ここに示されている技術は、エネルギー省（バイオサイエンス）と国立科学財団からの補助金によって資金で開発された。

パート1：成長と適切な植物材料を保存する<ol><li>我々の経験では、黄化キュウリの苗から若い胚軸は、これらの実験のための細胞壁物質の便利な供給源として働く。キュウリの種子は26で設定された恒温室で暗いキャビネットに保管され遮光ボックス、℃で湿紙に播種されていますC正常な状態になる22 °と30 °の間に何と正確な温度は、重要ではないが、温度は苗が開発の適切な段階に到達する速度が決定されます。周囲の温度になじませ、より高速な苗が開発します。我々は、典型的には3-4日播種後に到達する長さは5 cm程度に成長した苗を使用してください。光の少量であっても我々はこの手法で測定することを苗の開発の速度と細胞壁のプロパティの両方に影響を与えるとして、苗を暗所で栽培することが重要です。 3日目には苗の開発をチェックするために、薄暗い緑のフィルタリングライトを使用して、ボックス内のpeekすることができます。 </li><li>苗は、急速にカットし、小さなプラスチックの箱、箱当たり100〜150本の苗木に詰め、そして-80℃で保存されていますこの温度で​​は、彼らは数週間のための有用なままである。 </li></ol>第2部：準備の細胞壁のサンプル<ol><li>冷凍カット苗の小グループ（8-10）は、冷凍庫から-80冷凍ブロックを含む断熱容器に転送されます。 </li><li>胚軸をカバーするキューティクルはカーボランダムで摩耗しています。これは、繰り返しウェットカーボランダム粉末の厚さのスラリーでコーティングされている親指と人差し指の間の胚軸を描画することによって行われます。それは、使用する圧力の正しい量を知るには少し経験が必要です。すぎる圧力と表皮層が細断処理と引き裂かれたように開始します。少なすぎる圧力とキューティクルが浸透されることはありません。一つは、また、冷凍胚軸融解と、それが管理する弛緩性と、ハードになるのですぐに仕事をしている。 </li><li>残りの胚軸は同様の方法で調製されている間に磨耗した胚軸を氷水上に格納して付着カーボランダムとのほとんどを除去するために氷水に浸漬する。 </li><li>胚軸は、新しい単一の両刃カミソリの刃で、通常1.2センチメートル、希望の長さに切断し、スライドガラス上に整列されています。 </li><li>今、私たちは、細胞の樹液を削除するとクランプ容易にするために壁を平らにする必要があります。第2のガラススライドは、サンドイッチを形成する、8-10サンプルのグループの上に配置されます。重量（400-500 g）を5分間スライドガラスの上に配置されます。重量のために我々は日常的に適量の水を入れたビーカーを使用してください。 </li><li>オプションの手順：実験に応じて、胚軸はこの時点で短時間の熱処理で不活化されることがあります。これを行うために、我々は、ガラス、ゴムバンドのペアと一緒にスライド結合し、室温で脱イオン水100 mLの容器にアセンブリを配置し、フルパワーで電子レンジに入れてください。私たちの電子レンジで水は約50秒で沸騰し始め、沸騰が開始した後我々はマイクロ波の15秒を停止する。お湯はすぐにオフ注ぎ、変性を停止するために冷たい水に置き換えられます。あなたのマイクロ波が私達のものとは異なる可能性があるように、これらのタイミングを変化させる必要があります。過度の加熱により試料が弱くなり、簡単に壊れる。不十分な加熱で内因性のエクスパンシンが失活し、壁の試料は酸性pHにいくつかの応答性を保持されていません。 </li></ol>パート3：伸び計のセットアップ<ol><li>壁のサンプルは現在、一定の力の伸びにクランプされています。これは、壁のサンプルとサンプルの先端部に取り付けられた可動クランプの基端を保持するためのプレキシグラスのキュベットで構成されるカスタム構築されたデバイスです。可動クランプの位置センサのオープンコイルを通過するロッドの端にマウントされている、LVDTまたは電子的に小さな金属製のシリンダーの位置を検出する&quot;リニア可変差動トランス&quot;、、または&quot;コア&quot;は、ですロッドに装着。ロッドの上端部は、調節可能なおもりでレバーにリンクされています。このレバーは、壁のサンプル上に上向きの力の調整可能な量を発揮する。力は、レバーの遠端に校正された金属製のウェイトを追加したり削除したりして調整されます。 </li><li>後壁のサンプルへ - 胚軸サンプルの基端は、微細な鉗子でピックアップされており、先端部の2〜3 mmの可動クランプのオープンジョーとの間に配置されます。このクランプは、その金属ジョーが金属表面と緩衝液または壁のサンプル間の直接接触を防止するためにプラスチックでコーティングされているバネ仕掛けのワニクランプです。私たちの経験の金属イオンに延び、そして私たちは金属のカバーを維持するために壁の能力を阻害クランプから浸出がとができます。 </li><li>片方の手で可動クランプアセンブリを保持している、壁の試験片の基端は、現在プレキシグラスキュベットとキュベットの二つヒンジ片の間に操縦されているピースが結集し、それによりキュベットで壁のサンプルの下端をロック、タイトネジ止めされる。 </li><li>可動クランプアセンブリは、おもりの完全な力が壁の試料に転送できるように、今ゆっくりと放出される。我々は日常的にキュウリ胚軸から調製壁のサンプルを20 gの合計カウンターウェイトを使用してください。他の壁材や実験は、異なる重みを必要とする場合があります。 </li><li>キュベットは、バッファーで満たさ（200 UL）とキュベットの位置は、可動クランプがLVDT測定範囲の下端に持って来られるように、調節ネジで上下に移動されます。私たちのLVDTは、我々がコンピュータを介してLVDTの位置を監視するため、マイコンのデータ収集ユニットに接続され、。私たちは、私たちは同時に8壁のサンプルを実行することを可能にする一台のコンピュータに並列に接続した8 LVDTアセンブリを、持っている。コンピュータごとに1回LVDTアセンブリの各30秒の位置を記録</li></ol>パート4：酸性pHまたはエクスパンシンの測定の拡張レスポンス - 代表的な結果<ol><li> [最初の実験]酸誘発拡張子の測定には、我々は、ネイティブの壁のサンプルから始める（つまり、熱で失活しない）と中性緩衝液をキュベットに追加されます。壁のサンプルが追加された緊張に反応して、数分間延長するが、数分後に低レートへの拡張が減衰する。私たちのコンピュータは、私たちは、いずれかのサンプルの長さの変化を監視する（つまり、実験開始後、可動クランプの位置の変化）や我々が他の言葉で、拡張子の率を位置の時間微分を監視できることができます。伸長率は、時間と低い値で安定します。 </li><li> 〜20分後、中性緩衝液は削除されます。私たちは壁の軽微な障害または機械的なアセンブリと、すぐにバッファを削除するには、真空ポンプに接続されている大規模なゲージの注射針から作られた薄い金属パイプを、使用してください。酸性緩衝液は、酸性バッファーに完全な交換を保証する1-2クイック取引所で時々、追加されます。その後、我々は後ろに座ると壁の応答を監視します。一般的に我々は数分で拡張子のより速い速度を検出することができます。いくつかのケースでは測定が長時間に伸びるかもしれないが60分後、我々は通常、拡張子の応答を評価するのに十分な情報があります。 </li><li> [第二実験]エクスパンシン誘発性の細胞壁の拡張子の測定では、我々は、熱不活化の壁のサンプルと酸性緩衝で始まるをキュベットに追加されます。最初の実験と同様に、細胞壁の延長率は徐々にために機能的なエクスパンシンの欠如の低い値で安定します。 </li><li> 〜20分後、エクスパンシンタンパク質は、エクスパンシンソリューションの10-20 ULでキュベット内のバッファに&quot;スパイク&quot;でキュベットに追加されます。エクスパンシンは、急速に細胞壁のサンプルを貫通して数分以内に我々は、伸長率が増加していることがわかります。この拡張子の応答は、時間以上続けることができます。 </li></ol><img src="/files/ftp_upload/1263/fig1.png" alt="" width="600" height="469" /> 図1：酸誘発またはエクスパンシン誘発性の壁の拡張子を評価するために細胞壁を準備する手順の概要。

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+extension+of+isolated+cell+walls+from+growing+cucumber+hypocotyls.">Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls.</a> Planta. 177, 121-130 (1989).</li><li>Rayle, D. L., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acid+Growth+Theory+of+auxin-induced+cell+elongation+is+alive+and+well.">The Acid Growth Theory of auxin-induced cell elongation is alive and well.</a> Plant Physiol. 99, 1271-1274 (1992).</li><li>Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-induced+growth+of+Avena+coleoptiles+involves+two+mechanisms+with+different+pH+optima.">Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.</a> Plant Physiol. 99, 1556-1561 (1992).</li><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loosening+of+plant+cell+walls+by+expansins.">Loosening of plant cell walls by expansins.</a> Nature. 407, 321-326 (2000).</li><li>McQueen-Mason, S., Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+endogenous+proteins+that+induce+cell+wall+expansion+in+plants.">Two endogenous proteins that induce cell wall expansion in plants.</a> Plant Cell. 4, 1425-1433 (1992).</li></ol>

は、扱いやすい、それは良い感度で応答壁のサンプルの信頼できるソースであることが証明しているので、このデモのために我々は、キュウリ胚軸から細胞壁を使用。我々はまた、若いほうれん草の葉、セロリの茎などスーパーマーケットから他の苗だけでなく、いくつかの材料、から、壁との良好な成功を収めている。基本的に、若い、柔らかい、急速に成長している植物組織は簡単にこ?...

measuring plant cell wall extension (creep) induced by acidic ph and by alpha-expansin

成長する植物の細胞壁が特徴的に我々は彼らは低pH（&lt;5）1で、より拡張性のある意味これが&quot;酸成長&quot;、として知られているプロパティを示す。植物ホルモンのオーキシンは急速に酸-成長機構2、3が少なくとも部分的に若い茎と同様の組織において細胞の伸長を刺激する。オーキシンは細胞壁の溶液の酸性化を引き起こし、原形質膜のH +ポンプを活性化する。壁の酸性化は、細胞の膨圧によって作成された壁の緊張をもたらすために細胞壁を引き起こし、内因性の細胞壁緩み止めタンパク質4であるエクスパンシンを、活性化する。その結果、細胞は急速に拡大し始める。この&quot;酸成長&quot;現象は容易に分離された（非生物）の細胞壁の試料で測定されます。酸によって誘発される拡張子を受けるために細胞壁の能力は、単に細胞壁多糖類の構造配置（例えばペクチン）の結果ではなく、エクスパン5の活性に依存する。エクスパンシンは、既知の酵素活性を持っていないとエクスパンシン活性のアッセイへの唯一の方法は、細胞壁の拡張子のそれらの誘導を測定することです。このビデオのレポートは、詳細エクスパンシンアッセイに適した壁材を入手する方法と調製技術を、酸誘発性の拡張と成長しているキュウリ胚軸から調製壁のサンプルのエクスパンシン誘発性の拡張子を表示するために行く。 適切な細胞壁のサンプルを入手するには、キュウリの苗を暗所で栽培され、胚軸は-80で切断し、凍結されている℃に冷凍胚軸は、摩耗平らにし、伸びの測定のための特別なキュベットに一定の張力でクランプされています。酸によって誘発される拡張子を測定するために、壁には、最初にネイティブの細胞壁の成分であるエクスパンシンの活性が低い、その結果、中性のpHで緩衝化されています。酸性pHに緩衝液交換時には、エクスパンシンが活性化し、細胞壁は、急速に拡張されています。我々はまた、再構成アッセイにおけるエクスパンシンの活性を示す。この部分については、我々は、細胞壁のサンプルでネイティブエクスパンシンを変性させる簡単な熱処理を使用してください。これらの不活化細胞壁は、酸性緩衝液でも拡張しませんが、細胞壁のエクスパンシンの添加は、急速に拡張する能力を復元します。

Watch this Scientific Journal Video about Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin at JoVE.com

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

我々は、酸性バッファーとエクスパンシンタンパク質によって誘導される植物細胞壁の標本の長期延長（クリープ）を測定するために一定の力の伸びの使用例を示します。

酸性pHでとα-エクスパンにより誘起される植物細胞壁の延長（クリープ）を測定

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

measuring-plant-cell-wall-extension-creep-induced-acidic-ph-alpha

Research

JoVE Journal

Biology

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

13.9K ビュー.  Penn State University. 我々は、酸性バッファーとエクスパンシンタンパク質によって誘導される植物細胞壁の標本の長期延長（クリープ）を測定するために一定の力の伸びの使用例を示します。

ビデオ: Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

Title Cell Encapsulation by Droplets

滴タイトルセルのカプセル化

生物学

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

ウイルス誘導ジーンサイレンシング（VIGS）でタバコbenthamianaとトマト

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

好気性菌による制御植物組織の分解のための寒天ブロックの小宇宙

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

商業カセットを再利用することでタンパク質ゲルを注ぐと実行

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

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.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

結晶セルロース蓄積中の高解像度の定量化<em&gt;シロイヌナズナ</em&gt;ルーツは、組織特異的な細胞壁の変更を監視します

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

トマト毛状根の発生によるDNAの修飾のハイスループットCRISPRベクター構築と特性

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...