Siamo grati a Matthew Robert Weatherhead eccellente per il servizio tecnico e John Ralph, University of Wisconsin per preziosi consigli, discussioni, e il campione di legno di pioppo. Questo lavoro è stato finanziato dal US Department of Energy (DOE) dei Grandi Laghi Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494) e dalle Scienze Chimiche, Scienze geologiche e Bioscienze Divisione, Ufficio di Basic Sciences Energy, Office of Science , US Department of Energy (nessun premio. DE-FG02-91ER20021).

1. Isolamento della parete cellulare <ol><li> Grind circa 60-70mg di aria o liofilizzato materiale vegetale con 5,5 palle mm in acciaio inox in una ml 2 tappo della provetta Sarstedt vite utilizzando un iWall, un robot macinazione e dosaggio (30 s). L&#39;alternativa non-robotica a basso throughput usando una procedura di mulini a palle (retschmill) è presentata nella Parte II, 2. </li><li> Aggiungere 1,5 ml di etanolo al 70% acquosa al materiale terra erogato, e vortice accuratamente. </li><li> Centrifugare a 10.000 rpm per 10 minuti per far sedimentare il residuo insolubile alcool. </li><li> Aspirare o decantare il sopranatante. </li><li> Aggiungere 1,5 ml di cloroformio / metanolo (1:1 v / v) per il residuo del tubo e agitare accuratamente per risospendere il pellet. </li><li> Centrifugare a 10.000 rpm per 10 minuti e aspirare o decantare il sopranatante. </li><li> Risospendere il pellet in 500 ml di acetone. </li><li> Evaporare il solvente con una corrente d&#39;aria a 35 ° C fino a secco. Se necessario campioni essiccati possono essere conservati a temperatura ambiente fino al procedimento successivo. </li><li> Per avviare la rimozione di amido dal campione risospendere il pellet in 1,5 ml di sodio 0,1 M pH 5,0 tampone acetato. </li><li> Cap i tubi Sarstedt e calore per 20 min. a 80 ° C su una piastra riscaldante. </li><li> Raffreddare la sospensione sul ghiaccio. </li><li> Aggiungere i seguenti agenti al pellet: 35 ml di 0,01% di sodio azide (NaN3), 35 amilasi microlitri (50 mg / ml H 2 O, da specie Bacillus, Sigma), 17 pullulanasi microlitri (17,8 unità da acidopullulyticus Bacillus; Sigma) . Chiudere la provetta e miscelare accuratamente. </li><li> La sospensione è incubata per una notte a 37 ° C nello shaker. Orientare i tubi in orizzontale aiutanti migliorato miscelazione. </li><li> Sospensione di calore a 100 ° C per 10 minuti su una piastra riscaldante di interrompere la digestione. </li><li> Centrifuga (10.000 rpm, 10 min) e scartare il surnatante contenente amido solubilizzato. </li><li> Lavare il rimanente a pellet per tre volte con l&#39;aggiunta di 1,5 ml di acqua, vortex, centrifugazione e decantazione delle acque di lavaggio. </li><li> Risospendere il pellet in 500 ml di acetone. </li><li> Evaporare il solvente con una corrente d&#39;aria a 35 ° C fino a secco. Potrebbe essere necessario anche per rompere il materiale nel tubo con una spatola per una migliore essiccazione. Il materiale essiccato presenta parete isolata cella (legnocellulosa). Se necessario campioni essiccati possono essere conservati a temperatura ambiente fino al procedimento successivo. </li></ol> 2. Lignina Contenuto Questo metodo si basa su un metodo riportato da Fukushima e Hatfield 3. <ol><li> Pesare 1-1,5 mg di materiale preparato parete cellulare (vedi 1) in 2 ml matraccio lasciando un tubo vuoto di uno spazio vuoto. </li><li> lavare le pareti del tubo da 250 ml di acetone per raccogliere il materiale della parete cellulare nella parte inferiore del tubo, e far evaporare l&#39;acetone molto delicato sotto il flusso d&#39;aria. </li><li> Delicatamente aggiungere 100 ml di soluzione appena fatto bromuro di acetile (25% v / v bromuro di acetile in acido acetico glaciale) lungo le pareti del tubo per evitare schizzi. </li><li> Cap volumetrico matraccio e riscaldare a 50 ° C per 2 ore </li><li> Calore per un&#39;altra ora con vortex ogni 15 minuti. </li><li> Raffreddare in ghiaccio a temperatura ambiente. </li><li> Aggiungere 400 ml di idrossido di sodio 2M e 70 ml di preparati al momento 0,5 M idrossilammina cloridrato. Vortex matracci tarati. </li><li> Riempire pallone tarato esattamente a 2,0 ml segno con acido acetico glaciale, il tappo e capovolgere più volte per mescolare. </li><li> Pipettare 200 ml di soluzione in un UV specifico 96 pozzetti e leggere in un lettore ELISA a 280 nm. </li><li> Determinare la percentuale di lignina bromuro di acetile solubile (% ABSL) utilizzando un adeguato coefficiente (pioppo = 18,21; Erbe = 17,75; Arabidopsis = 15,69) con la seguente formula: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figura 1" /> Moltiplicazione delle ABSL% con 10 risultati in ug / mg unità della parete cellulare Aiuta a fare almeno 3 piatto legge in media l&#39;assorbanza (abs) dal particolato può causare una lieve variazione nei valori di assorbenza. Nota: 0,539 centimetri rappresenta il cammino ottico, ma a seconda del piatto questo potrebbe aver bisogno di essere determinato. </li></ol> 3. Lignina Composizione Questo metodo è adottato da un metodo di recente pubblicato da Robinson e Mansfield 5. <ol><li> Trasferire circa 2 mg di materiale della parete cellulare (vedi 1.) In una provetta di vetro con tappo a vite per thioacidolysis. </li><li> preparare con cura il 2,5% trifluoruro di boro dietil etherate (BF 3), 10% ethanethiol (EtSH) soluzione. È necessario utilizzare un pallone riempito di gas azoto per spostare il volume perso in bottiglia diossano con azoto. Diossano è molto pericoloso, non prelevare campioni o apparecchiature fuori dal cofano. Volumi necessari per la preparazione della soluzione per campione: 175 microlitri diossano, 20 EtSH microlitri, 5 microlitri BF 3. </li><li> Aggiungere 200 ml di EtSH, BF 3, diossano soluzione ad ogni campione. </li><li> Purge spazio di testa flacone con gas azoto e tappo immediatamente. </li><li> Calore a 100 ° C per 4 ore con dolce mescolando ogni ora. </li><li> Reazione fine di raffreddamento in ghiaccio per 5 minuti. </li><li> Aggiungere 150 ml di bicarbonato di sodio 0.4M, vortice </li><li> Per la pulizia aggiungere 1 ml di acqua e 0,5 ml di acetato di etile, vortex e lasciare separare le fasi (acetato di etile in alto, l&#39;acqua sul fondo). </li><li> Trasferire 150 ml di strato di acetato di etile in una provetta da 2 ml Sarstedt. Assicurarsi che l&#39;acqua non viene trasferito. </li><li> Evaporare solvente da un concentratore d&#39;aria. </li><li> Aggiungere 200 microlitri e far evaporare l&#39;acetone (ripetere per un totale di due volte rimuovere l&#39;acqua in eccesso). </li><li> Per la derivatizzazione TMS aggiungere 500 ml di acetato di etile, 20 ml di piridina, e 100 ml di N, O-bis (trimetilsilil) acetamide ad ogni provetta. </li><li> incubare per 2 ore a 25 ° C. </li><li> Trasferire 100 microlitri della reazione in un GC / MS fiala e aggiungere 100 ml di acetone. </li><li> Analizzare i campioni da GC dotato di un quadrupolo-spettrometro di massa o della ionizzazione di fiamma. Un sistema HP-5MS Agilent colonna è installato (30 mm x 0,25 mm X 0,25 spessore micron). Il gradiente di temperatura seguente viene utilizzato con un ritardo di 30 minuti e un solvente 1,1 ml / min Portata: tenere iniziale a 130 ° C per 3 minuti, un 3 ° C / min fino ad un rampa di 250 ° C e mantenere per 1 minuto; permettono equilibrio alla temperatura iniziale di 130 ° C. </li><li> Picchi sono identificati con tempi di ritenzione relativi con tetracosane standard interno (opzionale) o da ioni caratteristico spettro di massa di 299 m / z, 269 m / z, monomeri e 239 m / z di S, G e H, rispettivamente (vedi fig. 2). La composizione dei componenti lignina è quantificato impostando l&#39;area totale dei picchi al 100% </li></ol> 4. Rappresentante Risultati Un esempio di analisi muro è presentato in figura 2. In questo caso fusto di pioppo (legno) è stato analizzato mediante le varie procedure descritte nella sezione protocollo. Un esempio di cromatogramma la separazione della lignina componenti dopo thioacidolysis e TMS-derivatizzazione è mostrato. Chiaramente, l&#39;abbondanza relativa di syringyl-(S), guaiacyl-(G), e p-hydroxyphenol-(H) unità può essere determinato. Il contenuto di lignina solubile bromuro di acetile si spiega da sé, ci si può aspettare tra i valori del 20-50% del peso a secco della parete. Si noti che il bromuro di acetile non solubilizzare tutti i presenti lignina nella parete, e che il grado di solubilizzazione può variare a seconda del materiale. Tuttavia, questo metodo è relativamente facile da realizzare e rapida e fornisce una eccellente approssimazione del contenuto di lignina in un materiale ligno-cellulosica. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Figura 1" /> Figura 1:. Panoramica di analisi lignocellulosici pareti cellulari (legnocellulosa) sono isolati dal greggio materiale vegetale essiccato. Il materiale della parete viene poi pesata in aliquote e suddiviso per i vari metodi. Materiale della parete è trattato con bromuro di acetile e la lignina solubilizzato quantificato spettroscopia UV. Per la determinazione della composizione lignina, materiale della parete è sottoposto a thioacidolysis. I composti fenolici solubilizzato subire derivatizzazione TMS e possono quindi essere separati e quantificati mediante analisi GC-MS. La composizione polisaccaride matrice e cristallino protocollo contenuto di cellulosa è discusso nella Parte II, 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figura 2" /> Figura 2:. Completa analisi lignocellulosici di legno di pioppo Cippato di pioppo (Populus tremoloides) sono stati sottoposti ai protocolli descritti. Ligin composizione; H p-idrossifenil; G guaiacyl; S unità 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>

I metodi descritti permettono una rapida valutazione quantitativa del contenuto di lignina e la composizione della biomassa vegetale lignocellulosiche. Utilizzando il robot iWall circa 350 campioni di terreno e può essere erogato al giorno. La velocità dei vari metodi analitici per persona varia. Utilizzando i protocolli descritti qui, a 30 campioni possono essere trattati per contenuto di lignina, e 15 per la composizione della lignina al giorno. A causa della natura quantitativa della materia prima ottimale dei dati raccolti, varietà o genotipi può essere valutata in termini di idoneità per la produzione di biocarburanti.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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 necessità di fonti rinnovabili, carbon neutral, e sostenibile di materie prime per l&#39;industria e la società è diventata una delle questioni più pressanti per il 21 ° secolo. Questo ha riacceso interesse per l&#39;uso di prodotti vegetali come materie prime industriali per la produzione di combustibili liquidi per il trasporto<sup&gt; 1</sup&gt; E altri prodotti come materiali biocomposite<sup&gt; 7</sup&gt;. Biomassa vegetale rimane una delle più grandi riserve non sfruttate del pianeta<sup&gt; 4</sup&gt;. È principalmente costituito da pareti cellulari che sono composti da polimeri ricchi di energia compresa la cellulosa, emicellulosa vari (polisaccaridi della matrice, e la lignina polifenoli<sup&gt; 6</sup&gt; E quindi talvolta chiamate legnocellulosa. Tuttavia, le pareti delle cellule vegetali si sono evoluti per essere recalcitranti alla degradazione come pareti fornire resistenza alla trazione di cellule e le piante intere, allontanare gli agenti patogeni, e permettere all&#39;acqua di essere trasportati in tutta la pianta, nel caso di alberi fino a più del 100 m sopra livello del suolo. Grazie alle varie funzioni di mura, vi è una immensa diversità strutturale tra le mura di specie vegetali diverse e tipi di cellule all&#39;interno di una singola pianta<sup&gt; 4</sup&gt;. Quindi, a seconda di ciò che le specie coltivate, la varietà delle colture, o tessuto vegetale viene utilizzato per una bioraffineria, le fasi di lavorazione per la depolimerizzazione da processi chimici / enzimatici e successiva fermentazione degli zuccheri vari biocarburanti liquidi devono essere adeguate e ottimizzati. Questo fatto rafforza l&#39;esigenza di una caratterizzazione approfondita delle materie prime da biomassa vegetale. Qui descriviamo una metodologia completa di analisi che consente la determinazione della composizione del legnocellulosa ed è suscettibile di un mezzo di analisi high-throughput. In questa prima parte ci si concentra sull&#39;analisi della lignina polifenoli (Figura 1). Il metodo inizia con la preparazione di materiale destarched parete cellulare. Il legnocellulosa risultanti sono poi divisi per determinare il contenuto di lignina solubilizzazione acetylbromide<sup&gt; 3</sup&gt;, E la sua composizione lignina in termini di syringyl, unità guaiacyl-e p-idrossifenil<sup&gt; 5</sup&gt;. Il protocollo per analizzare i carboidrati nella biomassa lignocellulosica compreso il contenuto di cellulosa e polisaccaridi composizione della matrice è discusso nella 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 vegetale è uno dei principali carbon-neutral risorsa rinnovabile che potrebbero essere utilizzati per la produzione di biocarburanti. Biomassa vegetale è costituita principalmente delle pareti delle cellule, un materiale composito chiamato strutturalmente complesso legnocellulosa. Qui si descrive un protocollo per un&#39;analisi completa del contenuto e la composizione della lignina polifenoliche.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte I: lignina

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

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

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

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

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K Views.  Michigan State University (MSU). Biomassa vegetale è uno dei principali carbon-neutral risorsa rinnovabile che potrebbero essere utilizzati per la produzione di biocarburanti. Biomassa vegetale è costituita principalmente delle pareti delle cellule, un materiale composito chiamato strutturalmente complesso legnocellulosa. Qui si descrive un protocollo per un'analisi completa del contenuto e la composizione della lignina polifenoliche.

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

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

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

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

Isolamento delle isole pancreatiche umane: Parte I: Digestione e Collezione di tessuto pancreatico

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

Ricerca

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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 1

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

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

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

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

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Part 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 3

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.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte II: Carboidrati

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.

Senza etichetta In situ</emImaging> di lignificazione in pareti delle cellule vegetali

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 Glycan dei polimeri cellula vegetale da parete usando i 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.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

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

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

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

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

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

Un metodo efficiente per quantitativa, analisi singola cella della cromatina Cambiamento e architettura nucleare in tutto il montaggio Ovuli in<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 ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Isolamento e analisi della composizione delle piante cuticola lipidi poliestere monomeri

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...