这项工作是支持由美国国立卫生研究院/国家补充和替代医学（R21AT00978）中心（1 - P01 - 002048 - 01）（K01 - AT - 002166 - 01），国家研究资源中心（P41RR14075），精神疾病发现研究所（MIND）神经和脑计划资助（NS 34189）。

第1部分:知情同意并准备健康成人志愿者招募为研究对象，根据机构审查委员会批准的程序。开始研究前签署知情同意是主体。 第2部分:生理监测设备设置公元仪器PowerLab系统系统允许的生理数据同步功能磁共振成像扫描，通过使用功能磁共振成像的触发信号输入通道上记录。这是一台笔记本电脑，心电图，呼吸，皮肤电导反应，脉搏血氧饱和度，以及任何其他生理信号，将需要记录的投入为基础的系统。 <ol><li>如果正在使用呼吸带系统，传感器必须被插入一个电源插座在房间相邻的扫描仪，管领导带美联储通过入口进入扫描室和一个BNC连接器，导致在PowerLab系统扫描控制室。 </li><li>如果一个触发器被用来同步功能磁共振成像数据的生理数据，那么还必须连接BNC电缆的核磁共振成像扫描仪的触发信号，在扫描控制室PowerLab系统。 </li><li>收集监测装置。将需要4个心电电极，以及在体内系统rollcart心电图电缆连接到MR兼容。 </li><li>如果皮肤电导测量，皮肤电导连接器插入到扫描室的接线盒，并在扫描室准备皮肤电导凝胶。 </li></ol>第3部分:准备MR扫描的主题<ol><li>重要的是要排除不必要的焦虑和疲劳扫描当天，因为基本的状态可能会影响大脑的反应，以针灸刺激。建议受吃便餐之前的程序，这样可避免的弱点和不良反应。 </li><li>指示的主题在于佩戴耳塞机，以抑制扫描器噪音和仰卧头部固定的缓冲支持。由于针灸刺激扫描信号/噪声比低，与普通电机或视觉任务相比，数据往往更容易被损坏头部运动。一个支持是放在膝盖下，以保持高跟鞋担架的表面，以减少身体的运动。头线圈固定垫舒适的调整。平均扫描2个小时的会议，舒适性是不间断的扫描至关重要。 </li><li>如果需要生理监测，主题应根据上一节被放在扫描仪前，的准备。这是可取的主体觉醒与眼睛跟踪监测过程中如果有扫描。 </li><li>将4个心电图电极主体的左上胸（女科，采取隐私的预防措施，如百叶窗和降低扫描控制室湾的窗口，关闭扫描仪门）。连接电缆的电极，并在体内装置。对于安全电缆，导致这一问题的所有其他电缆应沿扫描表中直接运行，没有形成任何循环。在体内装置打开，并检查心电图信号质量。如果有必要调整电极定位。 </li><li>可控硅的电极与导电凝胶填充井。这种特殊的SCR凝胶应等渗皮肤。执行图软件打开电路零。可控硅电极连接两个相邻的主体的手的手指远端垫包装魔术贴周围紧紧是舒适的。 </li></ol>第4部分:成像针灸刺激量身定制的功能磁共振成像序列。 <ol><li>我们使用了1.5或3.0特斯拉西门子三星磁共振成像系统（西门子，德国埃尔兰根）配备一个标准的二次头部线圈平面回波成像。 </li><li>我们多年来的研究表明，针灸的功能系统，在大脑的多层次的广泛的行动。位于如大脑的杏仁核，海马，腹内侧前额叶皮层和subgenual地区的基础结构，特别容易受到易感性文物。重要的是要检查每个计划免疫功能扫描排除在这些地区的信号损失，我们发现了两种型号西门子扫描仪具有类似的磁场强度和类似的数据收集MRI序列之间的信号损失的差异。 </li><li>更薄的大脑切片最​​好减少易感性文物。片3毫米厚0.6毫米或0.75 mm的间隙在矢状面或轴向方向，用于覆盖整个脑包括脑干和小脑成像系统的内存容量而定。其他方向和切片厚度可用于局部脑成像。我们付出特别注意这些问题，有助于更基地在大脑结构上的针刺效应的一致结果。 </li><li>功能扫描获得一个T2 *加权梯度回波序列（TR / TE三十○分之四千MS，视场200毫米，64x64的矩阵，翻转角90 °，层厚3.0毫米，间隙0.6毫米）。我们收购了10分钟，共150个时间点。前4个虚拟扫描图像采集允许的fMRI信号的平衡。 TE较短的降低易感性文物。相对较长的TR允许高空间分辨率的全脑覆盖。 </li></ol>第5部分:最初的航向和解剖扫描<ol><li>先用针灸的功能扫描运行，航向扫描，高分辨率的解剖扫描，T1加权三维MP - RAGE收购，以保证正确的定位和解剖健全。在这一点上一般观察，可以保证不​​必要的扫描都无法运行。本地化，在很短的扫描，保证了全脑扫描，并允许你行的AC - PC线。如果有任何可疑的异常，研​​究人员将图像发送到一个受过训练的放射科医生，但不应该使任何索赔扫描科目是什么。 </li><li>最初的扫描大约需要20分钟。简介查看可以告诉研究者，如果这将是有益继续扫描。这是一个很好的时间与主题发言，并确保他们仍然是舒适的，没有遇到任何不良影响磁铁。 </li></ol>第6部分:针灸与功能磁共振成像监测治疗（针手动操纵和电刺激）两种形式，该视频将被限制在针灸手册，它的临床使用更长的历史。针灸刺激重复运行管理，古典取穴合谷的手，脚或腿部的足三里LV3，采用无菌，一次性使用针灸针。 <ol><li>指导受试者放松，保持自己的眼睛紧闭，避免在扫描的议案。为了避免伤害性刺激，我们特别指示的主题，以提高一个手指，如果针灸的感觉接近8分，并提高在任何程度剧烈的疼痛的情况下两个手指。 </li><li>在插入针，预试的题目的敏感性和耐受针操作，以帮助选择插入的深度和数据收集的力量操纵。 </li><li>在10分钟的功能成像，针是在货币供应量M1和M2的2分钟旋转，并留在原地的休息时间R1（2分）），R2（3分钟），R3（1分）期间。针灸使得一定不要接近或离开受直接刺激期之前或之后，即使该议案可能会影响结果。 （范式，图1） <img src="/files/ftp_upload/1190/1190fig1.n.jpg" alt="图1" /> 图1。针刺fMRI的范式。 </li><li>针灸刺激完成后，针灸记录针抗操纵的力量。在每次扫描结束时，另一位工作人员调查，询问受等级在1-10级经验丰富的感觉。列表，疼痛，酸痛，压力，丰满，沉重，麻木，刺痛，钝痛，锐痛，是对针灸社会和以前的实验数据，而不是在文学的疼痛问卷的经验为基础。心理物理学的反应是分类的德琪一个典型的德琪组和另一组根据这些记录， 剧烈的疼痛混合 。 </li><li>发言的科目之间的扫描，以确保他们感到舒适和他们保持清醒。如果他们的身体的任何部分开始抽筋或感到不舒服，在现场的科研人员应采取适当措施，以纾缓不适。主体所产生的变动会导致数据损坏。偶尔科目可能睡着了，在扫描过程中，这会影响血流动力学反应。 </li></ol>第7部分:数据处理<ol><li>多层次的分析可以运行。一般的线性模型利用休息时间作为基准，以确定％大胆致辞时大脑的每一个任务中的体素的信号变化。这也决定了这些变化的统计意义，并删除了大胆的数据没有在一个特定的P -值达到的3个连续体素的。 </li><li>执行种子为基础的交叉相关分析显示之一的神经系统地区和大脑的其余部分之间的功能连接。这是通过选择一个参考的地区和比较，与其他体素在大脑中的每一个的时间，当然。可应用于其他如独立成分分析，模糊聚类分析，结构方程模型，Granger因果关系分析。 </li><li>生理数据平均针灸针刺激的反应有关在Windows进行分析。例如，心电图数据可以为QRS波心率和心率变异性分析的时序注释。 </li></ol>第8部分结果: <img src="/files/ftp_upload/1190/1190fig2.n.jpg" alt="图2" /> 图2右合谷，足三里和LV3（52 runs/37科目）针灸德琪与触觉刺激的血流动力学反应。在MPFC（内侧前额叶皮质）集群停用结构（1），MPC（内侧顶叶）（2），MTL（内侧颞叶）（3），针灸更加突出（图A），比触觉刺激（图B）。广泛停用的权利BA22/21（4）在针灸没有观察到触觉刺激。感觉BA43（5）和结社皮质（6）显示，超过针灸触觉刺激激活（未显示）。有趣的是，右前脑岛（7）被激活了在针灸和触觉刺激。 （P &lt;0.0001）。 <img src="/files/ftp_upload/1190/1190fig3.n.jpg" alt="图3" /> 图3。 德琪 （52 runs/37科目）与德琪的血流动力学反应剧烈的疼痛（52 runs/29科目）混合右合谷，足三里和LV3，MPFC（1）突出的失活在针灸刺激，货币政策委员会（ 2）和MTL（3）疼痛（图A）与德琪缺席看到在疼痛的存在（图B）衰减。疼痛的感觉和结社皮质（4）激活变得更加突出，一个子集（如MIDC / SMA（5），postC_BA23（6），艾美（7），小脑蚓部边缘地区和paralimbic 8）成为激活。右前脑岛（9）显着激活在德琪 ，而前部和后部岛（10）较小的区域被激活，在痛苦中双边。 P &lt;0.0001。 <img src="/files/ftp_upload/1190/1190fig4.n.jpg" alt="图4" /> 图4针灸（LPNN）默认模式网络（DMN）的边缘，paralimbic新皮层网络的重叠;的LPNN大胆GLM分析（图A）和无模型的模糊聚类分析（图B响应）在针灸刺激合谷，足三里和LV3显示的DMN（Fig.C，舒尔曼等人，1997年改编）几乎相同的模式。相干信号减小的区域集群出现在内侧前额叶皮质（1），postero内侧皮质（2）和颞叶（3）。 P &lt;0.0001。

<ol>
	<li>Hui, K. K. S., Liu, J., Marina, O., Napadow, V., Haselgrove, C., Kwong, K. K., Kennedy, D. N., Makris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrated+response+of+the+human+cerebro-cerebellar+and+limbic+systems+to+acupuncture+stimulation+at+ST+36+as+evidenced+by+fMRI.">The integrated response of the human cerebro-cerebellar and limbic systems to acupuncture stimulation at ST 36 as evidenced by fMRI.</a> Neuroimage. 27 (3), 479-496 (2005).</li><li>Hui, K. K. S., Marina, O., Claunch, J. D., Nixon, E. E., Fang, J., Liu, J., Li, M., Napadow, V., Vangel, M., Makris, N., Chan, S. T., Kwong, K. K., Rosen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupuncture+mobilizes+the+brain's+default+mode+and+its+anti-correlated+network+in+healthy+subjects.">Acupuncture mobilizes the brain's default mode and its anti-correlated network in healthy subjects.</a> Brain Res. 1287, 84-103 (2009).</li><li>Napadow, V., Dhond, R. P., Purdon, P., Kettner, N., Makris, N., Kwong, K. K., Hui, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+acupuncture+fMRI+in+the+human+brainstem+with+heart+rate+variability.">Correlating acupuncture fMRI in the human brainstem with heart rate variability.</a> Conf Proc IEEE Eng Med Biol Soc. 5, 4496-4499 (2005).</li><li>Shulman, G. L., Corbetta, M., Buckner, R. L., Raichle, M. E., Fiez, J. A., Miezin, F. M., Petersen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+modulation+of+early+sensory+cortex.">Top-down modulation of early sensory cortex.</a> Cereb. Cortex. 7 (3), 193-206 (1997).</li></ol>

针灸fMRI数据的采集和解释，适当的技术，经验的针灸师和大样本大小的就业许可证无意伤害性刺激的混杂影响分离是重要的。几个经典的腧穴，我们的研究表明，针灸调节边缘网络，人类大脑的一个重要的内在的监管制度。网络显示了明显的重叠与休息大脑的默认模式。可以证明这些相关的自主神经功能变化的生理监测。

monitoring acupuncture effects on human brain by fmri

功能磁共振成像是用来研究针灸的效果就大胆的响应和人类大脑的功能连接。结果表明，针灸调动一个边缘paralimbic新皮层网络，其反相关的感觉/ paralimbic在大脑的多层次，网络的血流动力学反应是由心理物理反应的影响。生理监测，可进行探索的自主神经功能的外围的反应。本视频介绍合谷（合谷）进行的研究，足三里（足三里穴）和LV3（太冲），通常使用的调节和减轻疼痛的行动古典腧穴。指出fMRI来针灸调查的申请需要注意的一些问题。

Watch this Scientific Journal Video about Monitoring Acupuncture Effects on Human Brain by fMRI at JoVE.com

Monitoring Acupuncture Effects on Human Brain by fMRI

fMRI和生理监测是用来研究针灸对中枢和周围神经系统的影响。针灸调动一个边缘paralimbic新皮层网络，默认模式网络的重叠，调节神经活动，可能涉及到其周围神经系统的自主的影响。

通过功能磁共振成像监测针灸对人脑的影响

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

monitoring-acupuncture-effects-on-human-brain-by-fmri

Research

JoVE Journal

Biology

15.3K 次观看.  Massachusetts General Hospital and Harvard Medical School. fMRI和生理监测是用来研究针灸对中枢和周围神经系统的影响。针灸调动一个边缘paralimbic新皮层网络，默认模式网络的重叠，调节神经活动，可能涉及到其周围神经系统的自主的影响。

视频: Monitoring Acupuncture Effects on Human Brain by fMRI

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

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

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

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 ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

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 ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...