我们衷心感谢白厅基金会为斯克里普斯研究所开展这项工作提供的资金支持和启动资金。

1. 准备腹腔甘利亚，电生理测量，和隔离单一识别神经元从阿普利西亚加州的腹部刚利翁
<ol><li>在实验室水族馆中保持 阿普利西亚 ，在 12：12 光下在 16 °C 处循环人工海水 （ASW）：黑暗条件。</li>
	<li>腹部结肠分离。<ol>
<li>通过注射380毫米MGCl2 溶液5-10分钟（相当于动物体重的30-35%）麻醉动物。</li>
		<li>根据腹部在中枢神经系统中的位置24，28识别腹部结肠。</li>
		<li>通过手术切除黑帮，并立即储存在人工海水中，包括450立方米纳克、10米KCl、10米卡Cl2、55毫米MMMcl 2、2.5m NaHCO3和10m HEPES（pH 7.4）。</li>
	</ol>
</li>
	<li>蛋白酶治疗。<ol>
<li>在上面描述的 ASW 中稀释 0.1% 蛋白酶 （脱脂） 的培养皿中，在 34 °C ±0.5 处孵化腹部结节 30 分钟。 注意：使用的蛋白酶数量需要随着动物的年龄和体重而标准化。一般来说，年龄较大和较大的动物需要更多的蛋白酶。</li>
	</ol>
</li>
	<li>去除帮派护套。<ol>
<li>酶治疗后，将结膜固定到细胞室的 Sylgard 硅胶底座（+1 厘米;卷 = 0.3 毫升），并在室温下（18 °C ±2）与 ASW（流速：150μl/min）一起灌注。 注意：为了提高已识别神经元的可见性，将结核放在聚碳酸酯玻璃缸（图1）的顶部（直径+2毫米修改后的细胞室），使用双目立体显微镜，可轻松从底部亮起，放大20倍和40倍。</li>
		<li>使用钳子和微剪刀小心地从结石中取出护套。</li>
	</ol>
</li>
	<li>同时从两个神经元进行电子生理学记录。<ol>
<li>识别感兴趣的神经元。</li>
		<li>用一个具有电阻10-15 MΩ、充满3M KCl溶液的单细胞内锐利微电极来填充神经元。</li>
		<li>使用细胞内记录系统记录神经元活动（10 kHz 带通）。</li>
		<li>使用电子生理学软件（如轴电图）处理记录数据。 注：人们可以很容易地从多个神经元进行记录。神经元 L7、L11 或 R15 和 R2 很容易根据它们的位置进行识别。同时从两个L7和R15神经元（图2）进行记录需要两个放大器和两个微脉动器。</li>
	</ol>
</li>
	<li>药物应用。<ol>
<li>通过轻轻的管道或直接注射到神经元中，将选择的药物应用到细胞室中。</li>
		<li>进行电生理测量。 注：根据实验目标，可以测量神经元的不同电生理参数，如药物应用前、应用期间和之后膜电位 （MP） 或行动潜力 （AP） 的变化。</li>
	</ol>
</li>
	<li>单个神经元的隔离。<ol>
<li>在电生理实验结束时，停止ASW的灌注，用100%乙醇清洗结膜。 接触乙醇会使神经元石化，并使用细钳，在不损害神经元的情况下，很容易去除单个神经元。</li>
		<li>在立体显微镜下取出单个神经元，并转移到含有冰冷 500 μl RNA 提取试剂的小 Eppendorf 管中。孤立的单个神经元可存储在 -80 °C 的 RNA 提取试剂中，以便将来进行分析。</li>
	</ol>
</li>
</ol>2. 通过定量实时 PCR （qPCR） 与单一神经元和基因表达分析的 RNA 隔离
<ol><li>尽量减少RNA退化的预防措施： 核糖核酸（RNases）降解RNA，是非常稳定和活性酶。在处理RNA时采取以下步骤。<ol>
<li>用 RNase 停用剂清洁工作区域和仪器。</li>
		<li>在处理RNA时随时戴手套，并经常更换手套。</li>
		<li>仅使用无RNA的RNA。</li>
	</ol></li>
	<li>将RNA总数与单个神经元分离：<ol>
<li>RNA隔离的标准三佐尔协议可用于将RNA与单个神经元分离。简单地将 20% v/v 的氯仿添加到 Trizol 中，通过短暂的漩涡很好地混合。</li>
		<li>将管子放在旋转器上 10 分钟，在 4 °C。 在 12，000 x g 下旋转三氯仿混合，在 4 °C 下旋转 15 分钟。</li>
		<li>收集水相并转移到新鲜管。</li>
		<li>加入醋酸钠溶液（pH 5.5），最终浓度为0.3M、100 ng/ml的共生糖蓝和2.5卷100%乙醇沉淀RNA。</li>
		<li>将样品混合好，在-80°C的夜间孵化。</li>
		<li>在 4 °C 下以 12，000 x g 旋转样品 20 分钟，在管子底部可以看到一个小的蓝色颗粒。</li>
		<li>取出超自然剂，在 4 °C 下用 850 μl 的 75% 冰冷乙醇在 12，000 x g 下洗净颗粒 10 分钟。</li>
		<li>小心地取出超细剂，将RNA颗粒空气干燥7-10分钟，最大。 注意：确保RNA不会像过度干燥那样长时间干燥-干燥RNA颗粒会使溶解变得非常困难。
</li>
		<li>干燥后，将RNA颗粒在10微升的RNA酶无水中补充，并确定RNA浓度。 注：使用光谱仪确定RNA浓度。不立即使用时，将RNA存储在-80°C。</li>
	</ol></li>
	<li>单个神经元的 aRNA 合成：<ol>
<li>使用市售的线性RNA放大系统放大RNA（根据实验目标进行一到两轮）。 注：来自单个神经元的RNA总数在~10-60 ng之间。第一轮放大的预期收益率为+100-300 ng，第二轮放大为+0.8-1.5微克RNA。</li>
	</ol></li>
	<li>RNA 的反向转录：<ol>
<li>要从 aRNA 生成 cDNA，请在 20μl 的反向转录反应中使用 1.0μg 的 aRNA。为此目的，有几个套件可在商业上提供。 注：cDNA 是根据制造商的协议，使用热循环器上的以下设置合成的。 5 分钟，25°C 30 分钟，42 °C 5 分钟，85 °C 保持在4°C</li>
		<li>反向转录步骤后，将 cDNA 存储在 -20 °C 以进行长期存储。</li>
	</ol></li>
	<li>引言设计和标准化： 有几个优秀的软件程序是免费的，用于设计实时放大的引物。<ol>
<li>选择 18-24 mer 寡核苷酸，生产 70-110 bp 放大器，并使用商业来源合成引物。 注：应为每个感兴趣的基因设计两到三个引言对。每个引号集都需要通过 qPCR 进行标准化。用于基因CREB1（图4）的前向和反向引数对如下： 引号集 1 前进： 5'-塔加特加-3' 反向： 5 '- 阿卡格加加特加加 - 3' 引号集 2 前进： 5 '- 阿加特加特加加加 - 3' 反向： 5 '- 加卡卡加加塔特加卡 - 3'</li>
		<li>要选择用于下游分析的最佳引物，请监视 ct 值和 qPCR 中放大曲线的形状。 注：在 PCR 指数阶段，在 40 轮放大中给出 Ct（周期阈值）值在 10-35 之间的引物和平滑曲线，然后是平滑的高原，是基因表达分析的最佳选择。</li>
	</ol></li>
	<li>定量实时 PCR （qPCR）： 注意：使用与 qPCR 机器兼容的适当管子或板。<ol>
<li>用无核水将cDNA稀释至5倍。</li>
		<li>到稀释的 cDNA 的 2μl，加入 8 μl 的 qPCR 主混合，其中包含 2 μl 的 H2O、5 μl 的 2x SYBR 绿色主混合和 1.0 μl 的 10 μM（每个）前进和反向引物。</li>
		<li>关闭管子，并在管道 cDNA 和主混合后密封板。通过轻轻敲击混合内容，在 1，500 rpm 下旋转 30 秒。</li>
		<li>继续设置 qPCR 机器。</li>
	</ol></li>
</ol>

作者没有任何相互竞争的经济利益。

神经元R15参与调节心血管，消化，呼吸和生殖系统30。AP 的定期有节奏的爆裂活动是 R15 的一个功能。如结果部分所示，R15 和 L7 的配对记录显示，神经质制备保留了 R15 神经元的活性。R15和L7神经元对阿赫反应恰当。这种神经质制备可维持8-10小时，电生理活动可持续监测。因此，我们可以长期研究短暂神经递质暴露（局部暴露在特定神经元或洗澡中）的电生理效应。此外，人们还可以研究...

人脑极其复杂，有近1000亿个神经元和数万亿个突触连接。几乎有同样数量的非神经细胞与神经元相互作用，并调节其在大脑中的功能。神经元被组织成调节特定行为的电路。尽管我们对大脑功能和神经回路的理解有所进步，但对于控制特定行为的电路组件的身份却知之甚少。了解电路中各种成分的身份将大大促进我们对细胞和分子行为基础的理解，并有助于制定神经精神病的新治疗策略。
海洋蜗牛阿普利西亚卡利福尼卡一直是确定神经元电路的基础特定行为1-14的工作马。阿普利西亚神经系统包含大约20，000个神经元，这些神经元被组织成9个不同的神经质。Aplysia的神经元很大，可以根据它们的大小、电特性和在神经中的位置轻松识别。阿普利西亚有丰富的行为，可以研究。研究良好的行为之一是刺退出反射 （GWR）。这种反射的中心部分位于腹部结节。已绘制 GWR 电路的组件，并确定各种组件的贡献。重要的是，GWR电路经历关联和非关联学习5，6，15-19。数十年来对这种反射的研究也发现了几个信号通路，这些信号通路在学习和记忆20-24中起着关键作用。
几种不同的 阿普利西亚 制剂用于研究记忆存储的细胞和分子基础。其中包括完整的动物2，3，半完整的准备1，7，13，14，16 和重建神经电路的主要组成部分25-29。这里描述了为探索 阿普利西亚 神经元电路的电生理学和分子分析而减少的准备。研究了以下四个已识别的神经元。R15，一个爆裂的神经元，L7和L11，两个不同的运动神经元和R2，一个胆碱神经元被研究。R2是无脊椎动物神经系统中描述的最大神经元。简言之，该方法涉及结节蛋白酶治疗、药理治疗前后的电生理测量以及单个神经元的分离，用于基因表达的定量分析。这种方法使我们能够将分子分析与多个神经元同时记录相结合。这种方法通过配对细胞内记录成功地用于研究R15和L7神经元对乙酰胆碱（Ach）的反应。经过电子生理测量，R15和L7以及L11和R2等其他已识别的神经元被分离出来，用于定量聚合酶链反应（qPCR）对CREB1表达的分析，CREB1是记忆存储的重要转录因子。

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		<col />
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	<tbody>
		<tr height="101">
			<td height="101" style="height:101px;width:120px;">Aplysia</td>
			<td style="width:123px;">National Aplysia Resource Facility, University of Miami</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">NaCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 3014-1KG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">KCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">P 9333-500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">C5080- 500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:129px;">BP 214-501</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">NaHCO4</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 6297-250G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">HEPES</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">H 3375-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Protease</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:129px;">17105-042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Trizol</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">15596-026</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Chloroform</td>
			<td style="width:123px;">MP Biomedicals</td>
			<td style="width:129px;">2194002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">100% Ethanol</td>
			<td style="width:123px;">ACROS</td>
			<td style="width:129px;">64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">GlycoBlue</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9515</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">3 M NaOAc, pH 5.5</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9740</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Nuclease free water</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9737</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">MessageAmp II aRNA Amplification Kit</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM1751</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">qScript cDNA SuperMix</td>
			<td style="width:123px;">Quanta Biosciences</td>
			<td style="width:129px;">95048-100</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Power SYBR Green PCR Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">4367659</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">11252-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">15000-08</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Stainless Steel Minutien Pins&#160;</td>
			<td rowspan="2" style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">26002-10 or</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">26002-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Veriti Thermal Cycler</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">Veriti Thermal Cycler</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">5430R Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:129px;">5430R Centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">7900HT Fast Real-Time PCR</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">7900HT Fast Real-Time PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:120px;">Amplifier</td>
			<td>BRAMP-01R</td>
			<td style="width:129px;">NPI Electronics</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Digidata Converter</td>
			<td style="width:123px;">Instrutech ITC-18</td>
			<td style="width:129px;">HEKA ELEKTRONIK</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Micro Manipulator</td>
			<td style="width:123px;">Patch Star</td>
			<td style="width:129px;">Scientifica</td>
		</tr>
	</tbody>
</table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

神经生物学的一个主要挑战是了解控制特定行为的神经回路的分子基础。一旦确定特定的分子机制，就可以开发新的治疗策略，以治疗退行性疾病或神经系统老化引起的特定行为中的异常。海洋蜗牛 阿普利西亚卡利福尼卡 非常适合研究细胞和分子的行为基础，因为神经电路背后的特定行为可以很容易地确定，电路的各个组成部分可以很容易地操纵。 阿普利西亚的 这些优点导致了学习和记忆神经生物学的几个基本发现。在这里，我们描述了为个体神经元的电生理学和分子分析准备 的阿普利西亚 神经系统。简言之，从神经系统解剖的结膜暴露在蛋白酶中，以去除结膜护套，使神经元暴露，但保留神经元活动，就像在完整的动物中一样。这种制剂用于对单个或多个神经元进行电生理测量。重要的是，使用简单的方法进行记录后，神经元可以直接从神经中分离出来进行基因表达分析。这些协议用于同时进行L7和R15神经元的电生理记录，研究它们对乙酰胆碱的反应，以及在分离的单L7、L11、R15和 阿普利西亚R2神经元中对CREB1基因的量化表达。

本研究中使用的动物重量在100-200克之间。按照上述协议，我们对从2-5克到200-300克的动物分离的腹部神经元进行了电生理测量和分子分析。
蛋白酶治疗的标准化对于成功测量神经元的神经元具有重要意义。最初，使用多个蛋白酶（假发）浓度和持续时间，R15神经元的爆裂作用潜力记录为26，31。这些测量被比较成从完整的帮派中直接（没有蛋白酶治疗）记录。在随后的...

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

海洋蜗牛 阿普利西亚卡利福尼卡 已被广泛用作神经生物学模型，用于细胞和分子行为基础的研究。这里描述了一种探索 阿普利西亚 神经系统的方法，用于识别神经回路单神经元的电生理学和分子分析。

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

aplysia-ganglia-preparation-for-electrophysiological-molecular

Research

JoVE Journal

Bioengineering

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

9.0K 次观看.  The Scripps Research Institute, Florida. 海洋蜗牛 阿普利西亚卡利福尼卡 已被广泛用作神经生物学模型，用于细胞和分子行为基础的研究。这里描述了一种探索 阿普利西亚 神经系统的方法，用于识别神经回路单神经元的电生理学和分子分析。

视频: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

分子纠缠与生物大分子的Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜：含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

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 ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

在中短肛解剖，显微扫描和分析形态

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

阿普利西亚单一神经元电生理学和分子分析的甘利亚制备