrice et polysaccharides cristallins protocole de teneur en cellulose est discuté dans la Partie II 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figure 2" /> Figure 2:. Copeaux de bois complète l&#39;analyse lignocellulosiques à partir du bois de peuplier (Populus tremoloides) ont été soumis à des protocoles décrits. Ligin composition; H p-hydroxyphényl; G guaiacyle; S unités syringyl.

<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>

Les méthodes décrites permettent une évaluation quantitative rapide de l&#39;teneur en lignine et de la composition de la biomasse végétale lignocellulosique. En utilisant le robot iWall environ 350 échantillons peuvent être broyées et distribués par jour. Le débit des différentes méthodes analytiques par personne varie. En utilisant les protocoles décrits ici, 30 échantillons peuvent être traités pour le contenu en lignine, et 15 pour la composition de la lignine par jour. En raison de la nature quantitative des récoltes de données optimale les matières premières, de la variété ou des génotypes peut être évaluée en fonction de leur aptitude à la production de biocarburants.

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

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

La nécessité pour les énergies renouvelables, neutres en carbone, et durable des matières premières pour l&#39;industrie et la société est devenue l&#39;une des questions les plus pressantes pour le 21e siècle. Cela a ravivé l&#39;intérêt dans l&#39;utilisation des produits végétaux comme matières premières industrielles pour la production de carburants liquides pour les transports<sup&gt; 1</sup&gt; Et d&#39;autres produits comme les matériaux biocomposites<sup&gt; 7</sup&gt;. La biomasse végétale reste une des plus grandes réserves inexploitées sur la planète<sup&gt; 4</sup&gt;. Il est principalement composé de parois cellulaires qui sont composées de polymères riches en énergie dont la cellulose, les hémicelluloses diverses (polysaccharides matrice, et la lignine polyphénol<sup&gt; 6</sup&gt; Et donc parfois appelés lignocellulosiques. Toutefois, les parois des cellules végétales ont évolué pour être récalcitrants à la dégradation des murs de fournir résistance à la traction pour les cellules et les plantes entières, conjurer les agents pathogènes, et permettre à l&#39;eau pour être transportés à travers l&#39;usine, dans le cas des arbres à plus de 100 m au-dessus niveau du sol. Grâce aux différentes fonctions des murs, il ya une immense diversité structurale dans les murs de différentes espèces végétales et de types de cellules au sein d&#39;une seule plante<sup&gt; 4</sup&gt;. Ainsi, selon ce que les espèces cultivées, la variété des cultures, ou les tissus végétaux est utilisé pour une bioraffinerie, les étapes de traitement de dépolymérisation par des procédés chimiques / enzymatiques et la fermentation subséquente des sucres divers biocarburants liquides doivent être ajustés et optimisés. Ce fait de la nécessité d&#39;une caractérisation complète des matières premières de biomasse végétale sous-tend. Nous décrivons ici une méthodologie globale d&#39;analyse qui permet la détermination de la composition de lignocellulosiques et se prête à une moyenne d&#39;analyse à haut débit. Dans cette première partie nous nous concentrons sur l&#39;analyse de la lignine polyphénols (Figure 1). La méthode commence avec la préparation des destarched matériau de paroi cellulaire. Le lignocellulosiques qui en résultent sont ensuite divisés pour déterminer sa teneur en lignine par solubilisation acetylbromide<sup&gt; 3</sup&gt;, Et sa composition en lignine en fonction de son syringyl, unités guaiacyle-et p-hydroxyphényl<sup&gt; 5</sup&gt;. Le protocole pour l&#39;analyse des hydrates de carbone dans la biomasse lignocellulosique dont la teneur en cellulose et de la composition de polysaccharide matrice est discuté dans la Partie II<sup&gt; 2</sup&gt;.

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

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

La biomasse végétale est une grande neutralité carbone ressource renouvelable qui pourraient être utilisés pour la production de biocarburants. La biomasse végétale est principalement constitué de parois cellulaires, un matériau composite de structure complexe appelée lignocellulosiques. Nous décrivons ici un protocole pour une analyse complète du contenu et de la composition de la lignine polyphénolique.

Partie analyse complète de la composition des parois des cellules végétales (biomasse lignocellulosique) I: La lignine

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 vues.  Michigan State University (MSU). La biomasse végétale est une grande neutralité carbone ressource renouvelable qui pourraient être utilisés pour la production de biocarburants. La biomasse végétale est principalement constitué de parois cellulaires, un matériau composite de structure complexe appelée lignocellulosiques. Nous décrivons ici un protocole pour une analyse complète du contenu et de la composition de la lignine polyphénolique.

Vidéo: 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.

Isolement îlots pancréatiques humaines: Partie I: La digestion et la perception des tissus pancréatiques

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

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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.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 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.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 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.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 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.

Tri cellulaire activé par fluorescence des protoplastes végétaux

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.

Analyse de la composition globale de la paroi cellulaire végétale (biomasse lignocellulosique) Partie II: Glucides

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.

Sans étiquette In situ</emImagerie> de lignification dans les parois des cellules végétales

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.

Profilage Glycan des polymères végétaux de la paroi cellulaire en utilisant 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.

Marquage métabolique et la membrane de fractionnement pour comparative Analyse protéomique de<em&gt; Arabidopsis thaliana</em&gt; Cultures Suspension cellulaires

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.

Une méthode efficace pour quantitative, analyse unicellulaire de la chromatine Modification et architecture nucléaire dans l&#39;ensemble du montage ovules dans<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 ...