这项工作的经费由美国国立卫生研究院授予1R03AI090465。

1。获取数据<ol><li>手持GPS装置，GPS功能的智能手机跟踪应用程序，以及A-GPS（辅助GPS）设备，如我们的研究中，一个商业儿童跟踪器轨迹数据可以被收集。 </li><li>轨道数据通常保存在的时间，经纬度记录。所需的时间间隔应根据应用的需要。通常情况下，最频繁的时间间隔是需要时间的活动空间研究。 </li><li>将数据转换为以逗号分隔的值，或单独记录的id列。csv文件，经度，纬度，时间，分别。然后转换。CSV文件导入到常用的地理信息系统（GIS）文件格式（ 如 ESRI的shapefile 8）。 </li><li>加载在shapefile中的建筑物多边形的轨迹分析仪的研究领域的边界。正确设置"挤出"的建筑的3D二张开，并设置"挤出"和"透明度"的边界层适当地显示空间-时间立方体6，9的x，y尺寸代表空间和z维度表示时间。 </li></ol> 2。前处理<ol><li>有两个选项可用于前处理嘈杂的原始轨迹数据。人们可以从下拉列表中的前处理菜单中进行选择。 </li><li>如果选择"互动"，创建的二维投影的三维轨迹，便于查看和选择。操纵3D显示在空间和时间来检查原始轨迹。确定基于轨道段的形状，速度和/或拓扑的数据中的错误。通常不切实际的高速度或突然改变方向的轨道点（顶点）表示错误。选择并删除它们从原来的轨迹。选择并删除它们的三维轨迹或二维投影。 </li><li>集群中的沙尖的轨迹点PES（ 图1）空间和持续时间很长的时间表示，最有可能造成的室内GPS信号的地方是弱的错误。如果这些点的一组被选择时，该程序可以计算出的时空的选定点的质心和调整的轨道去通过质心。 </li><li>另外，如果选择"自动"的前处理菜单，设置的输入和输出的位置，以及经验参数确定的异常高的速度和突然转弯点。程序加载的轨迹数据的搜索算法，模拟的视觉误差检测方法的基础上并自动运行。 </li></ol> 3。轨迹分割的活动空间特征<ol><li>的轨迹分割需要建立层，确保建筑形状文件被加载。 </li><li>在工具栏上单击分割工具启动功能。设置的输入和输出，并位于文件作为参考层的建筑物形状。使用的的楼宇名称标签的分段轨迹。该算法识别的基础上设置或默认的标准，如速度，时间，轨迹点等 ，以及空间拓扑结构的建筑物的室内段。 </li><li>单击活动空间总结工具加载在分段的轨迹，并计算选定的的摘要属性表征的一个人的活动空间，如总的活动半径，半径在一定的时间期间，花费的总时间在室内与室外的比，和等。 </li><li>该属性可以被导出到电子表格定量建模使用。 </li></ol> 4。密度表面映射<ol><li>密度表面的活动空间与时间维的密度崩溃。有三个选项，从下拉列表中的密度表面映射菜单。 </li><li>如果选择了"轨道点密度"选项，填写对话框中的输入和输出信息，并选择3D或2D显示。从该轨迹数据的所有顶点被用来计算内核密度的点， 图2示出了密度表面。 </li><li>如果"轨道路径密度&#39;被选择时，该算法计算并显示单个路径行驶（ 图3）的密度。 </li><li>如果&#39;重新采样点的密度"选项被选择时，重新采样算法使用设定的时间间隔，和地图上的点的密度均匀地撒布在时间的轨迹数据。此选项被设计为，在不规则的时间间隔中，由于不同灵敏度的不同物理条件下的装置或分段轨迹收集跟踪点的跟踪设备， 图4示出了2D和3D的密度分段轨迹表面。 </li><li>如果&#39;时空聚焦&#39;被选择用于纽约州的上述认股权，时空聚焦10可以在不同的时间段进行检查的活动模式。例如，在不同的时间在一天的活动密度表面可能是可视化的，便于识别跨越时间的热点（ 图5）。 </li></ol> 5。密度蓄积量估测，容积再现<ol><li>密度体积可视化使用空间 - 时间立方体中的轨迹的可视化的概念。这种可视化的核心是分列的空间体素11。我们的可视化密度量的估计方法计数的时空轨道相交的体素数个人体素的密度量。此步骤可以点击"密度体积计算"下的密度体积可视化菜单。 </li><li>相同的三个选项可用于密度体积可视化密度表面可视化。 </li><li>下一步点击"音量渲染"推出的三维体积的可视化界面的交互式立体渲染12。通过设置沿每个轴的分割数，可以检查集群在不同的尺度。的Z-系数是用来设置垂直夸大，以便更好地观察。可以被加载的参考层，如建筑物，可视化以及帮助。的体绘制的结果，可以交互调整通过操纵控制映射的传递函数，从密度的颜色。 （ 图6）。 </li></ol> 6。探索性数据分析（EDA）和可视化<ol><li>过程是要显示在谷歌地球，可用于创建动画系列。在"其他"，点击"导出为KML为EDA访问此程序。它创建了一个KML 13在谷歌地球中打开的文件，互动式动画的轨迹。 </li><li>一个可以遵循的轨迹，下移动在谷歌地球沿时间线滚动升的环境。 </li><li>过程是可视化的地方利益之间的连接通过关联分析。例如，在大学校园的建筑物之间的连接是从分割的轨迹数据收集学生（ 图7）。 </li><li>根据上派生的连接，如楼宇最出站或入站流量和连接的枢纽，最繁忙的地方可能的热点。 </li></ol>

<ol>
	<li>Stoddard, S. T., Morrison, A. C., 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=The+role+of+human+movement+in+the+transmission+of+vector-borne+pathogens.">The role of human movement in the transmission of vector-borne pathogens.</a> PLoS Negl. Trop. Dis. 3 (7), e10 (2009).</li><li>Morens, D. M., Folkers, G. K., 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=The+challenge+of+emerging+and+re-emerging+infectious+diseases.">The challenge of emerging and re-emerging infectious diseases.</a> Nature. 430, 242-249 (2004).</li><li>Viboud, C., Bjornstad, O. N., 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=Synchrony,+waves,+and+spatial+hierarchies+in+the+spread+of+influenza.">Synchrony, waves, and spatial hierarchies in the spread of influenza.</a> Science. 312, 447-451 (2006).</li><li>Shoval, N., Isaacson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Application+of+tracking+technologies+to+the+study+of+pedestrian+spatial+behaviour.">The Application of tracking technologies to the study of pedestrian spatial behaviour.</a> The Professional Geographer. 58 (2), 172-183 (2006).</li><li>Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+GIS+design+for+exploring+interactions+of+human+activities.">Spatio-temporal GIS design for exploring interactions of human activities.</a> Cartography and Geographic Information Science. 33 (1), 3-19 (2006).</li><li>Kwan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+geovisualization+of+activity-travel+patterns+using+three-dimensional+geographical+information+systems:+a+methodological+exploration+with+a+large+data+set.">Interactive geovisualization of activity-travel patterns using three-dimensional geographical information systems: a methodological exploration with a large data set.</a> Transportation Research Part C. 8, 185-203 (2000).</li><li>Dem&#353;ar, U., Virrantaus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space-time+density+of+trajectories:+exploring+spatio-temporal+patterns+in+movement+data.">Space-time density of trajectories: exploring spatio-temporal patterns in movement data.</a> International Journal of Geographical Information Science. 24 (10), 1527-1542 (2010).</li><li>Kraak, M., Koussoulakous, A., Fisher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A visualization environment for the space-time cube. , 189-200 (2004).</li><li>MacEachren, A. M., Polsky, C., 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=Visualizing+spatial+relationships+among+health,+environmental,+and+demographic+statistics:+interface+design+issues.">Visualizing spatial relationships among health, environmental, and demographic statistics: interface design issues.</a> , 880-887 (1997).</li><li>Levory, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Display+of+surfaces+from+volume+data.">Display of surfaces from volume data.</a> IEEE Computer Graphics and Application. 8 (5), 29-37 (1998).</li><li>Drebin, R. A., Carpenter, L., 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=Volume+Rendering.">Volume Rendering.</a> Computer Graphics. , (1998).</li><li>Lee, W., Krumm, J., Zheng, Y., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trajectory+preprocessing.">Trajectory preprocessing.</a> Computing with Spatial Trajectories. , 3-34 (2011).</li><li>Han, B., Comaniciu, D., 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=Sequential+kernel+density+approximation+and+its+application+to+real-time+visual+tracking.">Sequential kernel density approximation and its application to real-time visual tracking.</a> IEEE Transactions on Pattern Analysis and Machine Intelligence. , (2007).</li></ol>

没有利益冲突的声明。

我们使用附加的机制的ArcGIS开发的接口。所有的交互操作，使用C + +。使用Python开发的自动处理和分析功能。 AGPS数据，或行人所收集的GPS数据的预处理，提出了独特的挑战，，由于邻接建筑物和频繁的室内站的错误可能是巨大的。此外，预处理的重点不应该是数据通常是车辆GPS轨迹数据由于稀缺的跟踪点已经明显减少。最明显的错误行人轨迹数据的模式，但是，提供独特的?...

<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> WorldTracker GPRS </td><td>跟踪世界</td><td></td><td></td></tr><tr><td>一台个人电脑运行分析</td><td></td><td></td><td></td></tr><tr><td> ArcGIS软件</td><td> ESRI </td><td></td><td></td></tr><tr><td>弹道分析仪扩展</td><td></td><td></td><td></td></tr></tbody></table>

trajectory data analyses for pedestrian space-time activity study

众所周知，人体运动的空间和时间维度，直接影响疾病传播的1-3。通常是通过感染者和易感个体重叠的活动空间之间的接触传播的传染病。因此，每日行动不便的活动信息可以用来作为一个指标来测量暴露感染危险因素。然而，一个主要的困难，因此产生的原因缺乏传染病传播的研究在微观尺度上缺乏详细的个人移动数据。此前在运输和旅游研究的详细时间的活动空间往往依赖于时间，空间日记的技术，它需要的主题，积极记录他们的活动在时间和空间上的数据。这是要求很高的从参与者参与者和协作极大地影响数据4的质量。 现代如GPS和移动通信的技术提供了可能的轨迹数据的自动采集。收集的数据，然而，这不是理想的模拟人类的空间 - 时间的活动，限制由现有设备的精度。也没有现成的工具是人类行为研究的数据的高效处理。在这里，我们提出了一套方法和一个集成的ArcGIS的轨迹数据的前处理和时空分析的基于桌面的可视化界面。我们提供的例子，这样的处理可以用来模拟人类的空间活动，特别是丰富的错误行人轨迹数据，这可能是有用的公共健康研究，如传染性疾病的传播模型。 程序包括前处理，轨迹分割，活动空间特征密度估计和可视化，和其他一些探索性的分析方法。前处理者g是喧闹的原始轨迹数据的清洗。我们引入交互式可视化前处理接口，以及一个自动的模块。弹道分割5涉及的室内和室外部分，从处理前的空间-时间轨迹的识别。同样的原因，交互式可视化分割和自动分割的支持。分段的时空轨迹进行分析，然后得出一个人的活动空间，如活动半径等密度估计和可视化的特点，是用来检查大量的轨迹数据模型的热点和互动。我们演示这两种密度的表面映射6和密度容积再现7。我们还包括一些其他的探索性数据分析（EDA）和可视化工具，如谷歌地球的动画支持和关联分析。一套分析以及视觉本文提出的方法可以适用于任何轨迹数据的s步伐活性的研究。

轨迹数据收集志愿本科学生肯恩大学（NJ，USA）在2010年春季。其目的是研究谁抓住了流感（医生诊断或自我诊断）相比，那些谁没有学生的活动模式。为了说明本文提出的方法和程序，我们把收集的轨迹在郊区的校园面积，产生具有代表性的结果。校园内的区域大多是行人的运动轨迹轨迹，只有一小部分驾驶之间的各种停车场和校园外的。 参照大学校园的建筑物的轨迹的空?...

Watch this Scientific Journal Video about Trajectory Data Analyses for Pedestrian Space-time Activity Study at JoVE.com

Trajectory Data Analyses for Pedestrian Space-time Activity Study

甲套件时空处理方法以分析人类的轨迹数据，如建模行人的空间 - 时间活动的目的，使用GPS设备收集。

轨迹数据分析行人时空活性研究

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

trajectory-data-analyses-for-pedestrian-space-time-activity-study

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Civil Engineering

Biology

Unit 1

Global Positioning System (GPS)

SCIENTISTS IN ACTION

13.4K 次观看.  Kean University. 甲套件时空处理方法以分析人类的轨迹数据，如建模行人的空间 - 时间活动的目的，使用GPS设备收集。

视频: Trajectory Data Analyses for Pedestrian Space-time Activity Study

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

被截肢者的智能植入种植体设计系统的生物电分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

体外sumo 法研究相扑 E3 连接活动

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...