この研究は、カナダ農務·農産食品省·ベースプロジェクト &quot;マーカー選抜における多変量品質形質phenomicギャップを減らすために肉サンプルのハイスループット技術の開発&quot;の一部だった。牛肉ユニットとラコンブ研究センターで食肉加工スタッフの熟練した援助は心から感謝しています。著者はまたクリスティーヌバービッジ·ボイド、フランコステロ、Glynnis Crokenとローナ·サッカー専用の技術支援に感謝したい。

1。ステーキコレクション<ol><li> 24時間で次の等級- 48時間後に屠殺、 最長筋 （どちら胸部および/ ​​またはlumborum端）牛肉の死骸から除去し、品質分析のために収集されています。 </li><li>筋肉は皮下脂肪と二乗オフのトリミングされています。筋肉は、その後、安全性と食品取り扱いプロトコルに従って2.5センチメートル（1）ステーキにカットされています。個々のステーキの切断に続いて、適当なラベル（動物番号、側面、筋肉の位置、または時間）は、それぞれのステーキに配置されている。 </li><li>ステーキは、その後、保存のために用意されている。その鮮度を保つべきである場合は、トレー上に置き、グリルに取られる。新鮮なステーキをすぐに調理することができない場合は、調理脱水を防ぐために、開始することができるまで、プラスチックで覆われる必要がある。ステーキは、クーラーで熟成、または凍結する場合、重量は酸素不透過性の真空パッケージBAに置かれる前に、小数点以下に記録されますgおよび真空包装機を使用して密封された。ステーキは、その後、それぞれの長期保存場所に配置される。 </li></ol> 2。調理手順<ol><li>サンプルが凍結されている場合は、一晩解凍する4℃クーラーフリーザーと場所から削除します。 </li><li>調理、予熱の当日平炉グリル210℃のサンプル調理用C、および走査装置のコンセントに熱電対を挿入することにより、温度ロガーシステムとコンピュータを準備。調理が始まる前に約10分短縮野菜と軽くグリースグリル。 </li><li>調理を始める前に、クーラーからステーキを削除します。そのパッケージからサンプルを取り出し、ペーパータオルでステーキから余分な水分を吸い取る。小数点第2位ステーキの生の重さを記録します。 </li><li>その縦軸に沿ってステーキの中点に熱電対を挿入します。調理情報を記録するために熱電対スキャンを開始する。 </li><li>置きグリルステーキは、ステーキとグリルの平らな調理表面に熱電対と平行を保つ。プローブは、平行に保持されていない場合は、不適切な温度測定値をもたらすことができる。 </li><li> 35.5の内部温度にステーキを調理°Cは、その後、サンプルを裏返して71の最終的な内部温度に調理続ける℃まで内部温度が71℃になったら、グリルからサンプルを削除して、ステーキの熱電対を引き出します。 </li><li>調理重量が必要な場合は、（1-2分）ステーキが少し冷まし、その後、余分な水分を除去し、小数点以下にサンプルを比較検討するペーパータオルでサンプルを吸い取る。 </li><li>調理情報が必要な場合は、熱電対スキャンデータを保存する。 </li></ol> 3。角度調整とサンプルの調製<ol><li>四角いエンドを作成するためにステーキの横端より2センチメートル - サンプルはまだグリルから暖かいですが、カットの1を行います。 </li><li> WID渡る5センチメートルセクションステーキのthはオリジナルのカットと平行に、5センチメートルでナイフノッチ &#39;サンプルサイジングボックス&#39;を使用することによって調製される。 </li><li>筋線維角の向きがステーキのナイフ切断面に分度器を配置することによって測定することができる。 </li><li>可変角度切断ボックスをダブルブレード付きナイフを案内するための底穴にカスタマイズされた下部金属板をスライドさせて試料の対応する角度に設定されている。対応する角度で下部金属プレートを固定するためにノブを締めます。 </li><li>筋線維の方向は二重ナイフブレード付きの角度と平行になるように可変角度切削ボックスの切断領域にステーキ5cmのセクションを配置。 </li><li>ダブルブレードナイフは以前にカット5センチメートルピース全体で1センチメートルスライスをカットするために使用されているので、ナイフは対応する角度で、サンプル全体を通過することを確認してください。ステーキを配置するとき、それに最も近いカッティングエリアの端に触れていることを確認カッティングモーションのようなユーザには、ユーザの方にステーキを引っ張ってくる。 </li><li> 、カットカッティングボックスに二重刃ナイフを挿入し、1センチメートルストリップに肉をカットするナイフを引っ張って。ユーザーに向けてナイフを引きながら肉の引き裂き防止するために、若干のソーイングモーションを使用しています。等しい切削プロポーションを維持するために、ガイドプレートの一辺に対してナイフの刃を保つ。 </li><li>その結果、繊維は一貫してスライスに平行に走ると、長さ5cmである暖かい、厚さ1cmのスライスにする必要があります。このスライスは、その後、肉テクスチャーのスライス剪断力解析のために使用される。 </li></ol> 4。剪断力解析<ol><li>せん断力解析を確認するには、スライスせん断力プロトコル（SSF）は可変角度切断ボックスを用いて得られたスライス5センチメートルを1センチ使用しています。平らなせん断スライスブレードでテクスチャー分析機を使用して、分析が暖かいスライス上で実行される。 </li><li> 50キロのロードセルとセットでテクスチャーアナライザーを準備500ミリメートル/分のクロスヘッド速度は48ミリメートルまで剪断距離。 </li><li>せん断は、スライス内せん断繊維をするために必要な最大の力の正確な分析を提供するために、筋線維に一度、垂直に実行されます。 </li><li>スライスが中心を通って完全にせん断された後、データを保存して記録します。 </li></ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+Texture+and+Juiciness.">Beef Texture and Juiciness.</a> Handbook of meat and meat processing. , 177-206 (2012).</li><li>Kerth, C. R., Montgomery, J. L., Lansdell, J. L., Ramsey, C. B., Miller, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+gradient+in+longissimus+steaks.">Shear gradient in longissimus steaks.</a> Journal of Animal Science. 80, 2390-2395 (2002).</li><li>Janz, J. A. M., Aalhus, J. L., Dugan, M. E. R., Price, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mapping+method+for+the+description+of+Warner-Bratzler+shear+force+gradients+in+beef+Longissimus+thoracis+et+lumborum+and+Semitendinosus.">A mapping method for the description of Warner-Bratzler shear force gradients in beef Longissimus thoracis et lumborum and Semitendinosus.</a> Meat Science. 72, 79-90 (2006).</li><li>Derington, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+slice+shear+force+and+Warner-Bratzler+shear+force+of+beef+strip+loin+steaks+as+related+to+the+tenderness+gradient+of+the+strip+loin.">Relationships of slice shear force and Warner-Bratzler shear force of beef strip loin steaks as related to the tenderness gradient of the strip loin.</a> Meat Science. 88, 203-208 (2011).</li><li>Shackelford, 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=Slice+Shear+Force.">Slice Shear Force.</a> Beef Facts. , (2009).</li><li>Henrickson, R. L., Mjoseth, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenderness+variation+in+two+bovine+muscles.">Tenderness variation in two bovine muscles.</a> Journal of Animal Science. 23, 325-331 (1964).</li><li>Ju&#225;rez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+relative+contribution+of+ante-+and+post-mortem+factors+to+the+variability+in+beef+texture.">Quantifying the relative contribution of ante- and post-mortem factors to the variability in beef texture.</a> Animal. FirstView, 1-10 (2012).</li><li>Shanks, B. C., Wulf, D. M., Maddock, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electric+belt+grill,+forced-air+convection+oven,+and+electric+broiler+cookery+methods+for+beef+tenderness+research.">Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research.</a> Meat Science. 58, 239-246 (2001).</li><li>Aalhus, J., Ju&#225;rez, M., Aldai, N., Uttaro, B., Dugan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+preparation+and+eating+quality.">Meat preparation and eating quality.</a> , 1058-1063 (2009).</li><li>Sa&#241;udo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+slaughter+weight,+breed+type+and+ageing+time+on+beef+meat+quality+using+two+different+texture+devices.">The effects of slaughter weight, breed type and ageing time on beef meat quality using two different texture devices.</a> Meat Science. 66, 925-932 (2004).</li></ol>

我々は、開示することは何もありません。

角度調整可能なボックスがSSFブレードがなく、常に一定の45°の角度のカット8で発生する可能性がほぼ垂直ではなく、真の垂直の角度で筋線維をハサミが保証されます。可変角度切削ボックスのアプリケーションでは、サンプル中の最大せん断力のより正確な描写を可能にし、それは可変角度切削ボックスが開発された客観的な品質分析技術を向上させるこれに基づいている。また、...

痛みは、肉1で最も重要な品質特性の一つである。牛肉の圧痛で矛盾は牛肉産業2が直面する主要な問題の一つとして同定されている。それは間違いなく最高を示しているインストゥルメンタルの方法であるとして、精密機械加工せん断プレートの三角形の穴が特徴ワーナー·Bratzlerせん断力（WBSF）テストでは、、、肉感覚優し3,4を示すために使用される最も普及している方法です肉タフネス5官能パネルのスコアとの相関。しかし、スライスせん断力プロトコル（SSF）は、標準WBSFプロトコル7に代わるものとして、筋肉の質感と優しさ6を分析するための重要な技術となっている。これは、迅速な分析やサンプル数が多いが、処理される必要がある場合において有益である。それがまだ暖かいときSSFの場合、1つのコアがステーキから取られているのに対し、複数のコア（3-6）は、通常、船尾ステーキから取らER WBSF 6冷凍の24時間。それは優しさは、平均WBSF最良の表現を持つステーキの中心と真ん中で、ステーキ9の内側側面に横方向の間で変化することが見出されているように、この1スライスから、SSFは、全体のステーキ8の平均の質感を分析。 SSFの崩壊はステーキ内に、横方向から内側側面に、剪断力の値9にばらつきがあるということであるが、常に5センチメートル部を作るためにサイジングボックスを使用し、それは来ることばらつきを低減することができるステーキの異なるセクションを使用してから。いくつかのステーキのサイズが異なるしかし、5センチスライスは次にせん断力値10に影響を与える可能性がステーキ、上の別の場所で発生する可能性があります。 一方、SSFプロトコルで分析するセクションを取得するために設計された元の切削ボックスは、2つの固定角度が、45°と90°を可能にするが、筋ステーキ内、筋肉11内の繊維配向が変化します。 Shackelfordとウィーラー12が平均最長角が43.8に近い45℃であった、°、サンプルは常に同じ場所から採取されたとき、したがって適切であると考えていたと述べた。しかし、Derington ら 11は 33.1°と53.9°の間で最長の筋に沿って繊維の角度の範囲を報告した。したがって、同じ動物からの複数のステーキを分析する必要がある場合、研究目的のために、45°又は90°のいずれかにすべてのファイバ角を割り当てると、潜在的に精度が低下する。切断角度を測定し、調整する能力を有するスライス一貫して筋線維と並列に実行することができるように可変角度切断·ボックスの使用は、最大せん断力のより正確な描写を提供することができる。

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Vacuum Packager</td>
			<td>Koch Equipment, Kansas City, MO, USA</td>
			<td>Model UV2100</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Winpak Ltd., Winnipeg, MB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocouples and Scanning Device</td>
			<td>Agilent / Hewlett Packard, Santa Clara, CA, USA</td>
			<td>34970A Data Acquisition Switch Unit</td>
		</tr>
		<tr>
			<td>Grill</td>
			<td>Garland Commercial Ranges Ltd., Mississauga, ON, Canada</td>
			<td>Model ED-30-B</td>
		</tr>
		<tr>
			<td>Crisco Vegetable Shortening</td>
			<td>The J.M. Smucker Company, Orrville, OH, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sample sizing box</td>
			<td>G-R Manufacturing Co., Manhattan, KS, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Angle adjustable box</td>
			<td>Innovation Centre, Red Deer College, Red Deer, AB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texture Analyzer Machine</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>Model TA-XT Plus</td>
		</tr>
		<tr>
			<td>Load Cell, 50 kg</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-XT Plus</td>
		</tr>
		<tr>
			<td>USDA Warner-Bratzler knife w/guillotine block</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7</td>
		</tr>
		<tr>
			<td>Flat rectangle Blade for Slice Shear Force</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7C</td>
		</tr>
	</tbody>
</table>

introducing an angle adjustable cutting box for analyzing slice shear force in meat

研究では、 最長筋の繊維角度がステーキ内および筋肉を通して場所に応じて変更することができることを示します。代わりに元の固定45°または90°の剪断力をテストするための切断角度を使用するナイフの角度は、各サンプルの繊維角度に対応するように、可変角度切断ボックスを調整することができる。 2分以内に71の内部温度に調理した後、210℃に設定平炉グリルC℃、5センチコア1 cmの2ナイフの刃を使用して、筋線維方向と平行に、ステーキから切断されて1セット離れてセンチ。このウォームコアは次いで、肉の質を評価するためにスライスせん断力プロトコル（SSF）に供される。可変角度カッティングボックスとSSFプロトコルの使用は、スライスや筋肉繊維が常に平行になるように、最大​​のせん断力の正確な表現を提供します。したがって、可変角度切断ボックスは、SSFプロトコルと併せて、高い番目として使用することができる他の筋肉の潜在roughput正確最長筋の異なる場所に肉の柔らかさを評価するための技術と、、。

分割された - 三十一商業牛肉牛が屠殺され、その死体（601キロ528）が終了しました。左側は2℃で3時間後、および、10℃で保持した一方優しさの変動を作成するために、左右を直ちに、2°Cで貯蔵した。左右両側から最長筋肉が虐殺24時間後に削除された。左サイドからのサンプル（ &quot;TENDER&quot;治療は）分析される前に6日間熟成させている間右側（ &quot;TOUGH&quot;処理）からのサンプル?...

Watch this Scientific Journal Video about Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat at JoVE.com

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

スライス剪断力は、牛肉のテクスチャ解析のための参照方法である。角度調整可能な切削ボックスを使用して、研究目的のためにその精度を高めることができる。内の別の場所からの結果<em&gt;最長</em&gt;筋肉がワーナー·Bratzlerせん断力の方法論とは異なる筋肉のための高い潜在的な適応性と高い相関を示しています。

肉のスライスせん断力を分析するための角度調整可能なカッティングボックスをご紹介します

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

introducing-an-angle-adjustable-cutting-box-for-analyzing-slice-shear

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JoVE Journal

Biology

12.8K ビュー.  Agriculture and Agri-Food Canada. スライス剪断力は、牛肉のテクスチャ解析のための参照方法である。角度調整可能な切削ボックスを使用して、研究目的のためにその精度を高めることができる。内の別の場所からの結果最長筋肉がワーナー·Bratzlerせん断力の方法論とは異なる筋肉のための高い潜在的な適応性と高い相関を示しています。

ビデオ: Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

生物学

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR -レポーターコンストラクトとマイクロRNAを使用してLightSwitchルシフェラーゼアッセイシステムによるヒトマイクロRNAの識別対象は付着細胞へのミミック

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

比色金ナノ粒子アッセイに適用される構造スイッチングアプタマーを選択するための方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

インフィールドアプリケーションの設計およびアプタマー金ナノ粒子基づく比色アッセイの開発

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

フローサイトメトリーによる生体内におけるタンパク質自己集合体の検出と特性特性

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

イエバエ複眼の視覚空間の自動チャート作成

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

実験室条件における葉切りアリ(atta)の収集と長期維持

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...