CIBC奖学金（JA）和NSERC补助金（RZ）。我们是感谢罗奇M.科莫（Brainsight）的红外摄像机对他的帮助，MR兼容的跟踪器和其他硬件支持。我们也感谢布赖恩，海因斯（Hybex创新公司）是谁设计的多节臂的线圈支架和提供的数字显示在视频。特别感谢所有的的MR技术人员和M.费雷拉从麦康奈尔脑成像中心的蒙特利尔神经学研究所的帮助我们优化设计的实验。

该协议为期两天的会议分为（不一定是连续的）。第一天由组成的解剖和功能磁共振扫描，以确定每个参与者的领域有针对性的TMS的功能磁共振成像定位。第二天由功能磁共振成像会议前和后TMS，TMS是应用在扫描仪内使用一个特殊的MR兼容TMS线圈（Magstim有限公司，威尔士，英国）和无框架立体定向系统（Brainsight）。后者用于在皮质区相对于每个参与者的解剖和功能的数据实时TMS线圈的位置。 1。本地化会议<ul><li>开始您的参与者获得高分辨率的解剖图像。 </li><li>然后，获取图像功能，使用梯度回波EPI脉冲和稀疏采样范式，以尽量减少任何大胆的效果或听觉掩蔽由于MRI扫描噪音14,15。在我们的例子中，功能磁共振成像输出D姚小萍Yao Xiaoping旋律声任务中，参加者，以确定是否两个连续的5个音符旋律是相同的或不同的2,16。一个非歧视性的听觉控制任务还包括，中，受试者听到两个相同长度的五个音符模式，所有的C5相同的间距，并指示点击鼠标左键后的第二个刺激。的静默期，也是中随机插入的任务在每次运行试验。共72试验随机顺序:24个试验的旋律歧视听觉对照试验，24和24期的沉默，总工期为12分16秒。 </li><li>定义刺激部位解剖和/或功能性的地标。一个人必须知道，TMS是有限的刺激部位的深度，因为深入的电场强度的衰减，并不能期望达到面积大于3厘米6,17更深。一个重要的步骤就是使用类似的标志性建筑的每个部分icipant，这可能是困难的，因为参与者之间的解剖结构和功能的差异。在这里，我们的目标Heschl的每一个参与者，位于回的解剖和功能的地标。我们所提供的哈佛，牛津结构的地图集（ <a href="http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html" target="_blank">http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html</a> ）和TMS目标Heschl的回用口罩单独定义的高峰期激活内的Heschl回2。此外，我们还定义了顶点的位置，将被用作控制现场控制非特异性作用的TMS如声学和体感的工件。顶点被定义作为一个点之间的枕外隆凸尖的鼻梁的中途解剖，并从右侧和左侧的intertragal槽口等距。刺激部位（ 即 Heschl的回或顶点）的顺序之间平衡个人。 </li></ul> 2。功能磁共振成像实验前和后TMS 前TMS功能磁共振成像会话<ul><li>准备去直接在扫描仪中的参与者。这包括去除金属和填充TMS和MR筛查形式。 </li><li>启动MR收购的解剖和功能扫描（相同的定位会话中进行，第1节）。 </li></ul>在MRI环境中的无框立体定向和TMS 无框立体定向系统是由一个红外相机（北极星光谱），一些工具和的纤夫（Brainsight）用于登记过程和一个计算机。计算机位于扫描器室之外，但位于扫描器室的入口和扫描仪的门保持打开期间TMS应用程序。的工具和跟踪器是MR兼容，以及支持红外相机三脚架（自制）和第erefore扫描仪的房间内使用。红外线摄像头是不是MR-兼容，因此扫描仪内部的室，扫描仪的门附近的在大约两米，从扫描仪床（见的安全过程​​的讨论）被定位。坐落在一个房间相邻的MRI扫描室的的TMS刺激器系统。我们使用的MRI兼容的TMS线圈位于扫描仪内部的室，并通过一个7-m的电缆连接到TMS系统通过一个RF滤波器管。 <ul><li>加载您参与者的解剖和功能影像和刺激目标到立体软件包（ 例如 Brainsight）。在这里，我们将目标瞄准正确的Heschl的回。 </li><li>前TMS功能磁共振成像采集后，去除上层的MR磁头线圈部分的32通道头线圈（如果使用西门子3T扫描仪，32通道头线圈结构）。 </li><li>接下来，向下滑动的参与者扫描仪的病床上。 </li><li>修复的头带和跟踪组的particip蚂蚁的头部。 </li><li>多节臂安装扫描仪的床和修复的MR兼容TMS线圈的手臂上。 </li><li>验证所有的跟踪和线圈是在该领域的摄像头。这里，相机略微移动到右侧的参与者，启用一个更容易跟踪线圈位移定位时的右半球。 </li><li>校准您的拍摄对象的头部立体定向工具（ 即指针工具）。这样做是通过coregistering参与者的解剖的数据具有相同的地标头（ 例如，在我们的例子中的鼻子，鼻根和两个耳朵的耳屏上的前端）上的多个标记。在此过程中，需要两个实验者，1接近到参与者的头部将指针定位工具，在参与者的头部，和其它实验在扫描器室入口，在计算机上执行的登记。 </li><li> MR兼容TMS线圈切到T的位置他头皮，和线圈纤夫朝向的红外摄像机。线圈与线圈手柄向后和平行指向中线2取向。使用多节臂的螺丝固定线圈位置。 </li><li>在房间相邻的MRI扫描仪，打开TMS系统，并开始刺激。 TMS图案协议， 即连续THETA突发刺激（CTBS）在3个脉冲组成，在50Hz时为40岁，在5Hz重复应用。我们使用的激励器输出18,19所定义的一个固定的刺激强度（41％）。我们选择了这个协议，因为它已被证明调节皮层可塑性，持续时间长达30分钟刺激停 ​​止后，在健康人群中20，（见讨论部分安全程序）。 </li></ul>后TMS功能磁共振成像会议<ul><li>刺激是完成后，重要的是得到的主题放回扫描器尽快。删除的T从扫描室，MS线圈和删除多节臂。参与者的头向后滑动的MR磁头线圈。请确保您的扫描仪的准备，蓄势待发。我们的建议是保持身体平台的整个TMS会话的过程中提出的，和定位扫描的次数和持续时间减少到最低限度。 </li><li>由于影响的经颅磁刺激是暂时的，最终的扫描会话的功能扫描开始。同样，我们进行了功能磁共振成像的旋律工作在一个12分钟的运行。 </li><li>在最终扫描完成后，完成与解剖扫描。 </li></ul> 3。代表性的成果前和后TMS功能磁共振成像会议的功能磁共振成像数据进行分析。对于每个功能磁共振成像会话（ 即前置和后置TMS），对比的旋律和听觉的控制任务，任务相关活动的左，右Heschl的脑回，颞上回，下额叶脑回和precenTRAL回（ 图1 A，B）。为了评估前和后TMS功能磁共振成像会话之间的差异，我们使用学生的配对t检验进行随机效应分析。意义被确定使用识别由AZ&gt; 2阈值和一个群集阈值校正为p = 0.05的群集。 图1 C表示的对比度后减去预CTBS为单个参与者。这些数据表明，针对正确的Heschl回（黑圈）的CTBS诱导增加（左）在对侧听觉皮层的功能磁共振成像响应，包括的左侧Heschl的回。功能磁共振成像反应的变化，也发现在左中央后回，左岛，并在外侧枕叶皮质双侧。然而，没有显着变化在fMRI响应下可见的线圈。此外，类似的合并的TMS-fMRI的协议重复刺激的顶点（控制场址）。比较CTBS施加超过顶点的前和后的fMRI会话没有显示任何标志意义nt的影响（数据未示出）。 <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="图1"/> 图1。分析的个人预TMS功能磁共振成像数据（A），后TMS fMRI数据（B）和后减前TMS的的fMRI数据（C）。 A.结果的对比旋律的歧视减去的听觉控制的试验，一个参与者在预TMS功能磁共振成像会议（A）和后TMS功能磁共振成像（B）会议。从左至右:轴向，冠状面和矢状面的看法。 （A）和（B），TMS线圈的目标是正确的Heschl回（黑圈）位于X = 54，Y = -13，Z = 1（MNI152标准的空间）。前和后TMS功能磁共振成像会议，坐标显示在x = -54，y = -13，z = 1的（MNI152标准空间）显示在左侧半球的变化，在现场的刺激（ 即右Heschl的回）。 C.结果的对比后减前TMS功能磁共振成像会使用学生的配对t检验。

没有利益冲突的声明。

我们描述了一个协议，结合当前离线TMS和功能磁共振成像调查的听觉皮层的功能组织。在接下来的章节中，我们将讨论的方法时，需要考虑的因素进行这样的方法。 收购及时间后TMS功能磁共振成像会话 顺序扫描采集和平衡的前和后TMS功能磁共振成像会议 关键的是要获得一个MR解剖扫描之前和之后，以TMS为内标，以得到一个鲁棒?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td></tr><tr><td>经颅磁刺激</td><td> Magstim超级Rapid2刺激，快速加一个模块</td><td> Magstim有限公司，威尔士，英国</td></tr><tr><td>线圈磁刺激</td><td> MRI兼容70 mm图8圈</td><td> Magstim有限公司，威尔士，英国</td></tr><tr><td>磁共振成像</td><td> 3-T的西门子三重奏扫描仪，32通道头线圈</td><td>西门子公司，德国</td></tr><tr><td>无框立体定向</td><td> Brainsight </td><td>盗贼研究公司，加拿大蒙特利尔</td></tr><tr><td>光学测量系统</td><td>北极星谱</td><td>北数码有限公司，安大略，加拿大</td></tr><tr><td>多节臂，线圈架</td><td>标准</td><td> Hybex创研离子公司，安茹，加拿大</td></tr><tr><td> MRI兼容的插入式耳机</td><td>型号S14（Sensimetrics，） </td><td> Sensimetrics公司，MA，USA </td></tr></tbody></table>

mapping the after-effects of theta burst stimulation on the human auditory cortex with functional imaging

听觉皮质涉及到的声音的处理，这是在语音或音乐相关的处理1的基础。然而，尽管有相当的最新进展，人类听觉皮层的功能特性和单侧远没有得到充分的理解。经颅磁刺激（TMS）是一种非侵入性技术，可以短暂或持久地调节大脑皮质的兴奋性，通过局部磁场脉冲的应用，并探索可塑性和连接是一个独特的方法。最近才开始被应用到理解听觉皮质功能2。 使用TMS的一个重要问题是，刺激的生理后果是难以确定的。虽然许多TMS研究使线圈的目标的区域是受影响的区域的隐含的假设，这不需要的情况下，特别是对于复杂的认知功能WHI的CH取决于许多大脑区域之间的交互3。这个问题的一个解决办法是用功能性磁共振成像（fMRI）技术，结合TMS。这里的想法是，功能磁共振成像的大脑活动的变化与TMS将提供一个指数。因此，功能磁共振成像将提供一个独立的手段，评估哪些地区受到影响的TMS和它们是如何调制4。此外，fMRI的允许功能连接的评估，这代表一个遥远的地区的时间之间的耦合措施。因此，它可以是有用的，不仅要测量的净活性调制引起的TMS在给定的位置，而且在何种程度上受TMS的网络性能，通过任何观察到的变化，在功能连接。 有不同的方法结合TMS和功能成像的时间顺序的方法。官能MRI可被施加之前，期间，之后，或者之前和之后都TMS。最近一些交错TMS和功能磁共振成像研究，以提供在线地图功能的变化引起的TMS 5-7。然而，这在线组合有许多的技术问题，包括静态的TMS线圈在扫描器室，或MR图像形成的过程中的TMS脉冲的影响的存在下，从所得的工件。但更重要的是，由TMS（与标准的使用，因为扫描器的谐振孔相比增加）和增加的TMS的线圈振动（由于MR扫描器的静磁场的强的机械力引起的诱导）的响亮的声噪声研究听觉处理时，构成一个关键的问题。 这是原因之一fMRI的满分，TMS为内标，本研究之前和之后进行。类似的方法已被用于针对运动皮层8,9，运动前皮层10，初级躯体感觉皮层11,12和语言相关领域的13，但到目前为止，还没有结合TMS-fMRI研究调查了听觉皮层。这篇文章的目的是提供有关的协议和注意事项要成功地结合这两种神经科学的工具来研究听觉处理的细节。 以前我们发现，在高，低频率（或10 Hz和1赫兹），在听觉皮层调制响应时间（RT）在旋律中的歧视任务2的重复TMS（经颅磁刺激）。我们还发现，RT调制在听觉功能连接网络评估与使用功能磁共振成像的功能左侧和右侧听觉皮层之间的连接执行任务的过程中，较高的易化作用（ 即降低RT）观察经颅磁刺激。然而，这些研究结果主要是相关性，功能性核磁共振造影前进行经颅磁刺激。在这里，功能磁共振成像进行前，后，立即TMS提供直接的措施听觉皮层的功能组织，并且更具体的塑料重组后发生的神经干预设置由TMS的听觉神经网络。 结合应用在听觉皮层的功能磁共振成像和TMS应该能够更好地理解大脑的听觉处理机制，提供生理功能的影响TMS信息。这方面的知识对于许多认知神经科学应用可能是有用的，以及用于优化的TMS的治疗中的应用，特别是在听觉相关的疾病。

Watch this Scientific Journal Video about Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging at JoVE.com

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

听觉处理是基础的语音和音乐相关的处理。经颅磁刺激（TMS）已经被成功地用于研究的认知，感觉和运动系统，但很少有人去试镜。在这里，我们调查了解听觉皮层的功能组织与功能性磁共振成像TMS相结合。

映射后的Theta突发刺激对人类听觉皮层功能成像

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

mapping-after-effects-theta-burst-stimulation-on-human-auditory

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K 次观看.  McGill University. 听觉处理是基础的语音和音乐相关的处理。经颅磁刺激（TMS）已经被成功地用于研究的认知，感觉和运动系统，但很少有人去试镜。在这里，我们调查了解听觉皮层的功能组织与功能性磁共振成像TMS相结合。

视频: Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

加固，Eyetracking，生理监测的功能成像

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...