Biz mükemmel teknik servis ve John Ralph, University of Wisconsin için değerli tavsiyeler, tartışmalar ve kavak ahşap örnek Matthew Robert Weatherhead için minnettarız. Bu çalışma Bilim Office US Department of Energy (DOE) Great Lakes Biyoenerji Araştırma Merkezi (DOE Bilim DE-FC02-07ER64494 BER Office) ve Kimya Bilimleri, Yerbilimleri ve Biosciences Bölümü, Temel Enerji Bilimler Dairesi tarafından finanse edildi ABD Enerji Bakanlığı (ödül no DE-FG02-91ER20021).

1. Hücre Duvarı İzolasyon <ol><li> IWall kullanarak bir 2 ml Sarstedt vidalı kapaklı tüp 5.5 mm paslanmaz çelik bilya hava dondurmak veya kurutulmuş bitki materyali yaklaşık 60-70mg Grind, öğütme ve dağıtım robot (30 s). Bölüm II 2 bilyalı değirmen (retschmill) kullanarak alternatif olmayan robot düşük verimlilik prosedürü sunulmaktadır. </li><li> 1.5 ml% 70 sulu etanol reçete zemin malzemesi, ve iyice vorteks ekleyin. </li><li> 10.000 rpm&#39;de 10 dakika pelet için alkol çözünmeyen kalıntı santrifüjleyin. </li><li> Süpernatantı aspire veya süzün. </li><li> Kalıntı kloroform / metanol (1:1 v / v) solüsyonu 1.5 ml ekleyin ve pelet tekrar süspansiyon tüpü iyice sallayın. </li><li> 10,000 rpm ve 10 dakika süpernatant aspirat veya süzün santrifüjleyin. </li><li> 500 ul aseton içinde süspanse edin pelet. </li><li> Kuruyana kadar hava akımı 35 ° C ile solvent buharlaşır. Gerekli kurutulmuş örnekleri daha fazla işlem kadar oda sıcaklığında saklanabilir. </li><li> Örnek nişasta kaldırılması başlatmak için 1.5 ml 0.1 M sodyum asetat tamponu pH 5.0 pelet tekrar süspansiyon. </li><li> 20 dakika Sarstedt boru ve ısı Cap. 80 ° C ısıtma bloğu. </li><li> Süspansiyon buz üzerinde soğutun. </li><li> Pelet için aşağıdaki maddeler:% 0.01 sodyum azid (NaN3) 35 ul 35 ul amilaz (50 mg / ml H 2 O; Bacillus türleri, Sigma); 17 ul pullulanase (Bacillus acidopullulyticus 17,8 birimleri; Sigma) . Ve iyice vorteks tüp kap. </li><li> Süspansiyon 37 gece boyunca inkübe ° C çalkalayıcı. Tüpler yönlendirmek yatay yardımcıları karışım geliştirdi. </li><li> Isı süspansiyon 100 ° C sindirim sonlandırmak için bir ısıtma bloğu 10 dk. </li><li> Santrifüj (10.000 rpm, 10 dakika) ve supernatant içeren çözünür nişasta atın. </li><li> 1.5 ml su ekleyerek vorteks, santrifüj ve çamaşır suyu şişeden kalan pelet üç kez yıkayın. </li><li> 500 ul aseton içinde süspanse edin pelet. </li><li> Kuruyana kadar hava akımı 35 ° C ile solvent buharlaşır. Daha iyi kurutma için bir spatula ile tüp içindeki malzemeyi kırmak için de gerekli olabilir. Kurumuş malzeme izole hücre duvarı (lignocellulosics) sunar. Gerekli kurutulmuş örnekleri daha fazla işlem kadar oda sıcaklığında saklanabilir. </li></ol> 2. Lignin İçerik Bu yöntem Fukushima ve Hatfield 3 tarafından bildirilen yöntem dayanmaktadır. <ol><li> 1 - 1.5 mg (1) hazırlanan hücre duvar malzemesi için boş bir tüp boş bir kalkan 2 ml.lik ölçülü balona tartılır. </li><li> tüp duvarları tüpün alt hücre duvarı malzeme toplamak için 250 ul aseton ile temizleyin, durulayın ve aseton çok hafif hava akımı altında buharlaşması. </li><li> Yavaşça sıçramasını önlemek için tüp duvarları boyunca taze yapılmış asetil bromür çözüm 100 ul ekleyin (% 25 v / v buzul asetik asit asetil bromür). </li><li> Cap hacimsel 50 balon ve ısı ° C 2 saat için </li><li> Her 15 dakikada bir vorteks ile ek saat için ısıtın. </li><li> Oda sıcaklığına kadar buz üzerinde soğutun. </li><li> 2M sodyum hidroksit ve 0.5 M hidroksilamin hidroklorür taze hazırlanmış 70 ul 400 ul ekleyin. Vortex hacimsel şişeler. </li><li> Buzul asetik asit, şapka ile tam 2.0 ml işareti ölçülü balona kadar doldurun ve karıştırmak için birkaç kez ters. </li><li> Pipet 200 UV özel 96 plaka çözüm ul ve 280nm ELISA okuyucu okumak. </li><li> (; Otlar = 17.75; Kavak Arabidopsis = 18,21 = 15,69) uygun bir katsayı ile asetil bromür çözünür lignin (% ABSL) oranını belirlemek için aşağıdaki formül ile : % ABSL Kireçlenme: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Şekil 1" /> 10 sonuç ug / mg hücre duvar ünitesi ile% ABSL çarpımı Bu parçacık absorbans değerleri, küçük bir değişime neden olabilir bu yana absorbans (abs) ortalaması en az 3 plaka okuma yapmak için yardımcı olur. Not: 0,539 cm yollu temsil eder, ama plaka bağlı olarak bu tespit edilmesi gerekebilir. </li></ol> 3. Lignin Kompozisyon Bu yöntem, Robinson ve Mansfield 5 tarafından yayınlanan yeni bir yöntem benimsenmiştir. <ol><li> Hücre duvar malzemesi yaklaşık 2 mg (1). Thioacidolysis için vidalı kapaklı cam tüp içine aktarın. </li><li> % 2.5 bor trifluoride dietil etherate (BF 3),% 10 ethanethiol (EtSH) çözümü, dikkatle hazırlayın. Dioksan şişe nitrojen ile kayıp ses yerinden azot gazı ile dolu bir balon kullanmanız gerekir. Dioksan çok tehlikeli, kaput numune veya ekipman yapmayız. 20 ul EtSH; 5 ul BF 3 175 ul dioksan: Hacimler örnek başına çözümün hazırlanması için gerekli. </li><li> EtSH, BF 3, her bir örnek dioksan çözüm 200 ul ekleyin . </li><li> Hemen azot gazı ve kapak Temizle flakon headspace. </li><li> Isı 100 ° C nazik karıştırma her saat ile 4 saat. </li><li> 5 dakika boyunca buz üzerinde soğutma sonu reaksiyon. </li><li> 150 ul 0.4M sodyum bikarbonat, girdap </li><li> Temiz su 1 ml ve 0.5 ml etil asetat, girdap ve aşamaları ayrı izin (alt üst, su üzerinde etil asetat). </li><li> 2 ml Sarstedt tüp içine etil asetat tabaka 150 ul aktarın. Hiç su transfer olduğundan emin olun. </li><li> Hava ile konsantratör çözücü buharlaşır. </li><li> (Iki kez olmak üzere toplam için tekrar aşırı su çıkarmak) 200 ul aseton ekleyin ve buharlaşır. </li><li> TMS türevlendirme için 500 ul, etil asetat, piridin 20 ul ve 100 ul N, O-bis (trimethylsilyl) her tüp asetamid ekleyin. </li><li> 2 saat boyunca inkübe 25 ° C </li><li> GC / MS şişenin içine reaksiyon 100 ul aktarın ve 100 ul aseton ekleyin. </li><li> Quadrupole kütle spektrometresi veya alev iyonizasyon dedektörü ile donanımlı GC örnekleri analiz edin. Bir Agilent HP-5MS sütun (X 0.25 mm X 0.25 mikron film kalınlığı 30 mm) monte edilir. Aşağıdaki sıcaklık gradienti 30 dakika solvent gecikme ve bir 1.1 ml / dak akış hızı ile birlikte kullanılır: 130 İlk tutun ° C için 3 dk; 3 ° 250 - C / dak rampa ° C ve 1 dakika boyunca tutun; izin 130 ° C başlangıç ​​sıcaklığı için dengeleme </li><li> Peaks tetracosane internal standart (isteğe bağlı) kullanarak göreceli tutma süreleri veya 299 karakteristik tayf iyonları tarafından tespit m / z, 269 m / z ve 239 m / z S, G ve H monomerler, sırasıyla (bkz. Şekil. 2). Lignin bileşenleri kompozisyon toplam pik alanı% 100 ayarı ile sayısal </li></ol> 4. Temsilcisi Sonuçlar Bir duvar analizi bir örnek Şekil 2&#39;de sunulmuştur. Bu durumda kavak kök (ağaç) protokol bölümünde belirtilen çeşitli işlemler analiz edildi. Kromatogram thioacidolysis sonra lignin bileşenlerinin ayrılması ve TMS-türevlendirme bir örnek gösterilmiştir. Açıkçası, göreceli bolluk syringyl-(S), guaiacyl-(G), ve p-hydroxyphenol (H) birimleri tespit edilebilir. Asetil bromür çözünür lignin içeriği kendi kendini açıklayıcı, bir duvar kuru ağırlığının% 20-50 arasında değerler bekleyebilirsiniz. Bir o asetil bromür duvardaki lignin mevcut çözünür ve çözünürleştirme derecesi malzemeye bağlı olarak değişebilir değildir dikkat etmelisiniz. Ancak, bu yöntem nispeten yürütmek kolay ve hızlı ve lignin içeriği mükemmel bir yaklaşım, lignocellulosic bir malzeme verir. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Şekil 1" /> Şekil 1: lignocellulosic analiz Genel Bakış Hücre duvarları (lignocellulosics) ham kurutulmuş bitki materyali izole edilmiştir. Duvar malzemesi, sonra alikotları içine ağırlıklı ve çeşitli deneyleri için bölünmüştür. Asetil bromür ve UV spektroskopisi ile nicel çözündüğünü lignin duvar malzemesi ile tedavi edilir. Lignin kompozisyonunun belirlenmesi için, duvar malzemesi thioacidolysis tabi tutulur. Çözünür fenolik TMS türevlendirme geçmesi ve daha sonra ayrılmış ve GC-MS analizi ile sayısal olabilir. Bölüm II 2 matris polisakkarit kompozisyon ve kristalin selüloz içeriği protokolü tartışılmıştır. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Şekil 2" /> Şekil 2: kavak (Populus tremoloides) kavak ahşap Kapsamlı lignocellulosic analiz Ahşap cips açıklanan protokollere tabi tutuldu. Ligin kompozisyon, H p-hidroksifenil, G guaiacyl S syringyl birimleri.

<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>

Açıklanan yöntemlerden lignin içeriği ve lignocellulosic bitki biyokütle bileşimi hızlı bir sayısal değerlendirme sağlar. IWall robot kullanarak yaklaşık 350 örnek zemin ve ücreti günlük reçete edilmesi. Kişi başına düşen çeşitli analitik yöntemlerin hacmi değişir. Protokolleri Burada anlatılan, lignin içeriği için 30 örnek işlenmiş ve lignin Hergün kompozisyon için 15 olabilir. Nicel veri en uygun hammadde bitkileri doğası nedeniyle, çeşitli veya genotipler, biyoyakıt üretimi için kendi uygunluk açısından değerlendirilebilir.

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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

Yenilenebilir, karbon nötr, sanayi ve toplum için sürdürülebilir hammadde ihtiyacını 21. yüzyıl için en önemli sorunlardan biri haline gelmiştir. Bu ulaşım için sıvı yakıtların üretimi için endüstriyel hammadde olarak bitkisel ürünlerin kullanımı ilgi başlattı.<sup&gt; 1</sup&gt; Biokompozit malzemeleri gibi ve diğer ürünler<sup&gt; 7</sup&gt;. Bitki biyokütle, gezegen üzerindeki en büyük enerji rezervlerinin biri olmaya devam ediyor<sup&gt; 4</sup&gt;. Bu daha çok enerji selüloz dahil olmak üzere zengin polimerler, çeşitli hemiselüloz (matris polisakkaritler, ve polifenol lignin oluşan hücre duvarlarının oluşur<sup&gt; 6</sup&gt; Ve bu nedenle bazen lignocellulosics olarak adlandırılır. Ancak, bitki hücre duvarları duvarlar hücreleri ve tüm bitkiler kopma mukavemeti sağlamak gibi patojenlerin savuşturma ve izin bitki boyunca su taşınacak, bozulmaya karşı inatçı gelişti; ağaçların durumunda yukarıda fazla 100 m zemin seviyesinde. Duvarları çeşitli işlevleri nedeniyle, büyük bir yapısal çeşitliliği, tek bir bitki içinde, farklı bitki türlerinin ve hücre tiplerinin duvarları içinde var<sup&gt; 4</sup&gt;. Bu nedenle, bitki türleri, bitki çeşitliliği, bitki doku biorefinery için ne bağlı olarak, kimyasal / enzimatik süreçleri ve sıvı biyoyakıtlar için çeşitli şekerler sonraki fermantasyon depolymerization işleme adımları ayarlanabilir ve optimize edilmesi gerekir. Bu gerçek temelini oluşturan bitki biyokütle hammadde tam bir karakterizasyonu için ihtiyaç. Burada lignocellulosics bileşimi tespitine olanak sağlar ve yüksek verimlilik analizi bir orta için uygun, kapsamlı bir analitik yöntem açıklanmaktadır. Bu ilk bölümde, polifenol lignin analizi (Şekil 1) odaklanmak. Bu yöntem destarched hücre duvarı materyalinin hazırlanması ile başlar. Ortaya çıkan lignocellulosics sonra acetylbromide çözünmüş lignin içeriğini belirlemek için bölünmüş<sup&gt; 3</sup&gt; Syringyl, guaiacyl ve p-hidroksifenil birimleri açısından ve lignin kompozisyon<sup&gt; 5</sup&gt;. Bölüm II lignocellulosic biyokütle karbonhidrat, selüloz içeriği ve matris polisakkarit kompozisyonu da dahil olmak üzere analiz etmek için protokol tartışıldı<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

Bitki biyokütle, biyoyakıt üretimi için kullanılan olabilir önemli bir karbon-nötr yenilenebilir bir kaynaktır. Bitki biyokütle esas hücre duvarları, lignocellulosics olarak adlandırılan bir kompleks yapılara sahip kompozit malzemeden oluşmaktadır. Burada içerik ve polifenolik lignin bileşimi kapsamlı bir analiz için bir protokol açıklar.

Bitki Hücre Duvarları kapsamlı Kompozisyonel Analizi (Lignocellulosic biyokütle) Bölüm I: Lignin

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

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

Unit 1

Plant Cell Structure and Organization

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32.0K Görüntüleme.  Michigan State University (MSU). Bitki biyokütle, biyoyakıt üretimi için kullanılan olabilir önemli bir karbon-nötr yenilenebilir bir kaynaktır. Bitki biyokütle esas hücre duvarları, lignocellulosics olarak adlandırılan bir kompleks yapılara sahip kompozit malzemeden oluşmaktadır. Burada içerik ve polifenolik lignin bileşimi kapsamlı bir analiz için bir protokol açıklar.

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.

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

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.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 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.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: 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.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 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.

Bitki protoplast Floresan Aktif Hücre Ayırma

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.

Bitki Hücre Duvarları Kapsamlı Kompozisyonel Analiz (Lignocellulosic biyokütle) Bölüm II: Karbonhidratlar

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.

Etiket- In situ</emBitki Hücre Duvarlar Lignification> Görüntüleme

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.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

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.

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

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.

Tüm montaj ovüllerindeki in Kromatin Modifikasyonu ve Nükleer Mimarlık Kantitatif, Tek hücreli Analizi için Etkin Bir Yöntem<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 ...