作者要感谢理查德·辛克先生和Todd Wiernicki方法学的培训和实验指导。我们还要感谢约翰·保罗·斯彭斯仔细阅读和编辑建议。这项工作是由卫生MH 060397，脑与行为研究基金会和美国礼来公司的研究所和公司通过礼来公司研究奖励计划（LRAP）的支持。

1。扩展含量就绪细胞和冷冻保存<ol><li>培养的CHO-K1-DRD 2L（CHO-D 2L）在15 cm 2的细胞培养皿中Ham氏F12培养基补充有1.0μML-谷氨酰胺，800微克/微升G418，300微克/微升潮霉素，100 U /微升细胞青霉素，100微克/微升链霉素和10％胎牛血清（FBS）。 </li><li>孵育的细胞在37℃下在湿润的培养箱中5％的CO 2，直至细胞为90-95％汇合。用10微升磷酸盐缓冲盐水（PBS）洗涤细胞，并通过加入3微升的细胞解离缓冲液5分钟，在37℃下收集细胞用12微升的培养基中重悬细胞，并使用台盼蓝排除法计数细胞。 </li><li>离心细胞悬浮液，在500×g离心5分钟，在室温下进行。吸出上清，重悬细胞沉淀于5μl冷冻介质（10％二甲亚砜，90％FBS）。稀释细胞悬液，以达到所需的细胞浓度（ 例如 1-20×10 6个细胞/微升）。 </li><li>分装1.0微升的细胞溶液到每个离心管。孵育在细胞冷冻集装箱的冷冻管在-80℃下过夜。转移至离心管的液体氮气罐长期贮存。 </li></ol> 2。电镀含量就绪细胞（和反向转股权而更改） <ol><li>快速解冻细胞的冷冻离心管在37℃水浴。一旦细胞被解冻，将细胞转移到15微升的锥形管含有9微升的Opti-MEM中，并通过反相管3到5倍混合。 </li><li>离心细胞，在500×g离心5分钟，在室温下进行。吸去上清，重悬的细胞在1微升的Opti-MEM。 </li><li>使用台盼蓝排除法测定细胞活力和稀释活菌根据需要（ 例如 ，3×10 5个细胞/μL的浓度）中计数细胞的Opti-MEM。使用多通道移液管板10微升的细胞在组织培养/孔处理的384孔板中。 </li><li>使cAMP作用中的Opti-MEM连续稀释以产生标准曲线估计的cAMP产生的细胞（根据生产商的建议 - 见第7节）。加入10μl的cAMP标准品/孔到板上注:一个单一的标准曲线可以在一个单独的盘或空白孔中而制备的测定板中的一个。 </li><li>离心板在100×g离心15秒，在室温下和在潮湿的培养箱中，用5％CO 2的1小时温育在37℃。 </li></ol> 3。反向siRNA转染选项* 这部分是可选的，相关的图3。 <ol><li>准备的siRNA的溶液中无RNA酶的双蒸2 O（ 例如 0.4皮摩尔/微升）。加入5微升至384 - 孔板各孔中，并离心板在100×g离心15本身C在常温。 </li><li>稀释脂质体2000中的Opti-MEM由0.006倍（ 例如加6μL脂质体2000〜1000μL的Opti-MEM），以及由上下吹打混匀。 </li><li>孵育5分钟，在室温下将脂质体2000/Opti-MEM溶液。加入5微升稀释的Lipofectamine 2000/Opti-MEM溶液至384 - 孔板各孔中已包含的siRNA。离心板在100×g离心15秒，室温下进行。 </li><li>孵育板30分钟，在室温下进行。 </li><li>平板测定细胞准备上述（第2部分）所描述的。离心板在100×g离心15秒，室温下进行。 </li><li>返回试验板到潮湿的培养箱中，在37°C（5％CO 2）为48-96小时所确定的靶基因敲下来（见讨论）。 </li></ol> 4。小分子筛选<ol><li>淡化感兴趣的药物（ 如 。小分子抑制剂）的Opti中-MEM 6倍的所需最终浓度。连续稀释可以使用手持式移液器或液体处理工作站完成。 </li><li>加试验化合物的2.5100μl/孔或缓冲液含有载体（ 如 DMSO）中，以使用多通道移液管的孔的侧面上。 </li><li>离心板在100×g离心15秒，在室温（潜伏期是可选的）。 </li></ol> 5。持久性激动剂治疗<ol><li>准备一个600纳米的的Opti-MEM quinpirole的溶液（浓度100nM 即 6倍所需终浓度）。 </li><li>添加2.5微升/孔的600 nM的quinpirole溶液用多道移液管的孔的侧面上。 </li><li>离心板在100×g离心15秒，在室温下和在潮湿的培养箱中，用5％CO 2下2小时，在37℃下。 </li></ol> 6。 cAMP的积累刺激<ol><li>在2小时培养，准备stimulati对的Opti-MEM溶液。刺激溶液是由40μM毛喉素，2μM的3 - 异丁基-1 - methyxanthine黄嘌呤（IBMX）和4μM螺哌隆（在步骤6中所有浓度4倍的所需最终浓度）。 </li><li>加入5微升/孔的刺激溶液用多道移液管的孔的侧面上。 </li><li>离心板在100×g离心15秒，在室温下和在室温下孵育1小时。 </li></ol> 7。淬火cAMP的积累和预测<ol><li>产生cAMP的细胞是使用的cAMP动态2试剂盒根据制造商的说明书测定。简言之，将重组抗-cAMP-穴状化合物和cAMP-d2的蒸馏水中。分装冷冻在-20℃下短期存储。 </li><li>根据制造商的说明，以使工作溶液稀释的抗-cAMP-穴状化合物的1等分试样和内cAMP-D2分开一个等份的裂解缓冲液。 </li><li>一西元10微升抗-cAMP-穴状化合物工作溶液和10μl内cAMP-d2的工作溶液到384孔板用多通道移液器/孔/孔。 </li><li>离心板在100×g离心15秒，在室温下和在室温下孵育1小时。 </li><li>读板中使用的337 nm光激发的荧光酶标仪（自动量程设定为灵敏度），并测量排放量为每620 nm和665 nm的制造商的说明。 </li><li>每申请比例分析，评估的cAMP标准曲线生产指令。使用所得到的值，推算估计的cAMP积聚在使用数据分析软件的测试井。 </li></ol>

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I., 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=Use+of+a+cAMP+BRET+sensor+to+characterize+a+novel+regulation+of+cAMP+by+the+sphingosine+1-phosphate/G13+pathway.">Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.</a> J. Biol. Chem. 282, 10576-10584 (2007).</li></ol>

作者什么都没有透露。

在努力促进外源过敏的研究中，我们广泛地修改了我们以前的方法实现一个精简的"组合和阅读"格式，易于进行高通量筛选和行动检查机制。主要的修改我们的协议可以概括如下:1）使用冷冻保存的细胞作为"测定就绪"试剂;测定到384 - 孔格式的2）的小型化，并在HTRF测定3）的利用cAMP的。 采用冷冻保存我们的基于细胞的测定，大大提高了效率和可重复性为我们日常的实验和筛?...

一种自适应的腺苷酸环化酶（AC）信号的响应被称为异源或超敏的诺贝尔奖获得者，马歇尔·尼伦伯格博士的实验室首次发现。尼伦伯格博士提出，所观察到的交流增加下列反应慢性δ阿片受体的激活是一个从事鸦片耐受性和依赖性1的机制。除了 ​​慢性δ阿片受体激活，交流信号的这种神经适应性反应也发生以下几种其他Gαi/ O偶联受体2的持续激活。值得注意的是，许多这些受体与疼痛，神经精神及神经系统疾病有关，包括μ/κ阿片，D 2/4多巴胺，5HT 1A和M 2/4毒蕈碱受体2。除了尼伦伯格博士的研究结果，大量的证据存在连接交流信号的宣传，以慢性阿片受体激活都<e米&gt;在体外和体内 3-7。交流的致敏也与各种涉及D 2类多巴胺受体包括精神分裂症和帕金森氏病的疾病有关（综述见参考文献2）。尽管致敏的潜在重要性，精确的机制（S）与持久Gαi/邻 - 偶联受体的激活，导致增加的AC响应在很大程度上仍然未知相关联。 这些研究为研究对腺苷酸环化酶的致敏机制作为一项重要的神经生物学指标的理由。同样，AC的信令8的生理相关性和重要性，个人交流亚型在此适应性反应保持还应认识2,9,10。在我们的研究的情况下，使用交流的重组同种型异源致敏相关联的一般功能平行那些特征德cribed学习交流的内源亚型。具体来说，以前的研究已经发现，Gαi/ O蛋白质和随后发布/重排βγ亚基的激活是所有交流亚型受体引起过敏的重要要求。此外，一些研究表明，由蛋白激酶和Gβγ亚单位的信令中涉及致敏2,11-13。个别的AC也显示独特而鲜明的致敏模式12。举例来说，D 2受体的持续暴露于激动剂与AC1和AC8的致敏关联到的Ca 2 + /钙调蛋白刺激14,15，而与之密切相关的AC3不致敏2。 AC2，AC4和AC7有密切的关系，但是，只有PKC刺激AC2活动是D 2受体的长期接触后强劲致敏激动剂7,14,16,17。此外，AC5和AC6显示海特的显着程度ologous宣传，以天然气和毛喉素刺激的cAMP积累下激活D 2受体14,18-20，但出现在他们的Gβγ亚单位交流的要求，以不同的相互作用21。虽然交流致敏的大多数研究中使用的模型的细胞系（ 例如 HEK293细胞表达个人交流异构体），看来这些结果转化为原生神经元细胞模型4,22。最近，在HEK293细胞中表达的AC亚型鉴定AC亚型选择性的小分子抑制剂的效果，也可以转换为在体内的行为研究23。 的可识别的分子机制的异源致敏的缺乏可能反映了自适应响应的复杂性以及各个AC亚型12的独特的调节特性。揭开这种复杂性是由使用繁琐的方法吨进一步复杂化帽子已经从用人公正的途径有限的学术研究。例如，我们以前的机理研究涉及使用连续培养的细胞模型使用24 -和48 -孔组织培养格式15。培养的细胞通常培养48小时，然后进行激动剂药物治疗（2-18小时），随后通过一系列细胞洗涤和温育（ 图1）。 AC-同工型特异性的cAMP积聚的协议当时采用随后的cAMP积聚的测量使用一个费 ​​时费力的[3 H] cAMP结合的方法15,24。从开始的持续时间来完成每一测定通常总共四到五天需要从细胞接种到数据分析（ 图1）。新技术和自动化的应用导致了显着的增强功能在工业和HTS中心设置致敏研究。例如，一组具有国家中心的化学工作CAL基因组报道进行了两天的HTS检测程序确定的μ阿片受体引起过敏的小分子抑制剂在1,536孔板格式25。 本文章介绍了我们的努力，开发利用技术，可在大多数学术研究机构异源致敏研究高温超导实验。这种策略通过配合使用冻存细胞的细胞模型异源表达的D 2多巴胺受体与内源性或个人重组腺苷酸环化酶亚型（CHO-D 2L或HEK-AC6 / D 2 L）的组合来完成。为了提高我们的产量，我们重新设计了48和致敏试验（ 约 &gt; 20级以上4-5天）到384孔的格式，基本上是"混读"五步，一天检测。新的格式使用市售的同质时间分辨荧光（HTRF）测定来衡量的cAMP ACCumulation在完整细胞与多模式读板器。化验是稳健经得起small分子筛选，并可有效应用于筛选异源致敏抑制剂。此外，我们提供数据，允许使用该测定的siRNA的反向转染靶向或全基因组的siRNA文库筛选，只有轻微的修改，一般的方法。

<table border="0" cellpadding="0" cellspacing="0" width="695">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">CHO-K1-DRD2L cells</td>
			<td style="width:144px;">DiscoveRx</td>
			<td style="width:148px;">930579C2</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Ham&#8217;s F12 media</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH30026fs</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Fetal Bovine Serum</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH3007003</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">G418</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A1720</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Hygromycin</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">50-230-5556</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Penicillin/ streptomycin</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">15070063</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">15 cm dishes</td>
			<td style="width:144px;">BD Falcon</td>
			<td style="width:148px;">353025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">DMSO</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">D2650</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cell dissociation buffer</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">13150016</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Phosphate Buffered Saline</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10010-049</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cryovials</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">976171</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Opti-MEM</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">31985070</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">TC treated 384 well plates</td>
			<td style="width:144px;">Perkin-Elmer</td>
			<td style="width:148px;">6007688</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:205px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:144px;">Cisbio Bioassays</td>
			<td style="width:148px;">62AM4PEB</td>
			<td style="width:199px;">http://www.htrf.com/camp-cell-based-assay</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Synergy 4</td>
			<td style="width:144px;">Biotek</td>
			<td style="width:148px;">H4MLFPTAD</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Prism 6</td>
			<td style="width:144px;">GraphPad Software</td>
			<td style="width:148px;">NA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">I5879</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">(&#177;)Quinpirole</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">Q111</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Spiperone</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S7395</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Clozapine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">C6305</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Haloperidol</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">H1512</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">S-(-)Sulpiride</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S112</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:205px;">Lipofectamine2000 transfection reagent</td>
			<td style="width:144px;">Invitrogen</td>
			<td style="width:148px;">11668019</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Trypan blue</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">T10282</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Water, DNase, RNase-free</td>
			<td style="width:144px;">MP Biomedicals</td>
			<td style="width:148px;">821739</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Gas siRNA</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">custom siRNA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Non-targeting siRNA control</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001206-14-20</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">siGlo Red</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001630-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and sirna screening applications

腺苷酸环化酶（AC）信号的致敏已经牵涉于多种神经精神和神经紊乱包括物质滥用和帕金森氏病。 Gαi/ O连接受体的急性激活抑制AC活性，而这些受体产生交流的异源致敏持续激活与细胞内cAMP水平的提高。以前的研究已经表明，这种增强的AC响应的观察在体外和体内以下几种类型Gαi/ O-连接的受体，包括D 2多巴胺和μ阿片受体的慢性活化。虽然交流的异源致敏首先报道四十年前，背后这一现象的机制（S）仍是未知。机械数据的缺乏可能反映的涉及本适应性反应的复杂性，这表明无偏差的方法可能有助于确定ING参与交流的异源致敏的分子途径。以前的研究已经牵连激酶和Gbγ信号的重叠，调节交流的异源致敏成分。标识与交流的过敏相关的独特的和额外的重叠的目标，一个可扩展的cAMP致敏试验开发和验证，需要更大的吞吐量。以前的方法来研究致敏一般都是笨重涉及连续细胞培养物保养以及一个复杂的方法用于测定cAMP的积累，涉及到多个洗涤步骤。因此，可在384孔的格式可用于高通量筛选（HTS）一个强大的基于细胞的测定的发展将有利于未来的研究。使用两个D 2多巴胺受体的细胞模型（ 即 ，CHO-D 2L和HEK-AC6 / D 2L），我们已经转换了48孔敏法（&gt; 20级4-5天），以五-STEP，在384孔板格式的单天测定。这种新格式是适合于小分子筛选，我们证明，该试验设计也可以容易地使用用于siRNA的反向转染在预期目标的siRNA文库筛选。

第一部分开发一个384以及异源致敏试验确定使用市售的细胞模型小分子抑制剂。 为了研究异源致敏的细胞模型，我们做了一些改进，使我们能够简化检测到一个"混合和阅读"格式。一些重要的修改包括以下各项，并且更详细地讨论所述。第一个关键步骤是从连续培养的细胞改变我们的细胞培养冻存细胞26。我们现在经常细胞培养批次可1-20×10 6个/?...

Watch this Scientific Journal Video about Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications at JoVE.com

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

持续激活的抑制性G蛋白偶联受体导致腺苷酸环化酶信令的致敏作用。以确定必需的分子途径，无偏差的方法是必要的，然而，这种策略需要一个可扩展的基于细胞的cAMP敏感测定法的发展。本文中，我们描述了一种致敏试验用于小分子和siRNA的筛选。

腺苷酸环化酶的药物引起的过敏性:含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

drug-induced-sensitization-adenylyl-cyclase-assay-streamlining

Research

JoVE Journal

Bioengineering

12.6K 次观看.  Purdue University. 持续激活的抑制性G蛋白偶联受体导致腺苷酸环化酶信令的致敏作用。以确定必需的分子途径，无偏差的方法是必要的，然而，这种策略需要一个可扩展的基于细胞的cAMP敏感测定法的发展。本文中，我们描述了一种致敏试验用于小分子和siRNA的筛选。 

视频: Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

科研

生物工程

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

硅纳米线场效应晶体管的研制化学生物传感与应用

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...