这种材料是以要求研究支持或部分支持由该技术商业化办公室，盐湖城UT斯达康，研究和发展办公室，康复ř＆D服务，DVA的SLC的卫生保健系统，盐湖市，UT斯达康，美国国防部PRMRP格兰特（号PR054520），阿尔伯特和玛格丽特霍夫曼主席和骨科部，美国犹他州大学医学院，盐湖城，UT斯达康提供了模拟的技术支持中心，结合生物医学计算科学计算及影像学研究所和部分可能由来自美国国立卫生研究院/ NCRR中心，结合生物医学计算P41 - RR12553 - 07软件 。 稿件准备和达斯汀威廉姆斯的援助附加感谢延长Gwenevere肖生物膜的形象。

第1部分：使用计算机断层扫描（CT）扫描，为截肢者重建<ol><li>回顾CT扫描收集从犹他州和部门获得IRB和HIPAA的批准后，退伍军人事务医院的大学。 </li><li> CT扫描，因为它们允许对X射线的吸光度为基础的组织类型之间的明确区分。 </li><li> CT的手动检查，包括在金属植入物的情况下，以防止图像神器的基础上研究。 </li></ol>第2部分：模型生成与Seg3D <ol><li> DICOM图像文件下载，并加载到Seg3D（版本1.11.0，software.sci.utah.edu）作为一个新的卷。 </li><li>中位数过滤器是用来顺利进口量前确定几何定义的组织结构。 </li><li>骨，骨髓，器官和脂肪组织的组织界限，所产生的阈值的CT文件交互方式（图1）。 <img src="/files/ftp_upload/1237/1237fig1.jpg" alt="图1" /> 图1：一个截肢者残肢的矢状截面阈值，并分隔成特定的组织类型。 </li><li>肌肉是通过手动设置阈值的肌肉组织内的种子点，并使用连接过滤器的信心，找到所有的连接到种子点的组织。这一步消除了错误的组织可能已经分组的基础上从CTS类似的吸水性肌肉。 </li><li>皮肤，这是无法可靠地辨别从CT图像，产生扩张的最外层组织2毫米，皮肤厚度平均产生一层均匀的厚度，包围整个模型1。 </li><li>分割手动检查，纠正，以确保准确性和有限元分析（图1）所需的地图到一个单一的标签的层次结构相结合。 <img src="/files/ftp_upload/1237/1237fig2.jpg" alt="图2" /> 图2：代表层次结构模型的创建与Seg3D双边截肢者。 </li></ol>第3部分：有限元分析的准备工作<ol><li>一个10厘米植入的目的是在Matlab作为电刺激植入的骨科器械和阴极和进口SCIRun（版本4.0，software.sci.utah.edu）。 </li></ol>第4部分：电极放置及设计<ol><li> SCIRun是利用电极设计，因为它支持交互式的电极放置和模拟。 </li><li>创建一个网络，并组织具有特定功能的模块来生成网格（图3）。模块被定义边界条件，组织电导率，网格细化，产生的matlab直方图，记录字段数据等（见表1）的重要。 <img src="/files/ftp_upload/1237/1237fig3.jpg" alt="图3" /> 图3：使用两个波段的外部电极配置的试点研究，从代表的网络形象。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 表1 </td><td></td></tr><tr><td colspan="2"> 分配到分段组织的导电性</td></tr><tr><td> 组织类型 </td><td> 导电性[S / M] </td></tr><tr><td>机构</td><td> 0.22 </td></tr><tr><td>皮肤</td><td> 0.26 </td></tr><tr><td>脂肪</td><td> 0.09 </td></tr><tr><td>肌肉</td><td> 0.25 </td></tr><tr><td>骨皮质</td><td> 0.02 </td></tr><tr><td>骨髓</td><td> 0.07 </td></tr></tbody></table></li><li>电极的配置包括一个补丁电极，两个电极补丁，一个连续的频段，并连续两个频段。 </li><li>外部电极带，适用于从病人的CT扫描生成的模型的残肢，分别为1.6厘米的厚度。 </li><li>电极贴片放置一个覆盖地带，大约一半的残肢的直径3厘米，厚度。 </li><li>代表种植体植入的内皮层植入设置内膜直径允许植入适合完美，并填写 2 。 </li></ol>第5部分：有限元分析<ol><li>模拟生成的假设，电动指标可以计算使用一个准静态的方法，没有时间依赖性。 </li><li>该模型是通过求解拉普拉斯Seg3D分割产生每个组织类型方程计算。 </li><li>由电极注入电流和指引​​，保持身体内形成的边界条件。 </li><li>由于电极植入了更大的电导率比周围组织，它是一个ssumed植入（阴极）是在一个恒定的潜力，同样的表面电极经皮植入一个恒定的电势差为蓝本。 </li><li>为了评估电极配置和尺寸的疗效，患者的具体型号是发达国家和种植体周围界面的电势来确定局部领域的优势。 </li><li>该模型是使用约180万分段均匀，欧姆和各向同性处理的元素组成一个六面体网格生成。 </li><li>这个实验的最佳模型选择一个相对差&lt;5％的电压梯度网格敏感性研究证实，以确保模型的准确度（见表2）。 <table border="1" cellspacing="0" cellpadding="0" width="305"><tbody><tr><td> 表2 </td><td></td><td></td><td></td></tr><tr><td colspan="3"> 网为截肢者模型的灵敏度研究</td><td></td></tr><tr><td> 网 </td><td> 元素 </td><td> 节点 </td><td> 相对差异 </td></tr><tr><td> 100 100 50 </td><td> 149089 </td><td> 161131 </td><td> 0.0995 </td></tr><tr><td> 125 125 75 </td><td> 350180 </td><td> 371472 </td><td> 0.0802 </td></tr><tr><td> 150 150 100 </td><td> 673032 </td><td> 706082 </td><td> 0.0545 </td></tr><tr><td> 175 175 125 </td><td> 1146778 </td><td> 1194044 </td><td> 0.0527 </td></tr><tr><td> 200 200 150 </td><td> 1796690 </td><td> 1860772 </td><td> 0.0439 </td></tr><tr><td> 250 250 200 </td><td> 3745038 </td><td> 3850202 </td><td> 0.0364 </td></tr><tr><td> 275 275 225 </td><td> 5097243 </td><td> 5226587 </td><td> 0.0301 </td></tr><tr><td> 300 300 250 </td><td> 6742588 </td><td> 6898729 </td><td> 0.0000 </td></tr></tbody></table></li><li>使用迭代求解器，电度量的有限元模型计算了电极的配置。 </li></ol>

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该技术在文章中披露，特别是利用作为一个功能阴极的骨科植入，增加骨骼的附着，减少康复和防止细菌粘附是由第一作者，在杂志上提交发明了一种新的想法。一项发明披露形式已被提交给犹他州立大学和一个美国临时专利保护所有披露的技术已提交。

了解电刺激模式在医疗和战场上的撤离战略的增强，导致幸存的勇士灾难性的战争工伤人数增加。虽然提高成活率是医疗进步，军人和妇女返回需要截肢打击密集的后续照顾退伍军人事务医疗保健系统3，漫长的康复过程和昂贵的假肢服务。美国国会报告的细节，作为一个持久自由行动（OEF）和伊拉克自由行动（OIF）的冲突4的结果发生了超过1000个与战争有关的截肢。 OEF和OIF退伍军人的情况下，约15％返回勇士已经失去了多个四肢，并有相当数量的军人和妇女返回残留四肢短套接字技术是不是一种选择，或已被患者拒绝。上肢假肢的使用报告的停产甚至超过50％，因为固定装置是繁琐和困难的舒适使用5的。下肢假肢是同样的问题，与软组织插座有关的常见问题，包括无法行走具有挑战性的terrain6， 有限的残肢长度为7，患者的不适感5， 非生理负荷8的关注，刺激异位骨化9和风险使人衰弱的疾病10。然而，骨整合技术是一种新型的手术技术可能减少11疼痛，皮肤红肿12，提高osseoperception 13，改善流动性6，减少与插座6相关的压疮，减少能源下地 7,14，更好地服务于有限的退伍军人和勇士残肢长度为15。 尽管众多的生理和心理优势的骨结合，相关的手术过程中需要更先进的预防感染治疗streategies 16，需要长期的康复计划，包括限制性的负重协议， 这可能长达1.5年术后17。由于宿主骨和残肢长度的可行性是非常重要的肌肉附着和功能，开发新的设备，以提高骨结合是军人和妇女返回的关键。因此，发展一个种植体植入智能设计（OIID）系统，它使用控制的电刺激可减少康复的长度，并增加对退伍军人和战士被截肢者的骨骼附件。然而，由于目前没有设备是市售和使用经皮种植体植入，该计划的动机是与有限元分析，以确认安全性和有效性。 了解电刺激在骨重塑的作用，特别是osteoids和矿化沉积，一直投机。然而，在骨观察的电活动可能是机械装载 18,19，因此，电刺激可能是一个有效的机制，诱导骨修复 19 。假设背后的逻辑是在骨折愈合模型的解释。当长骨被加载，在张力侧正电和压缩侧阴电20,21，然而，一次骨破裂，该网站将继续尊重周围环境的阴电直至愈合已经展开，动态平衡恢复 21 。电信号模拟自然愈合级联一直认为，以协助钙的沉积22日 ，中的氧含量和pH值23的轻微改变，招聘生长因子 22，并与成骨细胞的迁移和分泌更多的细胞外基质的24次助攻。 已经被重新定义的前提下，电刺激可以单独执政完整的骨修复和当前新的假说，形成完整的工会是由机械载荷和电刺激共同刺激19。压电变形的胶原蛋白或大的电动力学产生电流流动过去的骨基质 25的矿物部分的离子成分与体内观察到的电脉冲。事实上，自发的潜力已经在骨6毫伏和26骨矿化沉积率增加相关。 早期工作由布莱顿和Friedenberg 18,21,27,28用于骨再生的电刺激的概念在20世纪60年代和20世纪70年代和demonstrated直接电流可用于修复在较短的时间内，传统的治疗方法相比，非工会。其他型号进行了调查与限制负重骨形成和表现出成骨活性之间的控制和四肢电刺激25的增加了31 ％。 虽然在电刺激领域的研究铺平了道路为理解电刺激对成骨细胞外基质沉积的机制的方法，认识不足，限制了这项技术的扩张。虽然有许多情况下，非工会和骨折愈合模型，患者的不适感的例子和失败的尝试，成功的愈合是充满文献以及 29 。电刺激的问题发生，从科学家和临床医生控制错误的电气指标，仅仅集中在目前的程度。以前的研究人员研究过目前的“灵丹妙药”固定近似50万非工会每年发生 30 。然而，模型之间的可重复性已有限的31焦耳加热并发症，而不是确定的电流密度32。事实上，所有生产的生物医学设备必须限制电流密度小于2毫安/平方厘米作为国际电工委员会的概述，以防止局部组织坏死，患者的不适感33。 除了 ​​协助骨骼固定，控制电刺激也可阻止细菌粘附在骨科植入物和减少骨髓炎和生物膜形成34-37的风险。生物膜形成矫形器械导致病人的并发症和相当大的痛苦，那些依赖这些设备 38 。重点是放在上已经完全消毒仪器和植入手术39之前的必要性，但它往往难以诊断细菌粘附明显许多负面培养的情况下，这确实是感染了40。这个问题常常与事实生物膜缓慢增长40性质，不能增长准确地在体外培养 39，取决于细菌的细胞类型，表面的清洁度和免疫系统受影响的人 39 。欧洲oseeointegration技术transfemoral截肢者的调查显示，最常见的问题是感染（频繁浅表感染，1 / 3假体周围感染） 41。虽然有很大改进手术准备，消除细菌生物膜之间500-5000千倍更加困难，以消除由于非柏拉图的形式34,35,39改善骨结合的基本因素之一。因此，利用电刺激作为消除有害的细菌菌落和增加骨骼固定方式是确保保护病人的健康和OIID疗效的重要因素。 使用退伍军人和战士被截肢者的优点是相对的青年和其他好这些人的健康，使他们积极的恢复和经皮后，将作为日间援助和开发可作为阴极电刺激暴露的理想人口。种植体植入的存在并不需要额外的手术过程中插入电气元件，使设备外部控制，并防止进一步感染42的风险。因此，认识到退伍军人和战士被截肢者的残肢，1-10 V / cm时的幅度电场电流注入法可建立，控制和在种植体界面的测量。据推测，这将使交付电力安全水平，能够诱导成骨细胞迁移和改善骨骼附件。电场的这个学位将增加在种植体界面骨的数量和质量，并为加速截肢者的康复和骨骼固定改善的前景。没有被查处使用电刺激作为加快髓内假体植入骨结合的方式，并提出了许多机会转化研究，以改善病人护理。 实验结果患者的具体型号与经皮电刺激装置的必要性，在研究的支持。所建议的医学设备开发的模拟可能会加快增加成骨细胞迁移的骨骼附着和防止细菌粘附27,34,36,39的能力。比赛utation建模，有效地显示，1-10 V /厘米的电场和电流密度低于2毫安/平方厘米可作为一个功能阴极生成的植入，是最均匀分布使用两个频段外部电极。 OIID系统可与电刺激相关的经典问题解决的第一步，无法确定目前的途径，在人体 43 。因此，建立为提高骨骼附件的工具可以协助减少康复为种植体过程中所需的长度。 年纪较大的截肢者利用电刺激也是一个重要的方面，必须探索以及。骨量是最大的十年后骨骼生长停止，但明显减少第八届和第九届十年 44 。只要骨骼随着年龄的增长而变化，内膜直径往往更迅速，增幅比骨膜的直径，这可能会导致假体松动45。加之较弱的肌肉骨骼应变减少这个问题可能会导致使人衰弱的疾病，如骨质疏松症和骨量减少45，并需要额外的种植体植入患者的治疗方案。然而，控制电刺激和机械负荷可能会作为一个骨ongrowth协同催化剂，并保持与宿主骨床完整的老年患者使用OIID制度。

bioelectric analyses of an osseointegrated intelligent implant design system for amputees

预计美国截肢者的人数预计将上升到2050年的3.6万。许多这些人依赖于执行日常活动的假肢，但假体悬浮液使用传统的接口技术可以被证明是累赘和一个肢体残缺的人感到不舒服。此外，对于那些高近端截肢，有限的残肢长度可能会阻止所有exoprosthesis附件。种植体植入技术是一种新的手术方式，它允许公司之间的宿主骨和植入骨骼附件。在欧洲截肢者与种植体植入的初步结果表明允许负载直接转移到骨植入界面，改善临床结果。尽管骨结合的插座技术优势明显，目前的康复程序，需要长期的限制性承重之前，这可能与减少通过电刺激加速骨骼附件。智能植入种植体的设计（OIID）系统的目标是使电气系统，加速骨骼的附件，并有助于防止假体周围感染的植入部分。为了确定最佳的电极大小和位置，我们发起了概念证明，在截肢患者剩余四肢的电刺激产生的电场和电流密度的计算模型。为了保证病人的安全，具有追溯计算机断层扫描受试者选择创建定制的软件程序，以确保在IRB解剖的准确性（Seg3D和SCIRun）和HIPAA批准研究和三维重建。这些软件包支持患者的具体模式的发展和电极的位置和大小的互动操作所允许的。初步结果表明，电场和电流密度可达到均匀的电场诱导成骨细胞迁移，增强骨骼固定，并可能有助于防止假体周围感染的分布在种植体界面生成。根据电极与模型试验的配置，外部两个波段配置将在未来的主张。

Watch this Scientific Journal Video about Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees at JoVE.com

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

有必要开发替代假体附件因血管闭塞性疾病和创伤的断肢。这项工作的目标是引入智能植入种植体的设计系统，增加骨骼的内固定，减少病人需要种植体技术的假体周围的感染率。

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 ...

bioelectric-analyses-an-osseointegrated-intelligent-implant-design

Research

JoVE Journal

Biology

13.8K 次观看.  Department of Veteran Affairs. 有必要开发替代假体附件因血管闭塞性疾病和创伤的断肢。这项工作的目标是引入智能植入种植体的设计系统，增加骨骼的内固定，减少病人需要种植体技术的假体周围的感染率。

视频: Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

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 ...

科研

生物学

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

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 ...

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 ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

蝙蝠组织收集 -细胞分析和原细胞培养

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 &#176;C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

烟草角虫作为大麻素临床前研究的昆虫模型系统

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

大鼠 离体 肺灌注模型的设计和实现

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...