使用此协议的研究由心脏和中风基金会（G-18-0020952）、克雷格·尼尔森基金会（542675）、加拿大自然科学和工程研究理事会（RGPIN-2014-05498）和安大略省研究、创新和科学部（ER16-12-013）资助。

这项研究得到了大学健康网络研究伦理委员会的批准。在注册研究之前，从每个参与者那里获得了知情同意。还从录像中出现的任何照顾者或家庭成员那里获得了知情同意书。
1. 验证协议对个人的适用性
注:本协议适用于手功能受损但并非完全缺失的个人（具体标准可适应人口和/或兴趣问题）。
<ol><li>询问参与者受影响的手是否影响他们执行 ADL 的能力。 注:建议参与者举出一些他们能够且不能用受影响的手独立执行的任务示例。</li>
	<li>验证蒙特利尔认知评估 （MoCA） 的总分是否高于 21，以避免在理解和遵循协议程序方面出现潜在困难。</li>
</ol>2. 确定参与者的日常生活
<ol><li>请参与者回忆过去两周的日常生活。记录执行哪些日常任务、执行时间以及大约在什么时间执行。</li>
	<li>与参与者合作，选择 3 个时隙，每个时段 1.5 小时，在此期间录制视频。选择分布在一周中不同天数的时隙，并且通常按顺序执行涉及手的 ADL 时段。 注:选定的 ADL 必须代表每个参与者的典型活动，并被他们视为有意义的。在不同日期安排记录时间旨在增加记录的 ADL 的多样性，并促进捕获有用和有意义的数据。 注:录制时隙计划用于记录效率，但参与者应了解他们完全控制何时开始和停止录制。</li>
</ol>3. 与参与者就录制时间表和目标视频内容达成协议
<ol><li>在讨论他们可能遇到的任何顾虑后，获得每个参与者对录制时间表的一致意见。</li>
	<li>在两周内设定 3 小时视频的目标。告知参与者视频不足可能导致延长录制时间。</li>
</ol>4. 强调自然执行 ADL 的重要性
<ol><li>指示参与者专注于捕捉逼真的程序，而不是指定要记录的特定活动。该说明的目的是劝阻参与者人为地记录比他们通常记录的更多的特定活动。</li>
</ol>5. 通知家中录音过程中可能存在隐私问题
<ol><li>确保参与者明白，所有录音都应在家中进行，而不是在公共场所进行，以避免隐私问题。</li>
	<li>举一些可能引发隐私问题的例子，例如洗澡、穿衣/脱衣服和检查机密信息。提醒参与者注意镜子，镜子可能会在录音中显示他们的脸。</li>
	<li>建议参与者尽量避免其他人（如家庭成员或护理人员）出现在视频中。 注:在研究方面，在不可避免的情况下，应获得这些人的知情同意。</li>
</ol>6. 相机和平板电脑指令
注:如果参与者在初始接触期间表示，他们需要护理人员协助满足其许多日常需求，则鼓励护理人员也参加考察访问，并接受设备使用方面的培训，以便他们以后能够协助参与者。
<ol><li>向参与者演示如何使用以自我为中心的相机（材料表）。<ol>
<li>演示如何打开和关闭相机。</li>
		<li>演示如何使用相机控制录音（开始、暂停、停止）。</li>
	</ol>
</li>
	<li>演示如何使用平板电脑（材料表）与预装相机应用程序来控制录音，如果适用的话。 注:演示包括控制来自相机应用程序的录制，以及重播和编辑（例如，修剪或删除）录制的视频。相机遥控器最初被认为是（补充文件），但在实践中没有使用，因为参与者是舒适的使用相机或平板电脑开始和停止录音。<ol>
<li>演示如何打开和关闭平板电脑。</li>
		<li>演示如何通过相机应用程序将平板电脑连接到相机。</li>
		<li>演示如何控制来自相机应用的录音。</li>
		<li>演示如何查看相机应用中录制的视频。</li>
		<li>演示如何修剪或删除相机应用程序中的视频。</li>
	</ol>
</li>
	<li>演示如何使用可调节到参与者头部的弹性头带来穿和擦相机。 注:见图1。 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61898/61898fig01.jpg"> 图1。可穿戴相机设置。 （A）以自我为中心的相机的定位。（B）从相机查看角度。<a href="https://www.jove.com/files/ftp_upload/61898/61898fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
	<ol>
<li>将相机放在参与者的额头上。调整头带，舒适而稳定地佩戴相机。</li>
		<li>确保相机与额头的最佳角度。</li>
		<li>要求参与者录制一小段视频，同时移动他们面前的手并操纵对象（例如平板电脑）。</li>
		<li>查看录制的视频，并确保在执行操作任务时，两只手在场景的中心区域清晰可见。</li>
		<li>与参与者及其护理人员一起练习使用相机和平板电脑，直到他们表现出熟练程度。</li>
	</ol>
</li>
</ol>7. 赠送设备
<ol><li>将所有设备的套件交给参与者，以便在家里记录其 ADL。除了相机和平板电脑，该套件还包括额外的相机电池、用于相机和平板电脑的电池充电器、充电电缆、相机头带以及一套用于使用相机的打印指南（参见 补充材料）。</li>
</ol>8. 实验故障排除和后续工作
<ol><li>提供研究人员的联系方式，以帮助解决家庭实际录音过程中的障碍。一周后，研究人员打电话给参与者记录记录进度并解决任何潜在的技术问题。</li>
</ol>9. 检索设备和视频
<ol><li>通过预先付费的邮包，将参与者的所有设备和视频进行反驳。</li>
	<li>确保参与者同意共享返回的所有视频。鼓励参与者在将所有收集的视频返回给研究团队之前进行审查，并删除他们不想分享的任何部分。</li>
	<li>对于研究，请查看返回的视频，并检查是否有人在未经其同意的情况下出现在视频中。如果是这样，发送同意书或致电视频中出现的个人，以获得他们的同意使用视频。如果无法联系到这些个体，研究人员将删除他们出现的视频部分。</li>
</ol>

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作者没有什么可透露的。

我们提出了一个协议，用于在家中使用可穿戴摄像头在上肢损伤（如 cSCI 和中风）中录制 ADL 视频。该协议是灵活的，可以以目标为导向，以捕获特定 ADL 中的手功能性能，或远程跟踪居住在家中的人的康复进度。以自我为中心的视力模式在远程监测社区居民的手部功能以及一旦人们出院后优化康复方面具有巨大潜力。尽管在扩展使用期间（例如超过 1 小时）的舒适性存在一些限制，但以自我为中...

手部功能是决定上肢损伤1、2的临床人群的独立性和生活质量。掌握家庭手部损伤者的手部功能对于评估他们在康复期间和康复后开展日常生活活动的能力的进展至关重要。大多数临床手部功能评估是在临床或实验室环境中进行的，而不是在家里进行的。现有的临床手部功能评估，试图捕捉对国内ADL的影响是问卷，并依靠主观自我报告的评分5，6，7。目前尚无法进行客观评估，以评估康复对家庭手部功能的最终影响。
近年来，许多可穿戴技术已经开发和实施，以捕捉上肢功能在现实世界中的环境。可穿戴传感器（如加速度计和惯性测量单元 （IMUs） 通常用于测量日常生活中的上肢运动。然而，这些设备通常不能区分检测到的年代是否属于功能上肢运动8，9，定义为有目的的运动，旨在完成所需的任务。例如，一些可穿戴传感器对行走过程中上肢摆动的存在很敏感，而上肢不是功能运动。此外，虽然手腕磨损的加速度计可以捕捉上肢运动，但在真实环境中，它们无法捕捉到手部功能的细节。感应手套可以捕获有关手部操作的更详细的信息，但是对于手部功能和感觉已经受损的人来说，它们可能很麻烦。此外，亦有人提出可穿戴方法，透过磁力测量仪或手指磨损加速度计11、12、13捕捉手指运动，但这些运动的功能解释仍具挑战性。因此，虽然以前提出的可穿戴设备体小且使用方便，但不足以描述手部使用的细节和功能背景。
已建议可穿戴相机，以填补这些空白，并捕捉手功能的细节，在家里的ADL神经恢复应用15，16，17，18，19。使用计算机视觉对以自我为中心的视频进行自动分析，通过提供有关手本身和实际 ADL20中执行的任务的信息，具有相当大的潜力，可以量化上下文中的手功能。另一方面，根据电池、存储和舒适性考虑，连续录制的持续时间通常限制在大约 1 到 1.5 小时。在这里，在这些限制下，我们提出了一个以自我为中心的视频收集协议，旨在获得既能代表个人日常生活的数据，又能提供手功能评估的信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Egocentric camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>GoPro Hero 4 and 5</td>
			<td>A camera that records from a first-person angle.</td>
		</tr>
		<tr>
			<td>Battery chager and batteries</td>
			<td>GoPro Inc., CA, USA</td>
			<td>MAX Dual Battery Charger + Battery</td>
			<td>Extra batteries for the camera and battery charger</td>
		</tr>
		<tr>
			<td>Camera charger</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Supercharger</td>
			<td>This charger is connected to the camera directly without disassembling the camera frame.</td>
		</tr>
		<tr>
			<td>Camera frame</td>
			<td>GoPro Inc., CA, USA</td>
			<td>The Frame</td>
			<td>The hinge of the camera frame can be used to adjust the angle of view of the camera.</td>
		</tr>
		<tr>
			<td>Headband for the camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Head Strap + QuickClip</td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk, CA, USA</td>
			<td>32GB microSD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet</td>
			<td>ASUSTeK Computer Inc., Taiwan</td>
			<td>ZenPad 8.0 Z380M</td>
			<td>The tablet is installed with the GoPro App in order to connect with the camera.</td>
		</tr>
	</tbody>
</table>

capturing representative hand use at home using egocentric video in individuals with upper limb impairment

神经损伤后手部功能受损会对独立性和生活质量产生重大影响。大多数现有的上肢评估都是亲自进行的，这并不总是表明在社区中使用手。需要采用新的方法来捕捉日常生活中的手部功能，以衡量康复干预的真正影响。已建议将以Ego为中心的视频与计算机视觉相结合，用于自动分析，以评估家庭使用手部。但是，连续录音的持续时间有限。我们提出了一个协议，旨在确保获得的视频代表日常生活，同时尊重参与者的隐私。 
通过研究人员和参与者之间的协作过程选择具有代表性的录制时间表，以确保视频捕捉自然任务和性能，同时对手部评估有用。向与会者演示设备和程序的使用情况。总共安排了两周的3小时录像。为了减少隐私问题，参与者可以完全控制开始和停止录制，并有机会在将视频返回给研究团队之前编辑视频。提供提醒，如有必要，还提供帮助电话和家访。 
该方案对9名中风幸存者和14名颈椎损伤患者进行了测试。获得的录像包含各种活动，如准备膳食、洗碗和编织。平均获得3.11±0.98小时的视频。由于在某些情况下生病或意外事件，录制时间从 12-69 d 不等。从23名参与者中的22名成功获得数据，其中6名参与者在家庭录音期间需要调查人员的协助。该协议是有效的收集视频，包含宝贵的信息，手功能在家里后神经损伤。

参与者人口统计学和包容性标准 为这些研究招募了23名参与者的样本:9名中风幸存者（6名男性，3名女性）和14名患有csCI（12名男性，2名女性）。招聘样本的人口统计学和临床信息摘要报告在 表1中。
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<td rowspan="2"></td>
			<td rowspan="2">年龄（年）</td>
			<td rowspan="2">受伤后的持?...

Watch this Scientific Journal Video about Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment at JoVE.com

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

建议使用以自我为中心的相机捕捉手部损伤个体在日常生活中的自然手部功能。该协议的目标是确保录音代表个人在日常生活中通常使用的手。

捕捉代表手在家里使用以自我为中心的视频在个人与上肢损伤

Impaired hand function after neurological injuries can have a major impact on independence and quality of life. Most existing upper limb assessments are ...

capturing-representative-hand-use-at-home-using-egocentric-video

Research

JoVE Journal

Bioengineering

2.5K 次观看.  University of Toronto. 建议使用以自我为中心的相机捕捉手部损伤个体在日常生活中的自然手部功能。该协议的目标是确保录音代表个人在日常生活中通常使用的手。

视频: Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统:应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

基于家庭的盖特和活动分析监视器

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

使用微X射线计算机断层扫描与磷酸制备可视化人类纤维肌组织

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...