部分，这项工作已经资助，由加拿大创新基金会。

1。系统准备<ol><li>清除任何反射材料，可以观察到由摄像机捕获体积。这将减少实际的皮肤固定的背景标志相混淆的标记在运动过程中测试的机会，并提高整体精度的会议。 </li><li>校准摄像头的所有摄像机固定标记对准在实验室内的固定位置。扩展的静态标定，动态的动作，使用移动标记放置在已知的距离。一定要覆盖尽可能多的捕获量尽可能地优化校准。 </li><li>组织的所有材料（反射标记，测量装置，等）可用于患者准备。在测试过程中，提高了工作效率，降低了患者的负担。 </li></ol> 2。患者准备<ol><li>公开为尽可能拟被测定的关节和身体分部的皮肤。最小的耆老吨宽松的衣服和确保任何件衣服，可能会干扰的能力的摄像机的可视化的反射标记的约束。这可以通过使用磁带或剪辑。只要有可能，确保标记被直接固定到皮肤。 </li><li>准备在皮肤上标记固定。剃须或磨蚀的区域可能是必要的情况下，头发是本或在皮肤表面时，过分出汗或油性。擦拭面积使用外用酒精或类似液体可能是有用的。这些步骤是很重要的，以便最大限度遵守标记和皮肤之间的，并防止脱落的标记。 </li><li>触诊用于设置要使用的标志的基础上的关键的解剖标志。在实际的地标标记的皮肤会提高精度标记的位置，并提供必要的信息标记在评估过程中脱落的情况。 </li><li>根据解剖标志的粘贴反光标记规格的标记集。大多数标记设置将包括至少12-15标记在下肢和上身的各种解剖标志的双边。重要的是要注意，以重新创建实际骨骼运动的能力将取决于皮肤基于标记的定位。因此，必须认真考虑时，确定要使用的生物力学模型。 </li><li>测量重要的人体测量数据，如果需要的话。根据生物力学模型，这些数据可能需要计算段的长度，关节的旋转中心的位置，和整体的惯性特性的生物力学数据的脱机处理过程中的运动段和四​​肢。 </li></ol> 3。运动分析与提供的实时反馈<ol><li>具有主题捕捉卷在中间的立场初始静态试验持续约3秒。这项试验是必要的，以确保所有勒药效机理的标记是可见的，计算段的方向。 </li><li>使用数据采集软件，标签所有适当的标记，并创建一个模板，特定的人体特征的个人。标记放置到个人的机身尺寸相匹配，提高数据的实时跟踪和分析。尤其重要的是，创建一个模型的移动，可以将标记定位的冗余。在出现标记闭塞或落客的情况下，能够利用额外的标记，在适当的情况下，适当的运动特性，并保持实时显示的数据没有休息。 </li><li>执行初始的运动分析试验，持续10-30秒。这是必需的以获得基准数据，并且也可以用作提供反馈给病人的结果的第一机构。咨询与病人有关的调查结果，重要的是要帮助在电机李尔宁时，需要产生新的运动模式。 </li><li>有治疗师解释拟移动变形例的目的。这应该包括的修改和生物力学和临床的理论依据，它是唯一的，以给定的病理。示范的运动修改由治疗师将提高运动技能学习的病人。运动，通常会根据修改的病人在治疗过程中的生物力学和临床演示，或研究的问题进行检查，如果仅用于研究目的。 </li><li>开始运动再培训会议。如果使用的是一台跑步机，让被摄体选择自己喜欢的速度，并提供一，两分钟，以达到稳定状态。这也让病人熟悉和舒适的设备，实验装置和协议。 </li><li>提供反馈意见，在病人的运动性能。这可以采取了许多不同的形式，ferent方法，并结合这些在早期的训练是有益的。开始用较少的技术方法，如口头反馈和进度的实时生物反馈治疗。利用实时生物反馈应始终包括一个结果变量的最大的一次清晰的显示。 </li><li>为病人提供足够的时间来练习新的运动。学习有效的电机没有达到瞬间。相反，不断实践新的运动特性将有助于确保电机的程序，负责该运动的重新制定。一个典型的再培训干预可能需要8-10针对性的培训课程，每次持续30至60分钟。 </li></ol> 4。患者简报和后续的培训课程<ol><li>讨论会议与病人的重要发现和成果。应包括变异的重要因素集中在性能上，坚持规定动作modification，并进一步说明修改的理由和重要性。 </li><li>会议从患者处获得信息。鉴于每个病人的偏好可能会有所不同，这可能是必要的修改对于一个给定的个人的干预的交付。这些应及早发现，以优化效率。 </li><li>确定后续的培训课程的计划，如果必要的话。 ，如果多届干预的选择，后续的培训课程应该用一个褪了色的方法，以提高运动技能学习。提供整体的反馈和备用的反馈，没有反馈在今后的会议之间的时间段。 </li></ol>

<ol>
	<li>Ivanenko, Y. P., Poppele, R. E., Lacquaniti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+programs+and+walking.">Motor control programs and walking.</a> Neuroscientist. 12, 339-348 (2006).</li><li>Woodford, H., Price, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMG+biofeedback+to+improve+lower+extremity+function+after+stroke.">EMG biofeedback to improve lower extremity function after stroke.</a> Cochrane Database Syst. Rev. 2007, CD004585 (2007).</li><li>Moreland, J. D., Thomson, M. A., Fuoco, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromyographic+feedback+to+improve+lower+extremity+function+after+stroke:+a+meta-analysis.">Electromyographic feedback to improve lower extremity function after stroke: a meta-analysis.</a> Arch. Phys. Med. Rehabil. 79, 134-140 (1998).</li><li>Colborne, G. R., Wright, F. V., naumann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+of+triceps+surae+EMG+in+gait+of+children+with+cerebral+palsy:+a+controlled+study.">Feedback of triceps surae EMG in gait of children with cerebral palsy: a controlled study.</a> Arch. Phys. Med. Rehabil. 75, 40-45 (1994).</li><li>Binder, S. A., Moll, C. B., Wolf, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electromyographic+biofeedback+as+an+adjunct+to+therapeutic+exercise+in+treating+the+lower+extremities+of+hemiplegic+patients.">Evaluation of electromyographic biofeedback as an adjunct to therapeutic exercise in treating the lower extremities of hemiplegic patients.</a> Phys. Ther. 61, 886-893 (1981).</li><li>Tate, J. C., Milner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+kinematic,+temporospatial,+and+kinetic+biofeedback+during+gait+retraining+in+patients:+a+systematic+review.">Real-time kinematic, temporospatial, and kinetic biofeedback during gait retraining in patients: a systematic review.</a> Phys. Ther. 90, 1123-1134 (2010).</li><li>Miyazaki, T., Wada, M., 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=Dynamic+load+at+baseline+can+predict+radiographic+disease+progression+in+medial+compartment+knee+osteoarthritis.">Dynamic load at baseline can predict radiographic disease progression in medial compartment knee osteoarthritis.</a> Ann. Rheum. Dis. 61, 617-622 (2002).</li><li>Barrios, J., Crossley, K., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+retraining+to+reduce+the+knee+adduction+moment+through+real-time+visual+feedback+of+dynamic+knee+alignment.">Gait retraining to reduce the knee adduction moment through real-time visual feedback of dynamic knee alignment.</a> J. Biomech. 43, 2208-2213 (2010).</li><li>Hunt, M. A., Simic, M., Hinman, R. S., Bennell, K. L., Wrigley, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+a+gait+retraining+strategy+for+reducing+knee+joint+loading:+Increased+trunk+lean+guided+by+real-time+biofeedback.">Feasibility of a gait retraining strategy for reducing knee joint loading: Increased trunk lean guided by real-time biofeedback.</a> J. Biomech. 44, 943-947 (2011).</li><li>Simic, M., Hunt, M. A., Bennell, K. L., Hinman, R. S., Wrigley, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trunk+lean+gait+modification+and+knee+joint+load+in+people+with+medial+knee+osteoarthritis:+The+effect+of+varying+trunk+lean+angles.">Trunk lean gait modification and knee joint load in people with medial knee osteoarthritis: The effect of varying trunk lean angles.</a> Arthritis Care Res. , (2012).</li><li>Hunt, M. A., Simic, M., Hinman, R. 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没有利益冲突的声明。

实时反馈的性能在运动过程中，如散步，可以是一个有价值的辅助标准的运动分析方法。虽然其相对处于起步阶段，具体的研究和离散运动的修改肯定会从中受益的能力，以产生所需的准确性和实时修改。例如，如果病人需要一个特定的移动变形量，这个量可以被测量，并提供在实际运动。这里介绍的方法可以用于测试新的方法来运动变形例以及细化现有协议的范围广泛的患者人群。 <p class="j...

下肢神经肌肉或肌肉骨骼结构的任何显着变化将可能有的移动功能和相关的物理特性的影响。因此，改善身体功能，是任何康复干预的重要成果。正常的重复动作，如散步一般按电机程序是包含必要的控制信息，需要激活正确的强度和定时1的肌肉。这些运动项目是必要的运动，以提高自动化程度，从而减少了专门用于运动控制，并允许应注意的其他更高层次的任务。然而，由于在运动中和运动程序的作用，事实上，这些程序精制过了数年，后损伤或疾病是一个具有挑战性的企业不断变化的运动性能。 传统上，的运动再培训的干涉离子的运动性能提供了足够的反馈，以确保新的信息被纳入新的和不断变化的电机程序为前提。简单而有效的，方法包括口头反馈与全球的指令（ 例如，"弯多"，"让你的膝盖直"），以及如使用镜子或录像设备提供视觉反馈的机制。虽然这些间接的策略是有用的，尤其是在资源有限的临床设置，他们提供的运动变量的离散的，可量化的措施限制他们的困难。其结果是，与反馈的附加的更直接的方法补充这些技术可能会提高电机重新学习所需。 有很多接受的研究和临床社区提供反馈的离散的，可量化的结果的运动特性，可以提高性能，在运动过程中retraini<html> NG干预。例如，瞬间视觉或听觉的肌肉活化强度使用肌电生物反馈装置反馈的康复运动成为主流，尤其是在行程2-3，脑性麻痹4，或慢性偏瘫5人。的运动运动学的反馈（关节和段的角度）与此相反，已被证明是较少使用的由于快速，准确地评估和测量这些成果中的一个难点。事实上，尽管定量，以实验室为基础的分析，突出的运动特征的生物力学研究，并已开始被纳入临床上，绝大多数的运动分析使用离线分析测试后保留的。然而，有越来越多的研究文献中，使用新技术，提供反馈的步态措施为手段，提高运动的成效，再培训6。 ve_content"一个病理，目前正在研究使用标准的运动分析系统的实时生物反馈功能集成是膝关节骨性关节炎（OA）。最近的研究利用实时反馈的步态运动学设计，特别是通过减少负载膝关节，量化使用外部膝盖内收力矩OA进展7 -一个公认的危险因素。例如，研究利用实时生物反馈治疗的的大腿角8或主干角度9-10级。亨特等[11]提供了一个实时显示在与会者面前的躯干角度步行试验过程中表现出的能力，增加展示的躯干倾斜在一个单一的培训课程，并伴随着减少膝盖内收矩震级。相反，巴里奥斯等[8]进行了八个交易日修改动态冠状面膝关节行走训练干预的重点后的1个月的干预与基线相比，在立场和角度均显着减少膝内收力矩值。这些研究中，类似的研究，依赖的能力来衡量，分析和显示变量的兴趣在持续不断地向患者。这个新兴的研究领域，有各种疾病影响的运动特征的患者的临床意义。使用运动改变有关的膝骨性关节炎（OA）的例子，本文的目的是描述需要进行运动再培训的干预，使用实时生物反馈的行走性能的方法。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>反光标记</td><td> 3x3的设计</td><td></td><td>直径12毫米</td></tr><tr><td>标记磁带光盘</td><td>折扣耗材</td><td> TD-22电极环，8毫米</td><td>设计用途是作为电极铤</td></tr><tr><td>运动分析摄像机</td><td>运动分析公司</td><td></td><td></td></tr><tr><td> Biofeedtrak </td><td>运动分析公司</td><td></td><td></td></tr><tr><td> MATLAB </td><td> The MathWorks公司</td><td></td><td></td></tr></tbody></table>

movement retraining using real-time feedback of performance

运动 - 尤其是运动模式，已经过了数年的磨练 - 任何修改需要重新组织负责管理运动性能的神经肌肉模式。该电动机可以提高学习通过一些使用的方法，在研究和临床的设置一样。在一般情况下，在实时或后移动的结果的知识的性能口头反馈被临床上常用灌输运动学习作为一个初步手段。根据患者的意愿和学习风格，视觉反馈（ 例如，通过使用镜子或不同类型的视频）或本体指导，利用治疗师触摸，用于补充治疗师的口头指示。事实上，这些形式的反馈相结合的运动学习和优化的结果在临床上是司空见惯的。 以实验室为基础，定量运动分析提供准确，客观地分析了各种运动在健康和受伤的人群中一直在研究环境中的中流砥柱。虽然实际的动作捕捉机制可能会有所不同，目前所有的运动分析系统依赖的体节和关节的运动轨迹，并使用已建立的运动方程来量化关键的运动模式的能力。由于采集和处理速度的变动的，分析和描述的限制历来发生脱机完成后，一个给定的测试会话。 本文将重点介绍一个新的补充标准的运动分析技术，依赖于附近的瞬时评估和量化运动模式和显示特定的运动特征的患者在运动过程中分析会议。其结果是，这种新技术可以提供一种新的方法，提供的反馈，有优势奥雅纳R当前使用反馈方法。

在图2中示出一个例子，从单一的运动再培训会议聚焦增加横向躯干倾斜角在患者体内与膝关节OA。培训使用口头和基于镜像的性能的反馈的组合约15分钟后，病人被提供有关侧向躯干弯曲量的实时数据。使用此方法的训练持续了一个额外的10分钟。在正常（未修改）的试验中，患者表现出一个自我选择的横向干线约2°倾斜（见峰值的20％左右的立场虚线）。在修改试验过程中，患者被?...

Watch this Scientific Journal Video about Movement Retraining using Real-time Feedback of Performance at JoVE.com

Movement Retraining using Real-time Feedback of Performance

再培训异常的运动模式后，受伤或疾病，是身体康复的重要组成部分。最近技术的进步已批准的运动过程中准确地评估各种不同的任务，与附近的瞬间量化的结果。这提供实时修改错误的运动模式的新的机会。

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

movement-retraining-using-real-time-feedback-of-performance

Research

JoVE Journal

Medicine

13.2K 次观看.  University of British Columbia. 再培训异常的运动模式后，受伤或疾病，是身体康复的重要组成部分。最近技术的进步已批准的运动过程中准确地评估各种不同的任务，与附近的瞬间量化的结果。这提供实时修改错误的运动模式的新的机会。

视频: Movement Retraining using Real-time Feedback of Performance

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using biofeedback of real-time functional magnetic resonance imaging (rt-fMRI) data. Increased activity of this region is seen in relationship with contamination anxiety both in control subjects1 and in individuals with obsessive-compulsive disorder (OCD),2 a relatively common and often debilitating psychiatric disorder involving contamination anxiety. Although many brain regions have been implicated in OCD, abnormality in the orbitofrontal cortex (OFC) is one of the most consistent findings.3, 4 Furthermore, hyperactivity in the OFC has been found to correlate with OCD symptom severity5 and decreases in hyperactivity in this region have been reported to correlate with decreased symptom severity.6 Therefore, the ability to control this brain area may translate into clinical improvements in obsessive-compulsive symptoms including contamination anxiety. Biofeedback of rt-fMRI data is a new technique in which the temporal pattern of activity in a specific region (or associated with a specific distributed pattern of brain activity) in a subject's brain is provided as a feedback signal to the subject. Recent reports indicate that people are able to develop control over the activity of specific brain areas when provided with rt-fMRI biofeedback.7-12 In particular, several studies using this technique to target brain areas involved in emotion processing have reported success in training subjects to control these regions.13-18 In several cases, rt-fMRI biofeedback training has been reported to induce cognitive, emotional, or clinical changes in subjects.8, 9, 13, 19 Here we illustrate this technique as applied to the treatment of contamination anxiety in healthy subjects. This biofeedback intervention will be a valuable basic research tool: it allows researchers to perturb brain function, measure the resulting changes in brain dynamics and relate those to changes in contamination anxiety or other behavioral measures. In addition, the establishment of this method serves as a first step towards the investigation of fMRI-based biofeedback as a therapeutic intervention for OCD. Given that approximately a quarter of patients with OCD receive little benefit from the currently available forms of treatment,20-22 and that those who do benefit rarely recover completely, new approaches for treating this population are urgently needed.

Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.

实时功能磁共振成像生物反馈，针对污染焦虑的眶额皮层

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to assess human gait. The primary aim of this manuscript is to test the concurrent validity of kinematic measures recorded by a smartphone application in comparison to a 3D motion capture system in the sagittal plane. The secondary aim was to develop a protocol for clinicians on the set up of the smartphone camera for video movement analysis.
The sagittal plane knee angle was measured during heel strike and toe off events using the smart phone app and a 3D motion-capture system in 32 healthy subjects. Three trials were performed at near (2-m) and far (4-m) smartphone camera distances. The order of the distances was randomized. Regression analysis was performed to estimate the height of the camera based on either the subject&#39;s height or leg length.
Absolute measurement errors were least during toe off (3.12 &#177; 5.44 degrees) compared to heel strike (5.81 &#177; 5.26 degrees). There were significant (p &#60; 0.05) but moderate agreements between the application and 3D motion capture measures of knee angles. There were also no significant (p &#62; 0.05) differences between the absolute measurement errors between the two camera positions. The measurement errors averaged between 3 - 5 degrees during toe off and heel strike events of the gait cycle.
The use of smartphone apps can be a useful tool in the clinic for performing gait or human movement analysis. Further studies are needed to establish the accuracy in measuring movements of the upper extremity and trunk.

Video Movement Analysis Using Smartphones ViMAS: A Pilot Study

This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement.

视频运动分析使用智能手机（ViMAS）的初步研究

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

小鼠急性心肌梗死的实时压力容积分析

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...