这项研究是加拿大农业和农业食品部的A-基地项目“开发高通量技术对肉类的样品，减少phenomic的多元品质性状的分子标记辅助选择”差距。牛肉LACOMBE研究中心的单位和肉类加工人员熟练的帮助感到由衷的赞赏。作者还要感谢专门的技术援助，弗兰恭波比吉-博伊德·科斯特洛，Glynnis Croken和罗娜萨克尔。

1。牛排收藏<ol><li>分级后24小时- 48小时后屠宰， 背最长肌（ 胸椎和/或lumborum的的结束）将从牛肉尸体收集质量分析。 </li><li>肌肉修剪皮下脂肪和平方关闭。安全和食品处理协议后，肌肉，然后切成2.5厘米（1英寸）牛排。个别牛排的切断之后，适当的标签（动物数，侧面，肌肉的位置，或时间）被放置在各自的牛排。 </li><li>牛排，然后准备进行存储。如果他们要保持新鲜，他们都放在一个托盘上，采取的烧烤。如果不能立即烹煮新鲜的牛排，他们需要用塑料覆盖，直到可以开始烹饪，以防止脱水。如果要在一个凉爽岁或冻结的牛排，重量被记录到小数点后两位之前被放置在不透氧真空包装BAg和密封使用的真空包装机。牛排，然后放置在各自的长期存储位置。 </li></ol> 2。烹饪过程<ol><li>如果样品已被冻结，删除从冷冻库，在4℃冷却器解冻过夜的地方。 </li><li>烹饪天，平炉预热烤炉至210°C为样品烹调，并准备插入到网点的扫描装置的热电偶的温度记录器的系统和计算机。抹油烤蔬菜烹饪开始前约10分钟缩短。 </li><li>开始做饭前，取出牛排从冷却器。从包中取出的样品，从牛排用纸巾吸干多余的水分。记录的原始重量的牛排到小数点后两位。 </li><li>将牛排的中点沿其纵向轴线成一个热电偶。启动热电偶扫烹调数据记录。 </li><li>将牛排在烤架上，保持平行平烹调的牛排和烧烤表面热电偶。如果探头不能保持平行，它可以导致不正确的温度读数。 </li><li>库克牛排内部温度为35.5°C，则翻转样品和继续烹调末期内部温度为71°C。一旦内部温度是71°C，取出样品从烤架拉热电偶的牛排。 </li><li>如果烹饪的重量，让牛排略有降温（1-2分钟），然后涂抹样品用纸巾去除多余的水分和称量到小数点后两位。 </li><li>如果烹调数据是必需的，节省热电偶的扫描数据。 </li></ol> 3。角度调整和样品制备<ol><li>虽然样品仍然是温暖的烧烤，切1 - 2厘米的牛排从外侧端创建一个正方形端。 </li><li>整个WID部分5厘米次的牛排的制备是使用样品尺寸框中，然后用小刀在5厘米的槽口，平行于原来的切割。 </li><li>肌纤维角度的方向放置量角器牛排刀切割表面上可以测量。 </li><li>的可变角度切割方块然后被设置为相应的角度的样品通过滑动与底部的孔，用于指导双刃刀定制的下部金属板。拧紧旋钮下部金属板固定在相应的角度。 </li><li>将5厘米的边的牛排进入切削区的可变角度切割框中，然后使肌纤维方向平行的双刃刀的角度。 </li><li>双刃刀是用来切1厘米的片，跨越先前切成5厘米的一块，所以在相应的角度，确保刀将通过整个样本。放置时，牛排，确保其接触最靠近切削区的边缘用户，切割运动将拉动向用户牛排。 </li><li>切割，切割框中插入双刃刀，拉刀，切肉成1厘米的条状。为了防止撕裂的肉，用轻微的锯切运动，拉刀的同时向用户。保持对的导向板的一个边缘的刀片，以保持相等的切割比例。 </li><li>结果应该是一个温暖，1厘米厚5厘米长的片，与纤维始终运行平行的切片。此片，然后用切​​片纹理肉的剪切力分析。 </li></ol> 4。剪切力分析<ol><li>要确定剪切力分析，切片剪力协议（SSF）使用1厘米5厘米切片获得可变角度切割盒。使用一个纹理分析仪一台剪切机，薄刀片，温暖切片进行分析。 </li><li> 50公斤称重传感器和一套准备纹理分析器剪切距离48毫米，500毫米/分钟的十字头速度。 </li><li>剪切被执行一次，肌纤维垂直剪切切片中的纤维所需的最大力，提供准确的分析。 </li><li>一旦已将片完全通过中心剪切，保存并记录数据。 </li></ol>

<ol>
	<li>Jayasooriya, S. D., Torley, P. J., D'Arcy, B. R., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+power+ultrasound+and+ageing+on+the+physical+properties+of+bovine+Semitendinosus+and+Longissimus+muscles.">Effect of high power ultrasound and ageing on the physical properties of bovine Semitendinosus and Longissimus muscles.</a> Meat Science. 75, 628-639 (2007).</li><li>Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+proteinases+and+meat+aging.">Muscle proteinases and meat aging.</a> Meat Science. 36, 93-104 (1994).</li><li>Lepetit, J., Culioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+meat.">Mechanical properties of meat.</a> Meat Science. 36, 203-237 (1994).</li><li>Honikel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+methods+supported+by+OECD+and+their+use+in+Mediterranean+meat+products.">Reference methods supported by OECD and their use in Mediterranean meat products.</a> Food Chemistry. 59, 573-582 (1997).</li><li>Monin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+methods+for+predicting+quality+of+whole+meat.">Recent methods for predicting quality of whole meat.</a> Meat Science. 49, S231-S243 (1998).</li><li>Shackelford, S., Wheeler, T., Koohmaraie, M. <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+slice+shear+force+as+an+objective+method+of+assessing+beef+longissimus+tenderness.">Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.</a> Journal of Animal Science. 77, 2693-2699 (1999).</li><li>Ju&#225;rez, M., Hui, Y. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+The+effect+of+freezing+on+Warner-Bratzler+shear+force+values+of+beef+longissimus+steaks+across+several+postmortem+aging+periods.">Technical note: The effect of freezing on Warner-Bratzler shear force values of beef longissimus steaks across several postmortem aging periods.</a> Journal of Animal Science. 80, 2122-2125 (2002).</li><li>Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., Dikeman, M. 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>

我们什么都没有透露。

角度可调箱，确保社保基金理事会刀片总是剪的肌肉纤维，而不是一个真正的垂直角度近似垂直，这可能发生在一个固定的45°角切8。可变角度切割中的应用可以更准确地描绘样品中的最大剪切力，它是在此基础上改善客观的质量分析技术，可变角度切割盒的开发。此外，提出的角度可调箱（30°〜60°）的范围广泛的角度允许样品制备的从肌肉背以外的SSF分析。然而，双刃刀，设置1...

嫩度是肉1中的最重要的质量属性之一。牛肉嫩度不一致已确定为牛肉行业面临的主要问题之一。在Warner-BRATZLER剪切力（WBSF）试验，其特征在于在一个精密加工的抗剪板由一个三角孔，是最普遍的用于指示的肉感觉压痛3,4的方法，因为这是仪器的方法，该方法可以说是表现出最好的肉韧性5品评分数的相关性。然而，切片剪切力协议（SSF）已成为一个重要的技术用于分析肌肉纹理和触痛6，作为一种替代的是标准WBSF协议7。在快速分析大量的样品需要进行处理的情况下是有益的。对于社保基金中，只有一个核心，从牛排时，它仍然是温暖的，与多个内核（3-6）从牛排通常船尾ER 24 ​​小时制冷了WBSF 6。从这个片，SSF分析整个牛排8的平均纹理，因为它已被发现，压痛牛排9之间的外侧向内侧的侧变化，具有最适合表示平均WBSF牛排的中心和中间。社保基金的垮台是，在牛排，从外侧到内侧的两边，有变化的剪切力值9;然而，一贯使用的大小方块，使5厘米的部分，它可以减少变异，能来从使用不同路段的牛排。然而，由于一些牛排的大小不同，有5厘米的片，可能会出现在不同的位置上的牛排，这可能会影响剪切力值10。 另一方面，原来的切割设计的框中，然后取得部分中待分析的SSF协议允许只有2个固定角度，45°和90°，但是，肌肉纤维取向的变化，牛排，内和肌肉内的11。 12沙克尔福德和惠勒说，平均背角为43.8°，这是接近45°，因此认为适当时，总是从同一地点采集样品。然而，Derington 等 11报道沿最长肌纤维33.1°和53.9°之间的角度的范围中。因此，当用于研究目的，从同一动物的一些牛排需要加以分析，分配的所有光纤的角度为45°或90°准确性可能降低。使用一个可变的角度切割中可能提供一个更精确的描述的最大剪切力，具有的容量测量和调整的切削角，允许切片始终如一地与肌肉纤维平行运行。

<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

研究指示的最长肌纤维角度可以不同，取决于内的牛排和整个肌肉的位置时。 ，而不是使用原有固定的45°或90°的切割角度剪切力测试中，一个可变的角度切割方块的刀的角度可以调整，使对应于各样品的纤维角度。蒸煮后内部温度为71℃下在2分钟内上设置平炉格栅，在210℃，1厘米×5厘米的芯被切断的牛排，肌纤维方向平行，使用2个刀片组1除了厘米。这个温暖的核心，然后进行切片的剪力协议（SSF）评估肉质。可变角度切割方块和SSF协议的使用提供的最大剪切力的精确表示，作为切片和肌纤维始终平行。可变角度切割中，因此，在与SSF协议一起，可以用作高第roughput技术来准确地评估在不同的位置的最长肌肉的嫩度，并可能在其他的肌肉。

三十一成品商业牛肉肉牛屠宰和分割它们的尸体（528 - 601公斤）。为了创造变异压痛，右两侧立即被存储在2°C，同时举行了3小时，然后在2°C在10°C左右两侧左，右两侧的背肌肉被拆除屠宰后24小时。从右侧（“TOUGH”的待遇）的样品进行分析的那一天，而从左侧的样本（“招标”的待遇）账龄分析前6 D。制备了两对牛排前（5 日 -6 日 胸椎 ），中（12 日 -13 <...

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

切片剪切力是牛肉的纹理分析的参考方法。使用角度可调切削中，可能会增加其准确性用于研究目的。内的不同位置的结果<em&gt;背</em&gt;肌肉表现出很高的与华纳BRATZLER的剪切力的方法和针对不同的肌肉潜在的适应性高的相关性。

介绍一个角度可调切割盒切片肉的剪切力分析

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

Research

JoVE Journal

Biology

12.8K 次观看.  Agriculture and Agri-Food Canada. 切片剪切力是牛肉的纹理分析的参考方法。使用角度可调切削中，可能会增加其准确性用于研究目的。内的不同位置的结果背肌肉表现出很高的与华纳BRATZLER的剪切力的方法和针对不同的肌肉潜在的适应性高的相关性。

视频: 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. 

器官型E18大鼠脑切片文化

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

科研

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.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

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.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

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.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

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 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

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.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

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.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

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.

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

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.

家蝇复眼视觉空间自动制图

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.

在实验室条件下收集和长期维护切叶蚁（Atta）

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