作者感谢芭芭拉Berli提供的示例数据和加拿大野生生物联合会的盛情资金。

1。捕捉和驯化<ol><li>使用无结网，收集他们的保温水箱的个人鱼。最小采集时间，避免收集多种鱼类和不持有超过净几秒钟的鱼，因为这些因素可以增加压力。压力可以影响游泳的表现。 </li><li>放置鱼在麻醉或直接向游泳隧道呼吸计（“游泳隧道”）转让坦克。如果鱼被麻醉后，无论是tricaine磺酸（MS222），或可以用丁香油。可接受的浓度将取决于许多因素，包括物种的灵敏度和温度，但通常范围从10-100毫克/ L为一麻醉1,2。麻醉药浓度应诱导第3阶段的麻醉（即对外界刺激的反应迟钝），并允许快速恢复（即秒）。使用淡水MS222时，应使用碳酸氢钠等份（质量）作为缓冲。无缓冲，MS222将大大降低pH值，低pH值可能会损害鳃组织。缓冲是在海水中的必要，因为它包含足够的缓冲能力。 </li><li>可以绝育手术，麻醉鱼的质量和长度的测量，并给出一个粗略的外部考试对​​自己的病情。长度可被视为总长度（TL），叉长（FL）或身体的长度（BL）。这些不同的指标通常是根据标准的做法，种形态（如没有分叉鳍）或鱼的条件（如尾鳍磨掉）。 </li><li>麻醉和非麻醉鱼类应尽快和流动的水，因为这将有助于恢复到在游泳隧道引入。流速通常是这样，鱼将能够在底部休息而勉强游泳。一个成年红鲑率是0.3 BL / s的</li><li>鱼需要留在游泳隧道驯化。驯化期间可以设置传统的周期（如12小时），或可高达30分钟的简短的最新研究中所使用的。要确定一个理想的驯化期，对于一个给定的鱼，达到一个稳定的，最好是低，代谢率可使用（详细下一节）的时间。 </li></ol> 2。测量代谢率<ol><li>在游泳隧道内氧气的变化应当记录在游泳试验的持续时间（S）。氧探头可以直接进入游泳隧道被封闭，但是，它必须首先确定水流量超过探头是否会影响探头的功能。如果探头是由水的速度，在测试过程中可能出现的影响，探头将需要被放置在隧道外的自己的商会，并与隧道水由泵提供。 </li><li>为了补充氧气和减轻废物堆积，游泳隧道可能会需要定期刷新。氧饱和度不得低于70％下降为大多数物种，作为游泳的性能可能会降低或有可能是无氧代谢的转变。法拉盛隧道水，也可能有助于降低水温的任何增加，但是，水的温度应举行由耦合游泳隧道冷却器不断。温度的变化会影响代谢率。 </li><li>常规和最大代谢率，缩写“，RMR”和“孕产妇死亡率”，通常是确定的（图1A）。这些费率可以在体积（V O 2）或质量（M O 2）每单位质量的氧气表示，单位时间内，如mg O 2 /公斤/小时。可以采取两种税率之间的差异“活动的范围”。 “氧债”下面的练习中，被称为“过量运动后耗氧量（EPOC），也可确定和等于在运动后返回，RMR所需要的氧气。 </li></ol><ol><li>注：许多氧探头，其输出会自动提供溶解氧温度校正的变化。那些不将需要予以纠正。此外，一些值需要加以纠正，如果在盐碱条件下进行测量。 </li></ol>游泳的性能测量各种游泳表现能力，可以评估在游泳隧道。最常见的方式来刻画游泳的表现是它可以维持3的时间。速度越慢，它可以维持，反之亦然。速度一般被称为“爆裂”，“长期”或“持续”，它们分别对应秒的持续时间，以小时（MAX = 200分钟）分钟，小时，超出（&gt; 200分钟），分别。要确定这些速度，鱼类受到用户定义的“步骤”，设置“高度”，即水流的速度增加，和“长度”，即一步期限。台阶高度最大化和最大限度地减少步长将捕捉到的最大的连拍速度，同时做反向，将捕获的最大持续速度。在步骤测试，传统的方式来报告所取得的最大速度是相等的全面完成的最后一步加的决赛中，未完成的步骤（图1A）的时间部分的速度。 突发游泳绩效评估 <ol><li>为了捕捉突发游泳或冲刺能力，下面的驯化，估计最高（如两次ü 暴击带来的流动速度，见下文），并激励游泳的鱼。鱼应休息室后部流动的速度迅速增加。鱼可能需要动机游泳。动机可机械（如触摸）或电（如适用于游泳隧道背面冲击电网充了一小）。 虽然没有特别的突发能力的测试，鱼也可以得到一个“不断加速测试（CAT 或 UΔV）组成的几个步骤，持续时间短（如1 分钟 ） 4,5。 </li><li>记录游泳时间。个别突发的数量和他们的距离，也可以记录 6。速度可以得到最大达到或按图1a中的方程计算。 长期和持续的游泳绩效评估 使用最频繁的措施，估计长期或持续的游泳能力被称为“临界游泳的表现”（U暴击）。步长为 U 暴击历来被设置在20分钟，虽然也被用于较短的时间，如10分钟。一个一般的规则是，至少有十个步骤之前，应采取疲劳，这可能会导致在长时间的测试，如3小时/鱼。为了缩短美国 暴击测试，最初的七个步骤可缩写为5分钟（测试，然后是所谓的“斜坡- U 的暴击” ）。 </li></ol><ol><li>经过驯化和/或做法游泳，增加流动的速度，根据步长。 </li><li>要设置适当的步骤为 U 暴击高，它可能会是必要的，估计一个被测试的鱼样本 ü暴击。这可以通过使用文献值。另外，鱼类可以得到一个“实践游泳”后引入隧道。对于这一点，给鱼短，用户定义的步骤，直到疲劳。使用作为一个早期的 U 暴击估计达到最终速度。每条鱼的调整步长大小（高度和长度）。 </li><li>为了测试恢复能力或性能确定的个体差异，鱼可给予第二次游泳测试后， 回收期 7-9 。 </li><li>鱼也可以在学校进行测试，虽然鱼往往在不同的时间的疲劳，在测试过程中可问题，他们从隧道中取出和消除氧气的吸收能力来衡量。 </li><li>一旦疲劳，鱼应该允许恢复或组织取样立即删除。 游泳相关的一些性能指标可以收集在测试过程中或以后使用视频分析（图1 C，D）。 </li><li>要测量通风率和尾部摆动频率，鳃盖抽水率和尾巴拍应超过几秒钟的时期（图1B）10,11采样。 </li><li>要衡量尾击败幅度最大挠度，尾鳍可以进行测量（图1B）12。 </li><li>以下测试，鱼可以安乐死，给予了验尸检查和收集，以确定血细胞比容（盒装红细胞体积），应激激素的浓度，肌肉能量储备（如磷酸肌酸，糖原），酶的活动（如乳酸脱氢酶，乙酰胆碱酯酶的组织），或其他生理参数。 </li></ol> 3。代表性的成果<img src="/files/ftp_upload/2572/2572fig1.jpg" alt="图1" /> 图1 A）的一个例子斜坡- U的暴击测试少年鲑鱼孵化场提出的虹鳟鱼（Oncorhynchus mykiss）的大规模，6.3厘米，身体长度（BL）和3.5克。测试进行了修改布雷特式呼吸仪（枪乌贼系统，丹麦，一个12升<a href="http://www.loligosystems.com/">www.loligosystems.com</a> ）。常规和最大代谢率计算斜坡（毫克/小时）×容积（L）/鱼质量（kg），分别为198和961 毫克 O 2 /公斤/小时，分别为（为活动范围= 4.84） 。这些费率由Brett报道少年红鲑（O. nerka）13。注：氧摄取量计算，水的体积等于呼吸计数量少，鱼量。交叉表示点里的鱼拒绝进一步游泳， 其中 U 暴击计算。二）尾摆动频率值均录得超过10秒六倍;尾击败振幅值均录得三倍;值显示是平均值± SEM。 c）在扩大后的侧视图和D）的内游泳室的鱼游泳的顶视图。 d）显示EthoVision软件（Noldus，荷兰追踪鱼; <a href="http://www.noldus.com/">www.noldus.com</a> ）。缩写：ü 暴击 =临界游泳速度; ü F =速度全面完成的最后一步，U S =步的速度，T F =最后一步花的时间 ，T S =步的时间。

<ol>
	<li>Anderson, W. G., McKinley, R. S., Colavecchia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+clove+oil+as+an+anesthetic+for+rainbow+trout+and+its+effects+on+swimming+performance.">The use of clove oil as an anesthetic for rainbow trout and its effects on swimming performance.</a> North Am. J. Fish Manage. 17, 301-307 (1997).</li><li>Cho, G. K., Heath, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+tricaine+methanesulphonate+(MS222)+and+clove+oil+anaesthesia+effects+on+the+physiology+of+juvenile+chinook+salmon+Oncorhynchus+tshawytscha+(Walbaum).">Comparison of tricaine methanesulphonate (MS222) and clove oil anaesthesia effects on the physiology of juvenile chinook salmon Oncorhynchus tshawytscha (Walbaum).</a> Aquacult. Res. 31, 537-546 (2000).</li><li>Beamish, F. W. H., Hoar, W. S., Randall, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swimming capacity in Fish Physiology. , 101-187 (1978).</li><li>Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+swimming+performance+in+rainbow+trout+using+constant+acceleration+and+critical+swimming+speed+tests.">Comparisons of swimming performance in rainbow trout using constant acceleration and critical swimming speed tests.</a> J. Fish Biol. 72, 693-710 (2008).</li><li>Tierney, K. B., Casselman, M., Takeda, S., Farrell, A. P., Kennedy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+cholinesterase+inhibition+and+two+types+of+swimming+performance+in+chlorpyrifos-exposed+coho+salmon+(Oncorhynchus+kisutch).">The relationship between cholinesterase inhibition and two types of swimming performance in chlorpyrifos-exposed coho salmon (Oncorhynchus kisutch).</a> Environ. Toxicol. Chem. 26, 998-1004 (2007).</li><li>Martinez, M., Bedard, M., Dutil, J. -. D., Guderley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+condition+of+Atlantic+cod+(Gadus+morhua)+have+a+greater+impact+upon+swimming+performance+at+Ucrit+or+sprint+speeds.">Does condition of Atlantic cod (Gadus morhua) have a greater impact upon swimming performance at Ucrit or sprint speeds.</a> J. Exp. Biol. 207, 2979-2990 (2004).</li><li>Marras, S., Claireaux, G., McKenzie, D. J., Nelson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+and+repeatability+in+aerobic+and+anaerobic+swimming+performance+of+European+sea+bass,+Dicentrarchus+labrax.">Individual variation and repeatability in aerobic and anaerobic swimming performance of European sea bass, Dicentrarchus labrax.</a> J. Exp. Biol. 213, 26-32 (2010).</li><li>Kolok, A. S., Plaisance, E. P., Abdelghani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+in+the+swimming+performance+of+fishes:+an+overlooked+source+of+variation+in+toxicity+studies.">Individual variation in the swimming performance of fishes: an overlooked source of variation in toxicity studies.</a> Environ. Toxicol. Chem. 17, 282-285 (1998).</li><li>Tierney, K. B., Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationships+between+fish+health,+metabolic+rate,+swimming+performance+and+recovery+in+return-run+sockeye+salmon,+Oncorhynchus+nerka+(Walbaum).">The relationships between fish health, metabolic rate, swimming performance and recovery in return-run sockeye salmon, Oncorhynchus nerka (Walbaum).</a> J. Fish Dis. 27, 663-671 (2004).</li><li>Webber, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upstream+swimming+performance+of+adult+white+sturgeon:+Effects+of+partial+baffles+and+a+ramp.">Upstream swimming performance of adult white sturgeon: Effects of partial baffles and a ramp.</a> Trans. Am. Fish. Soc. 136, 402-408 (2007).</li><li>Farrell, A. P., Gamperl, A. K., Birtwell, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+swimming,+recovery+and+repeat+swimming+performance+of+mature+sockeye+salmon+Oncorhynchus+nerka+exposed+to+moderate+hypoxia+and+pentachlorophenol.">Prolonged swimming, recovery and repeat swimming performance of mature sockeye salmon Oncorhynchus nerka exposed to moderate hypoxia and pentachlorophenol.</a> J. Exp. Biol. 201, 2183-2193 (1998).</li><li>Flammang, B. E., Lauder, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speed-dependent+intrinsic+caudal+fin+muscle+recruitment+during+steady+swimming+in+bluegill+sunfish,+Lepomis+macrochirus.">Speed-dependent intrinsic caudal fin muscle recruitment during steady swimming in bluegill sunfish, Lepomis macrochirus.</a> J. Exp. Biol. 211, 587-598 (2008).</li><li>Brett, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+metabolism+and+swimming+performance+of+young+sockeye.">The respiratory metabolism and swimming performance of young sockeye.</a> J. Fish. Res. Board Can. 21, 1183-1226 (1964).</li><li>Bla&#382;ka, P., Volf, M., &#268;epela, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+type+of+respirometer+for+the+determination+of+the+metabolism+of+fish+in+an+active+state.">A new type of respirometer for the determination of the metabolism of fish in an active state.</a> Physiol. Bohemoslov. 9, 553-558 (1960).</li><li>Peake, S. J., Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatigue+is+a+behavioural+response+in+respirometer+confined+smallmouth+bass.">Fatigue is a behavioural response in respirometer confined smallmouth bass.</a> J. Fish Biol. 68, 1742-1755 (2006).</li></ol>

在自然环境中，如捕食逃生和迁移的行为取决于能够在不同时期的具体强度游泳。在实验室环境中，游泳隧道的呼吸和加强在速度增加，可用于估计鱼的能力，以执行众多的行为。代谢率和其他组织水平的评估（如乳酸生产）的措施可以包括游泳测试，以帮助确定潜在的生理机制不同的游泳方式。 游泳隧道的设计，可以大大影响游泳性能。有几种类型的游泳隧道，用于确定游泳性能，大多数的特点是要么布雷特 13 -或Blazka 14类型。无论是设计，主要考虑因素包括水流均匀度“（这是多么的直线），最大流量速度和游泳室的长度。均匀度是很重要的，因为鱼会本能地发现，在低流量区游泳，以尽量减少工作量。高潜在的流量可能是必要的测试突发游泳能力或快速游泳的物种（如金枪鱼）。鱼游泳室的长度可以是非常有意义的小长度，即鱼的长度&gt;腔长度的10％的商会，可能无法利用所有游泳模式。具体来说，小管，可能会限制能力从一个稳定的鱼类过渡到不稳定游泳门，即爆裂和海岸游泳。因此，鱼可能会停止在大约相同的速度游泳，不论步长和 U暴击可能是类似于“门过渡的速度”15 。 所有游泳隧道需要使用，如流的探头或粒子成像测速（PIV）设备的设备进行校准。理想的情况下游泳隧道将有直线流量，但如果鱼被发现有利于一个地区，它们的游泳速度可以调整的基础上在流动的地方差异。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>tricaine methanesulfonate (MS222)</td>
<td>&nbsp;</td>
<td><a href="http://www.argent-labs.com">www.argent-labs.com</a></td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
<tr>
<td>Clove oil</td>
<td>&nbsp;</td>
<td>Pharmacy or <a href="http://www.sigmaaldrich.com">www.sigmaaldrich.com</a></td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
<tr>
<td>Swim tunnel respirometer</td>
<td>&nbsp;</td>
<td>Custom made or <a href="http://www.loligosystems.com">www.loligosystems.com</a></td>
<td>&nbsp;</td>
<td>Required</td>
</tr>
<tr>
<td>High speed cameras</td>
<td>&nbsp;</td>
<td>Numerous sources</td>
<td>&nbsp;</td>
<td>optional</td>
</tr>
<tr>
<td>Video capture card</td>
<td>&nbsp;</td>
<td>Numerous sources</td>
<td>&nbsp;</td>
<td>optional</td>
</tr>
<tr>
<td>Behaviour analysis software</td>
<td>&nbsp;</td>
<td><a href="http://www.noldus.com">www.noldus.com</a> <a href="http://www.cleversysinc.com">www.cleversysinc.com</a> <a href="http://www.loligosystems.com">www.loligosystems.com</a> <a hre</td>
<td>&nbsp;</td>
<td>optional</td>
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swimming performance assessment in fishes

鱼游泳的性能测试已经不可或缺的肌肉能量的研究，游泳力学，气体交换，心脏生理，疾病，污染，缺氧和温度。本文介绍了一种灵活的协议，以评估鱼游动的性能，使用的设备，可以控制水的流速。该协议涉及到一个加强流动速度的目的是造成鱼类疲劳增加。步速度和持续时间可以设置捕捉到不同的生理和生态相关的游泳能力。最常见的步长的大小，以确定临界游泳速度（U 暴击 ），其目的是捕捉到的最大持续游泳能力。传统上，这个测试包括约十个步骤，每20分钟持续时间。然而，持续时间较短（例如1分钟）的步骤是越来越多地被用来捕捉加速能力或突发游泳性能。无论游泳测试步长，可重复随着时间的推移来衡量的个体差异和恢复能力。端点相关的代谢率，鱼翅使用，通风率和行为，如鱼群之间的距离，措施，如游泳，通常包括之前，期间和之后，游泳测试。鉴于鱼类物种的多样性，一些未开发的研究问题，许多物种的重要性，对全球生态和经济的健康，鱼游动性能的研究仍将在可预见的未来的流行和宝贵的。

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Swimming Performance Assessment in Fishes

游泳的鱼大部分生活的前提。这个协议描述技术，捕捉游泳个人和鱼群的模式可供选择的范围，包括游泳的生理和行为相关的指标。

鱼类游泳绩效评估

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

swimming-performance-assessment-in-fishes

Research

JoVE Journal

Biology

25.3K 次观看.  University of Alberta. 游泳的鱼大部分生活的前提。这个协议描述技术，捕捉游泳个人和鱼群的模式可供选择的范围，包括游泳的生理和行为相关的指标。

视频: Swimming Performance Assessment in Fishes

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

科研

生物学

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

果蝇的耐力训练协议和纵向性能检测

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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 ...

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 ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...