この生産と実験的な作品がここに親切にリナアギーレ、ジルコルドバ、ウィリアムメラ、Ximenaボニーヤ、そしてダリオViverosでサポートされているレイナルドパレハの献身と熱狂的な支援を反映して。我々はまた、私たちはビデオと原稿を向上させる助けコメントのジョンマイルズ三匿名の校閲者に感謝する。

1。植物<ol><li>成熟した植物から単幹挿し木は実験台として使用されます。挿し木は植え材料の均一性を確保するために10 cmにトリミングされています。 </li><li>植物の病原体による汚染を防ぐために、挿し木は2分間、3％次亜塩素酸ナトリウム溶液で洗浄し、その後、水道水でthoughroughlyすすぎ洗いされています。 </li><li>それぞれのカットは、CAに植えられている。円筒状の塩ビ管の滅菌土壌36 gは（6.5センチ×高さ外径5.3センチメートル）発泡スチロールのカップと下端部に密封し、PVCのブッシングで​​キャップ。カットは、ブッシングの中央開口部に配置された泡のリングで固定されています。 </li><li>十分な土壌水分を維持するために、必要に応じて植物は受精と水やりしている。 </li><li>植物は根、木部授乳アワフキムシの幼虫のためのサイトを餌として表面的な根、の開発を可能に、昆虫が挑戦される前に一ヶ月のために成長します。 </li><li>根挿しによるPVCチューブは、部分的にルートのアーキテクチャに固有の差異を調整し、さらに表面的な根の成長を刺激する前に出没〜8 Dに反転されています。この手順では、24ヘクタールの日のために提供する光の人工光源を必要とする。この反転処理は実験から除去された後、草食動物の抵抗を持つ草食から交絡エスケープを避けるために、個々の植物はほとんど、またはまったく表面的なルーツを複製します。この除去は、それらが表す遺伝子型の排除につながることはない、まれなはずです。 </li></ol> 2。昆虫<ol><li>成人spittlebugsは、フィールドから集めた種を同定し、それらを餌に影響を受けやすいbrachiariagrassesの葉を提供している産卵ケージに導入されています。女性は、土壌中のそれらの卵は、ケージの底部に配置浅いリムーバブルトレイに含まれて横たわっていた。 </li><li>数日後、土は水に懸濁させ、卵を収集するふるいのシリーズ（42、60、150メッシュ）を介して渡されます。 30パーセント生理食塩水でふるいにかけた材料​​を中断すると、未成熟卵と、残留有機物を残し、成熟卵が浮くようになります。 </li><li>昆虫病原体による汚染を防ぐために、卵は5分間0.5％次亜塩素酸ナトリウム溶液で消毒してから蒸留水で洗浄されています。 </li><li>各試験プラント（実験的ユニット）は、6成熟卵で、土壌表面で出没です。孵化に近いだけ成熟した卵が蔓延のために選択されています。昆虫の腹部に対応する彼らの後部にピンクのマーク、;と完全に拡張された蓋それらは2つの赤虫の目に対応する彼らの前方部分のドットの存在によって識別することができます。 </li><li>成功したハッチングを任意まだ孵化していない卵が我々のコロニーから新生児アワフキムシにより置換されている時点で、4dと、後で確認されている。 </li><li>試験植物は、遺伝子型ごとに6つの複製を持つ無作為化完全ブロックデザインに配置されています。 </li></ol> 3。評価<ol><li>実験は、CAが評価されます。侵入後の30 D（正確な時間は勉強昆虫種によって異なります）。 </li><li>ダメージは1が死んだ植物に目に見える損傷および5に相当する（図1）に対応する1から5までの視覚のスケール、で採点される。植物の損傷を獲得する耐性の推定値を提供します。 </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="図1" /> Brachiaria属のアワフキムシ草食への耐性を評価するために使用される図1。被害の規模。 1 =検出可能な損傷、2 = 0から25パーセント壊死葉、3 = 25から50パーセント壊死葉、4 = 50から75パーセント壊死葉、5 = 75から100パーセント壊死葉。 <ol start="3"><li>昆虫の生存は、その最終的な齢、大人のステージ（図2）のどちらかに到達する昆虫を数えることによって評価される。昆虫の生存のためにスコアリングする抗菌の推定値を提供します。 </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="図2" /> 一齢幼虫（左）から大人（右）に図2。アワフキムシの開発。評価日で少なくとも最終的な齢（矢印）に到達する唯一の昆虫は、次のように数えている&quot;生存者。&quot; 4。代表的な結果アワフキムシAeneolamia reductaへの抵抗のための代表的なアッセイのスクリーニングのBrachiariaのハイブリッドからの結果を図3に示されている。 <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="図3" /> 図3。アワフキムシAeneolamia reductaへの抵抗のためにBrachiariaの雑種のスクリーニングの結果。草食性への応答は、このプロットの下半分に低下した場合我々は、植物が耐性を考慮してください。右から左への抗生作用の増加と上から下へ耐性が増加する。矢印は、私たちの耐性チェックを指す。

<ol>
	<li>Painter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

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草食動物の耐性機構の間で判別することは作物の育種への取り組み3を照らすことができる。強力な抗生作用に基づく抵抗は、ある状況下で、より積極的な害虫の生物型のための選択圧を作成することができます。一方、許容範囲に基づいて抵抗値が許容誤差が最終的には圧倒されるまで、害虫が増加する可能性があります。耐久性のある抵抗のために繁殖、したがって、どのような耐性機構は、特定の作物/病害虫システムのために最も安定性を付与するという[ケネディらを参照して慎重に検討する必要があります。 （1987）トピック] 3に関する包括的な議論のため。 このビデオデモでは、抗菌とbrachiariagrasses 6-9のアワフキムシの抵抗に耐性の相対的な寄与を推定するために設計されたいくつかの以前の研究に基づいて構築。選択テストやフィールド実験とは対照的に、非選択のテストはすべての植物がこのように草食動物の行動（例えば、ホストの優先順位、アグリゲーション）から生じる相違点のための制御を提供する、同じ草食動物の圧力を受けることを確認してください。このような理由から、しかし、非選択のテストはantixenosisで抵抗を評価するためには不向きである。 人為的に昆虫のコロニーを飼育する抵抗のスクリーニングアッセイ10用のテストの昆虫を得るために好ましい方法である。コロニーは、11年のいつでも実施する実験のために知られている年齢のspittlebugsの信頼性と均一なソースを提供しています。それは、しかし、定期的にコロニーは、関連するフィールドの個体数10から遺伝的に逸脱しないことを保証するために植民地に野生の個体を注入する必要があるかもしれません。これらの人工的に育てられた草食動物の適応度を評価するために、そして時間をかけて彼らの積極性の変化の可能性を監視し、それはすべての上映は、既知の抵抗のレベルの適切なチェックを含めることが重要です。 複数のパラメータを慎重に適切な抵抗のスクリーニングアッセイを設計するために定義する必要があります。我々は我々のテストを設計すると考えているいくつかの重要なパラメータは、宿主植物の年齢、種とspittlebugsの発達段階、寄生のレベル、および植物-昆虫の接触8,9,12の期間が含まれています。実験およびプロトコルの改良のいくつかのラウンドは、適切にフィールドに草食動物の抵抗を予測する迅速かつコスト効率、信頼性、アッセイに達するために必要な場合があります。

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

antixenosis、抗菌及び公差1：植物は3つの大まかなメカニズムを介して草食動物の損傷に抵抗することができます。 Antixenosisは、草食動物は、他の植物2を選択することができるときに植物が回避される程度です。抗菌は、植物が1日に摂食草食動物の適応度に影響を与える程度です。許容範囲は、植物が草食動物の成長と繁殖1を損なうことなく耐えられるか、草食動物によって引き起こされた損傷を修復できる程度です。農業環境における草食動物の抵抗の耐久性は、作物育種の取り組み3で支持抵抗機構程度にかかっています。 我々は、抗菌及びBrachiaria属のアワフキムシ抵抗に対する耐性の相対的な寄与を推定するために設計された非選択実験を示しています。属Brachiariaのアフリカの草のいくつかの種は新熱帯区の貴重な飼料と牧草ですが、それらは深刻なspittlebugsのいくつかの在来種（半翅目：アワフキムシ科）で挑戦することができます。spittlebugsに彼らの抵抗を評価4に、植物は、栄養、伝播されます茎の切断によってとspittlebugsが供給可能な上表面的な根の成長を可能に、約一ヶ月間成長させた。その時点で、各試験プラントは、個々に孵化に近い6アワフキムシの卵に挑戦しています。蔓延は、植物の損傷や昆虫の生存率を評価する前に、一ヶ月間進行されている。昆虫の生存を採点しながら、植物の損傷を獲得する耐性の推定値を提供する抗菌の推定値を提供します。このプロトコルは、商用brachiariagrases 5アワフキムシの抵抗を高めるために私たちの植物の品種改良の目標を促進してきた。

Watch this Scientific Journal Video about Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example at JoVE.com

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

このビデオでは、草食性に宿主植物の抵抗性のメカニズムを説明し、のアワフキムシ抵抗に抗生作用と耐性の相対的な寄与を推定するのない選択式テストを示しています<em&gt; Brachiaria</em&gt;属。

特徴付ける草食動物の耐性機構：上Spittlebugs Brachiaria属。例として

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

characterizing-herbivore-resistance-mechanisms-spittlebugs-on

Research

JoVE Journal

Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K ビュー.  CIAT. このビデオでは、草食性に宿主植物の抵抗性のメカニズムを説明し、のアワフキムシ抵抗に抗生作用と耐性の相対的な寄与を推定するのない選択式テストを示しています Brachiaria属。

ビデオ: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

生物学

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

草食動物によって誘導されたブルーベリー揮発性物質とイントラ植物のシグナリング

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

浸透水透過係数（Pの測定<sub&gt; F</sub&gt;）球状細胞の：例として単離された植物プロトプラスト

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

植物の葉からのアポプラスト液の抽出のための浸透·遠心分離技術の使用<em&gt;インゲンマメ</em&gt;例として

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

規制エキソサイトーシスの記者として FITC デキストランをイメージング

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

22 Tで 医薬トランカチュラ ルーツで実証MRMマイクロコイル性能キャリブレーションと使用

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

ウイルス感染時のm6Aおよびm5Cエピトランスクリプトームの探索:HIVの例

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

原子間力顕微鏡を用いた生きた植物器官の初代細胞壁の力学特性評価

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

リ ケッチア 属菌を蛍光タンパク質発現シャトルベクターでダニ細胞株に形質転換するエレクトロポレーション法

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

FluoZin-3アッセイによる哺乳類細胞における亜鉛輸送メカニズムの調査

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

著者スポットライト:ZnT1機能による細胞亜鉛制御の探索 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

In Vitro亜鉛輸送アッセイを用いた哺乳類亜鉛トランスポーターの特性評価(英語)

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...