この作品は国立科学財団 (NSF) によってサポートされていた許可なし。IOS 1354956。博士アンドリュー d. ウォーレン (鱗翅目、生物多様性、フロリダ州フロリダ大学自然史博物館・ マクガイア センター) 蝶の画像を使用する許可を感謝します。

1. 昆虫種、ソリューションおよび供給ステーションのセットアップの準備
注: 流体吸収能力、口器形態22,23に関する以前の研究の使用のため、キャベツ蝶 (モンシロチョウL.、シロチョウ科) は代表チョウ目の種として選択されます。家 (イエバエL., イエバエ科) や青いボトル蠅 (オオクロバエ vomitoria L., クロバエ科) は、彼らは多孔性の表面の13の餌、多く観察されるために使用されます。
<ol>
	<li>それら人工のダイエット (材料の表を参照してください) 蛹環境室の大人として浮上するまで昆虫のサプライヤーとリアから幼虫として順序モンシロチョウは 23 ° C、18 L: 6 日長に設定。順序M。イエバエと蛹としてC. vomitoriaとモンシロチョウと同じ環境条件でリアのそれら。彼らは供給実験前蛹から出てくる後は、チョウ類成虫、ハエを給紙されません。</li>
	<li>20% ショ糖液 (コントロール) と流体吸収をテストする 20% ショ糖ナノ粒子溶液を準備します。蛍光磁性ナノ粒子を追加することによってナノ粒子溶液を準備 (鉄酸化物ポリアクリル酸コーティング、約 20 nm 径の) 20% ショ糖液 (1 Mg/ml と dH2O ナノ粒子と 20% スクロースを24ソリューションには、1:1)。リン酸緩衝生理食塩水 (PBS) の 1 x ソリューションの準備 (10 x dH2O、pH 7.4 で 1 倍に希釈)、解剖に使われます。</li>
	<li>手動式マニピュレーターのクランプと別の餌ステージ (平らなプラットフォーム) で構成される供給ステーションの設定 (図 1)。摂食の段階に凹面のスライドを配置、毛穴サイズ直径 1、5、8、11、20、30、41、または 60 μ m、膜フィルターの孔のサイズ径ナイロン ネット フィルターまたは供給実験の近くに 10 μ m。</li>
</ol>
2. 餌のプロトコル
<ol>
	<li>昆虫の体、足と翼を折りたたんだティッシュ ペーパーにラップします。虫の位置を頭と口器のみが公開されます。昆虫、昆虫摂食の段階 (図 1) 上記の中断は、マニピュレーターをクランプによってまとめられる 2 つの顕微鏡のスライド間の翼に配置します。</li>
	<li>凹面のスライドの中央に 20% ショ糖溶液のマイクロ ピペットで 20% ショ糖ナノ粒子液 30 μ L の液滴を管理します。ナノ粒子溶液の液滴とフィルター紙の中心が揃うように、凹面のスライドに特定の細孔径の単一のフィルター紙を置きます。液滴とろ紙の間お問い合わせは満ちる気孔 (図 1)、フィルター紙に沿って広がっているソリューションの結果します。 注: フィルター ペーパーは、昆虫がフィード フィルター紙の上にナノ粒子が存在する可能性を最小限に抑えるため、周りの他の方法ではなく、液滴の上部に配置されます。</li>
	<li>口器の遠位領域のみの摂食の段階 (図 1) 接液部のフィルター ペーパーを連絡することができるように、マニピュレーターを持つ昆虫を配置します。虫ピンを使用してフィルター紙の上に口器を拡張し、45 のフィードに昆虫を許可する s。</li>
	<li>フィルター ペーパーの表面に存在するかもしれない液体の膜にする昆虫のチャンスを最小限に抑えるために口器の位置 (すなわちスライドの平らな部分に触れているろ紙の部分と接触しているので。、スライドの凹の部分の上に直接ではなく)。虫餌に興味を表現していない、口器は餌の時間の期間のろ過紙、虫ピンに保持することができます。</li>
</ol>
3. 解剖
<ol>
	<li>昆虫の体をカバーする十分な解決策があるので、50 ミリメートル時計ガラスに PBS のソリューションを配置します。時計ガラス、万国実体写真の横にある装置 (春マイクロはさみ、昆虫ピン、鉗子を切り裂く細かい点を解剖) を解剖位置と昆虫の下に配置します。</li>
	<li>授乳後ティッシュ ペーパーから昆虫を削除し、翼を閉じた状態でそれを保持します。春の微小解剖はさみで頭、足、および昆虫の翼を取り外し、時計ガラス (図 2) PBS 溶液で昆虫を置きます。</li>
	<li>必要な場合は、解剖前に昆虫を麻酔します。腹部遠位近傍キューティクルによって昆虫を保持するために鉗子を使用します。支配的な手で胸部に達するまで後端から始まる、腹部の側面に沿って前方方向でキューティクルをカットするのにはさみを解離性春マイクロを使用します。表皮だけをカットし、(図 2) が破損している昆虫の中の消化管ではないことを確保するための特別な注意を取る。</li>
	<li>消化管内部 (図 2) を明らかにする腹部を開く解剖はさみでキューティクルの追加カットを作る。昆虫ピンの支援を受けて腹部キューティクル、脂肪体と他の構造を削除し、時計ガラスの胸部と消化管だけを残して後の処分のため時計のガラスの外にそれらを再配置します。 注: 解剖胸部と腹部の合流点近くに位置する嚢のような構造 (消化管の延長) である作物が表示されます。</li>
	<li>作物が公開されていない場合は、作物を明らかにするまで、はさみで胸郭に追加切開を確認します。作物が表示、時計ガラス (図 2) で作物と消化管だけを残して昆虫の残りの部分を切り取る。 注: 鱗翅目昆虫の作物はほぼ透明で流体がないかどうかと、展開を認識することは困難かもしれない性質または解剖中切ってかどうかセロファンのような。</li>
	<li>その後画像 (図 2) の coverslip (24 mm × 24 mm) の上に作物を配置するのに鉗子を切り裂く細かい点を使用します。</li>
</ol>
4. 摂取されたナノ粒子の定量
<ol>
	<li>位置、細かい点を解剖鉗子使用して coverslip の作物は作物の破裂を防ぐために注意します。20 倍の倍率で画像を倒立顕微鏡で CY3 チャネル (または位相) を使用します。乾燥からそれを防ぐために郭清後すぐに作物をイメージします。</li>
	<li>顕微鏡の動作段階の制御ではない手で電磁攪拌バー (41.3 mm の長さと直径 8 mm) を保持します。</li></ol>前後それぞれモーション約 1 秒 (図 2) では、作物 (作物から約 10 mm 距離) の近くに来たり、電磁攪拌棒を振る。
	<li>電磁攪拌棒が振られる間は、ナノ粒子の作物を検査します。ほぼ透明な作物内でナノ粒子の動きのための顕微鏡の接眼レンズをのぞきながら、前後営業ステージをゆっくりと移動します。ナノ粒子が作物である場合肯定的な給餌を示す彼らは応答と波磁気と一体で攪拌棒 (図 2)。</li>

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著者が明らかに何もありません。

昆虫の口器機能は形態学からみた歴史的に (e.g、鱗翅目のテング機能関連飲むわら25,26);。ただし、実験的証拠が組み込まれた最近の研究は、昆虫の口器と構造機能関係2,12,13の複雑さの私達の理解のパラダイム シフトで起因しました。,22,...

多くの昆虫のグループ (proboscises) の口器がある適応流体を供給するため、花の蜜、果実の腐敗など sap フロー (例:双翅目1、鱗翅目2膜翅目3)、木部 (半翅目4)、涙 (鱗翅目5) と血 (目, シラミ目6ノミ7、双翅目7、8半翅目、鱗翅目9)。流体を餌に昆虫の能力は生態系の健全性 (例えば受粉10)、病気伝達4,11生物2,12、および研究に関連します。収斂進化13。多彩な食料源、にもかかわらずいくつかの流体餌昆虫の中でテーマとマイクロやナノ液滴、液膜または多孔性の表面に限定されるが、液体の少量を取得する機能です。
流体食性昆虫 (すべての動物種の14,の15の 20% 以上) と様々 な食料源を餌に能力の膨大な多様性を与えられた彼らの餌を理解動作流体吸収機構の解明、多くの分野で重要であります。昆虫の口器の機能、たとえば、バイオミメティック技術、例えば少量の流体が用いられるものと同様の方法を使用しての取得などのタスクを実行できるマイクロ流体デバイスの開発の役割を果たしています。昆虫16。流体吸収メカニズムの研究における根本的な問題は、流体、昆虫のフィード方法のみを決定するが、メカニズムをサポートして実験的証拠を取得します。のみ動作を使用して (例えば、テング12,17プロービング) 流体の正常な吸収を確認しませんも、それがルートを決定するための手段を提供するため、供給のための指標が十分なこと流体の旅行昆虫を通過するとき。また、少量より流体の実験を実行する流体が制限リソース2,12自然の供給シナリオを表します。
位相コントラスト イメージング多孔性の表面12から液体の少量で蝶がどのようにフィードを評価するオオカバマダラ (オオカバマダラl.) を併用した x 線します。モナルカ蝶フード運河に小さな毛穴に閉じ込められた流体をもたらすテングに沿って毛細管を介して表皮突起 (背 legulae) の間にスペースを使用します。着信の流体は、成長し高原不安定12,18, 頭に吸引ポンプの作用で、蝶の腸を運ばれる液体の橋に崩壊食品管壁のフィルムを形成します。X 線位相コントラスト イメージング昆虫12,19,20,21内の流体の流れを可視化するための最適なツールですが、テクニックはすぐに利用できるより便利メソッドは、昆虫の能力吸収液の迅速な評価のために必要ですし、摂取します。
D. plexippusの供給メカニズム適用他の鱗翅目に、ハエ (双翅目)、(両方のグループが多孔性の表面の液体のフィード) かどうかを決定する Lehnertら13には、ここで詳細に報告されている多孔性の表面の液体の少量を餌に昆虫の能力を評価するための手法が適用されます。ここで説明したプロトコルは接液部を使用して研究と多孔質の表面、プール供給メカニズムのアドレスなど、他の研究方法論を変更できます。また、アプリケーションは、マイクロ流体システムと材料技術など、あらゆる分野に拡張します。

<table>
	<tbody>
		<tr>
			<td>20% sucrose solution</td>
			<td>Domino Sugar</td>
			<td></td>
			<td>Sugar needed to produce the sucrose solution with dH2O</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X concentration diluted to 1X in dH2O for insect dissections</td>
		</tr>
		<tr>
			<td>Single depression concave slide</td>
			<td>AmScope</td>
			<td>BS-C6</td>
			<td>Slide is necessary for feeding stage setup</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY6004700 (60 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY4104700 (41 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY3004700 (30 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY2004700 (20 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY1104700 (11 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TCTP04700 (10 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TETP04700 (8 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TMTP04700 (5 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>RTTP04700 (1 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Iris microdissecting scissors</td>
			<td>Carolina Biological Supply Company</td>
			<td>623555</td>
			<td>Scissors used for dissections</td>
		</tr>
		<tr>
			<td>Insect pins (#1)</td>
			<td>Bioquip Products</td>
			<td>1208B1</td>
			<td>Pins used during dissections and feeding trials</td>
		</tr>
		<tr>
			<td>Extra-fine point dissecting forceps</td>
			<td>Carolina Biological Supply Company</td>
			<td>624684</td>
			<td>Dissecting equipment</td>
		</tr>
		<tr>
			<td>Leica M205 C Stereoscope</td>
			<td>Leica Microsystems</td>
			<td>M205 C</td>
			<td>Stereoscope used for dissections</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>Fluorescent microscope used to detect magnetic nanoparticles</td>
		</tr>
		<tr>
			<td>Fisherbrand PTFE Disposable Stir Bar</td>
			<td>Fisherscientific</td>
			<td>S68067</td>
			<td>Magnet used to detect nanoparticles</td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td>Tissues used to secure insects during feeding trials</td>
		</tr>
		<tr>
			<td>House fly (Musca domestica) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Blue bottle fly (Calliphora vomitoria) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Cabbage butterfly (Pieris rapae) larvae</td>
			<td>Carolina Biological Supply Company</td>
			<td>144102</td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Finnpipette F1&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4641080N</td>
			<td>micropipette for dispensing liquids</td>
		</tr>
		<tr>
			<td>Finntip 250 pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>9400250</td>
			<td>micropipette tips</td>
		</tr>
		<tr>
			<td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td>
			<td>AmScope</td>
			<td>CS-S24-100</td>
			<td>coverslips for viewing the insect&#39;s crop on confocal microscope</td>
		</tr>
	</tbody>
</table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

流体食性昆虫を摂取する環境では、プール、映画、または小さな毛穴に閉じ込められた液体の様々 な。口器の構造と機能の関係を評価する液体の獲得の研究が必要とただし、流体の吸収機構の解明は歴史的構造のアーキテクチャは、実験的証拠と時々 一人旅の観察から推論されます。蝶 (鱗翅目) とハエ (双翅目) の液体の少量を使用して流体吸収能力を評価する手法を報告する.昆虫は、特定の細孔径のフィルター ペーパーから蛍光、磁性ナノ粒子と混合 20% ショ糖液が供給されます。作物 (流体を格納するために使用される内部構造) は、昆虫から削除され、共焦点顕微鏡上に配置。磁石は昆虫が水分を摂取することができるかどうかを示すナノ粒子の存在を確認するための作物で手を振った。この手法を使用して、機構の解明、広範な供給 (毛細管と液体ブリッジの形成) 多孔性の表面からを供給するとき可能性のある鱗翅目、双翅目の間で共有されます。さらに、このメソッドは液餌昆虫、病気の感染、バイオミメティックス、ナノまたはマイクロ導管を含む可能性のある他の研究で重要なを含むさまざまな機能の研究使用できます、液体の輸送には、検証が必要です。

液餌昆虫の中で流体吸収能力のパターンの研究には、餌が発生したときの決意が必要です。ここで説明したプロトコルを使用して、鱗翅目、双翅目の13の間制限の細孔サイズ仮説をテストしています。制限の細孔サイズ仮説は、細孔径が供給管12の直径より小さい場合は、液体充填孔から液餌昆虫を与えることができない状態します...

Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

流体食性昆虫は、多孔性の表面の液体の微量を取得する能力を持っています。このプロトコルでは、直接蛍光、磁性ナノ粒子供給溶液を用いた多孔質表面から液体を摂取する昆虫の能力を決定する方法について説明します。

蛍光灯、摂取昆虫における流体吸収能力を決定するための磁気ナノ粒子

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

the-ingestion-fluorescent-magnetic-nanoparticles-for-determining

Research

JoVE Journal

Biology

6.3K ビュー.  Kent State University at Stark. 流体食性昆虫は、多孔性の表面の液体の微量を取得する能力を持っています。このプロトコルでは、直接蛍光、磁性ナノ粒子供給溶液を用いた多孔質表面から液体を摂取する昆虫の能力を決定する方法について説明します。

ビデオ: The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

咀嚼昆虫にシロイヌナズナの抵抗をテストするための選択肢と無選択アッセイ

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

カウントと培養細胞の生存率の決定

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

運動時のエネルギーシステムの貢献を決定する

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

白蝶貝からの内生菌の抽出方法<em&gt;ベミシア・タバチ</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

二重陽電子放出断層レントゲン写真撮影のため68Ga コアをドープした酸化鉄ナノ粒子の合成/(T1) 磁気共鳴イメージング

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

昆虫の臨界熱限界のハイスループットアッセイ

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

蛍光灯、摂取昆虫における流体吸収能力を決定するための磁気ナノ粒子

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