以下议定书是接受马里兰大学的机构动物保育和使用委员会以及机构审查委员会。这些议定书遵循联邦、州和地方标准, 以及马里兰大学生物安全政策规定的指导方针。
1. 眼部标本解剖
<ol><li>
从当地屠宰场或组织银行获得一份眼科样本。如果整个眼睛地球仪获得, 立即提取晶状体, 附着 zonules, 和玻璃体。 注意: 下面描述的具体细节与猪和人的眼睛有关。<ol>
<li>使用消毒手术剪刀和镊子, 切除和清除周围的巩膜所有多余的组织。</li>
		<li>牢牢地握住眼睛在它的一侧, 并使用剃刀刀片, 使一个小切口沿眼睛一侧3毫米远离角膜。使伤口深到足以达到玻璃内的眼睛。</li>
		<li>使用剪刀, 小心地沿着切口绕着眼睛的圆周切开。避免刺穿镜片。有代表性的图像显示在图 2A中。</li>
		<li>一旦眼睛的外圆周被切开, 用镊子去除眼睛的后部组织。将晶状体、zonules、睫状体和附着的玻璃体与镊子隔离。有代表性的图像显示在图 2B中。</li>
		<li>使用剪刀和镊子, 取出多余的玻璃, 使透镜可以平放在 MLS 上。 注: 在角膜移植的病例中, 角膜按钮用于外科手术, 而其余的地球则可供研究用。然而, 这部分地球仍然可以用于组织准备的镜头担架设置。如果只获得后部, 只执行步骤 1.1. 4–1.1. 5。</li>
	</ol>
</li>
	<li>在15% 漂白剂溶液中消毒所有使用过的设备30分钟。</li>
</ol>2. 手动镜片担架的试用装配
<ol><li>将 10 mm 的鞋底和相应的鞋顶插入到 MLS 的底部板上, 这样在鞋的缩进和鞋的后壁之间仍然有5毫米的空隙。</li>
	<li>将顶部和底部板对齐, 将板材合在一起;设备现在处于无拉伸位置。</li>
	<li>将板材插入板壳中, 塞入螺钉进入底部板侧的孔中。</li>
	<li>将板壳插入底座, 并将扳手置于所对齐的缩进中。</li>
	<li>顺时针扭扳手, 直到它到达停止螺丝, 以收缩鞋, 并扭转回逆时针返回原来的无拉伸位置的鞋。</li>
</ol>3. 镜头的安装
<ol><li>将10毫米的鞋底插入到 MLS 的底部板中, 使5毫米的间隙保持在鞋缩进和鞋本身的后壁上。</li>
	<li>使用弯钳, 将所提取的透镜面向上放置在底部板的中间, 使鞋在中心孔上支撑透镜。</li>
	<li>把相应的鞋顶放到位, 只修剪 zonules 和玻璃。视觉上确保镜头保持在底部板尽可能居中。</li>
	<li>重复步骤2.3–2.4。</li>
</ol>4. 镜片的测量
<ol><li>将成像系统直接放在设备的上方, 以便捕捉视频和拉伸过程的图片。一定要在图片的框架中包括一个标尺, 以便在后处理中准确地调整图像大小和缩放。 注: 任何合适的成像系统都足以进行此步骤;在这里, 我们使用一个12像素, 调焦智能手机一脚从样本。</li>
	<li>牢固而平稳, 旋转扳手在顺时针方向伸展透镜。图 3在无拉伸和拉伸状态下显示具有代表性的图像。</li>
	<li>拍摄拉伸透镜后, 旋转扳手在逆时针方向, 以恢复样品到其静止状态。 注意: 必须及时进行镜片的测量, 以尽量减少镜片的脱水。</li>
	<li>清晰地拍摄镜头的最后静止状态。</li>
</ol>5. 数据分析
<ol><li>将图像上载到 ImageJ, 并使用 "点" 功能在透镜圆周周围至少选择40点, 如图 4A所示。使用 "分析" → "度量" 选项来产生每个选定点的位置。</li>
	<li>适合 (使用软件例如MATLAB) 位置点, 以便产生一个半径和卡方的配合, 如图 4B所示。使用拍照的标尺将像素半径和错误转换为度量值。</li>
	<li>执行配对两个尾 t 测试, 以比较一个单独的镜头之前和之后, 从 MLS 伸展。</li>
</ol>

<ol>
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AB 公司拥有 Bioniko 咨询有限责任公司的所有权权益。

我们设计了一种新的方法, 以提供一个准确和有效的方法来研究镜头的住宿能力, 利用双件夹紧机构, 以夫妇的担架到样品。在住宿期间, 透镜放松, 并且直径减少为响应悬韧带纤维的放松 1, 2, 4, 19.该方法通过夹紧和控制悬韧带纤维的张力来研究这种现象。因此, 必须对 zonules 内的鞋类进行严格的护理, ?...

住宿是人类的眼睛能够动态调整其晶体透镜形状的过程, 可以看到物体在远距离或接近焦距的尖锐聚焦。住宿是一种内在的生物力学过程。在神经刺激作用下, 睫状肌产生一力到睫状体和 zonule 纤维连接到透镜胶囊的圆周上1,2。虽然有不同的理论背后的生物力学的住宿, 最广泛接受的是亥姆霍兹假说。根据该假说, 透镜处于自然拉伸状态, 与透镜最薄的形状相对应, 它最适合于远距离物体的聚焦。为了将焦点转移到更接近的物体, 睫状肌收缩, 悬韧带纤维松弛。反过来, 透镜变厚, 增加前和后表面曲率。这对应于屈光度功率的增加, 这是近视力所必需的, 因此, 较短的焦距为1。
通过一个名为 "视时间" 的条件, 可容纳的能力受到影响。在50岁时影响每个人, 老视使眼睛无法动态地改变焦点从远到接近距离3。为了对抗视镜, 目前的方法是被动的, 包括矫正镜片和双光眼镜。虽然在少数几架飞机上提高了对接近对象的专注能力, 但这种被动处理无法恢复透镜4、5的动态焦点能力。为了有效地治疗大视地, 或可能阻止它, 目前需要更好地了解住宿。
为了研究透镜的住宿, 已经开发了许多设备来模拟这种现象 "体内"4,6,7,8,9。首先引入旋转圆盘, 通过离心力监测透镜的拉伸情况8。为了更忠实地再现这种现象, 镜头拉伸装置逐渐被引入和创新。使用透镜担架, Manns et al。特征是在与透镜功率和赤道直径9相关的情况下容纳透镜所需的力。目前的理解是, 透镜僵硬的年龄, 导致透镜形状的变化, 以响应从睫状体的同等力量3,10,11,12。
目前的镜头担架往往涉及复杂的设置, 实施电子和可编程伸展率, 并要求整个尸体眼球6,7,10,13。这一要求增加每项实验的成本超过每只眼睛500.00 美元, 并减少样品的可用性。在这里, 我们提出了一种以低成本复制镜头住宿的方法, 因为眼睛的后部总计约为200.00 美元。虽然比今天使用的许多设备都要复杂得多, 但这项技术在不影响结果的情况下更具成本效益和收养。此方法以图 1中描述的手动透镜担架 (MLS) 为中心, 并在悬韧带光纤上使用独特的夹紧系统, 并采用径向扭转方法来扩展透镜的直径。贝尔纳et al的研究结果验证了该协议的生理准确性, 研究了前、后悬韧带纤维连接到透镜胶囊14的途径.采用自定义鞋的设计, 只需要透镜、zonule 和睫状体, 我们的目的是通过复制生理调节来研究透镜的生物力学。

<table border="0" cellpadding="0" cellspacing="0" style="width:856px;" width="856">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Manual Lens Stretcher</td>
			<td style="width:233px;">Bioniko</td>
			<td style="width:132px;">MLS</td>
			<td style="width:300px;">Different animal species will require different shoe sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Porcine Eye Samples</td>
			<td>George G. Ruppersberger; slaughterhouse</td>
			<td>N/A</td>
			<td>Whole eyeballs were obtained</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Eye Samples</td>
			<td>The National Disease Research Interchange</td>
			<td>N/A</td>
			<td>Posterior poles without corneas were ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors (5 1/2&#39;&#39; Straight)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Forceps (4 1/2&#39;&#39;)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iPhone 6s</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Any imaging system with ~0.1 mm resolution will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hypochorite</td>
			<td>Clorox</td>
			<td>Clorox Regular-Bleach</td>
			<td>Any disinfectant will work</td>
		</tr>
	</tbody>
</table>

simulating the mechanics of lens accommodation via a manual lens stretcher

本议定书的目标是以成本效益高、实用性强的方式模仿生理调节的生物力学。住宿是通过睫状体收缩和 zonule 纤维松弛而实现的, 这导致了近视力所需的透镜的增厚。在这里, 我们提出了一个新的, 简单的方法, 通过绷紧的 zonules 连接到镜头胶囊通过一个手动镜头担架 (MLS) 的住宿复制。这种方法监测透镜在承受一致力时所达到的径向拉伸, 并允许比较可伸展的镜片, 使其不能伸展。重要的是, 担架夫妇对 zonules 直接, 而不是对巩膜的眼睛, 因此只需要透镜, zonules, 和睫状体, 而不是整个地球样本。与获得整个地球相比, 这种差异可以显著降低获得捐赠尸体镜片的成本约62%。

猪眼, 一个共同的样本, 研究通过透镜拉伸4,15, 获得了, (n = 10) 从一个地方屠宰场和这个协议被用来观察的住宿能力的镜片。图 5A显示通过 MLS 拉伸前后猪晶状体的比较。在拉伸 (p < 0.001) 时, 透镜半径平均增加了 0.19 0.07 毫米, 相当于原来半径的4.2 英寸1.62% 的增加。住宿与透镜弹性相关, 因...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

用人工晶状体担架模拟透镜体的力学性能

我们提出了一个有效的方法来研究透镜的住宿使用人工晶状体担架。该协议模拟生理调节, 通过拉 zonules 连接在透镜胶囊, 从而, 伸展透镜。

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

simulating-mechanics-lens-accommodation-via-manual-lens

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.7K 次观看.  University of Maryland. 我们提出了一个有效的方法来研究透镜的住宿使用人工晶状体担架。该协议模拟生理调节, 通过拉 zonules 连接在透镜胶囊, 从而, 伸展透镜。

视频: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

创伤性脑损伤研究神经细胞的脉冲增压

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

科研

生物工程

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

使用光学镊子探测肌动蛋白丝束中的肌球蛋白系综力学

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

寡核苷酸的化学三磷酸化

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Cerebral organoids provide unprecedented opportunities for studying organ development and human disease pathology. Although great success has been achieved with cerebral organoid culture systems, there are still operational difficulties in applying this technology. The present protocol describes a cerebral organoid procedure that facilitates medium change and organoid transfer.

通过侧向柔光照明促进脑类器官培养

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...