第1部分：设置和编制皮肤<ol><li>设置的呼吸带，肌电图，心电图，皮肤电导，温度，地面以下传感器; </li><li>准备的皮肤，轻轻揉搓用酒精擦拭在下列地点：肌电图的眼睛额头以上，里面心电图双腕，地面，左手（接地电极的位置可能会有所不同取决于二手设备）; </li><li>连接传感器的皮肤在下列地点：呼吸带在腰上，肌电图上的每只眼睛上方的额头，心电图上的每个手腕内侧，皮肤电导左手中指上，左手的指针温度计，地面左手背; </li><li>选择呼吸+心率变异性的会议; </li><li>确保阻抗是接受的所有传感器和传感器响应客户端的运动和生产合理​​的读数。 </li></ol>第2部分：录音<ol><li>解释程序到客户端。 </li><li> （0-2分钟）指示睁着眼睛静静地坐下来与客户端。记录两分钟的基线。 </li><li> （2-4分钟），在两分钟结束，开始两分钟的认知应激-算术问题，逐步在不使用笔和纸或计算器（解决的难度增加，如果算术特别是客户端的困难，要求他们反复减去从100开始，即“串行七人榄球赛”）7。下面的说明给客户端：“在接下来的2分钟内，我将要求你解决一些数学问题。请尽快可以告诉我答案“。不要提供以客户的回答是否正确或不正确反馈，并提供最低限度的鼓励（如，“只是做最好的，你可以”），以免消除轻度应激诱导这次演习。 </li><li> （4-6分钟）让客户知道，这部分的评估，并要求他/她静静地坐2分钟打开眼睛。 </li><li> （6-8分钟）开始身体应激：打开响亮的有害噪声（婴儿的哭声，车祸等），并要求客户没有调整的声音听出。 </li><li> （8-10分钟）关闭噪音，并要求客户端静静地坐下来与眼睛2分钟打开。 </li><li> （10-12分钟）开始情绪应激：要求客户谈紧张的事件，他们清楚地记得，包括事件的细节，他们的想法和感受。提供最低限度的移情的反应，以免减轻应激反应。 </li><li> （12-14分钟）中断客户端的故事，即使他/她还没有完成，并指示睁着眼睛静静地坐下来与客户端。 </li><li>停止录制，保存数据，并让客户知道，评估完成。 </li></ol>第3部分：述职和治疗计划<ol><li>询问关于他们的经验在评估过程中的客户端。 哪些部分是特别紧张，为什么呢？ 他/她觉得任何生理上的变化发生呢？ </li><li>根据附录一所述的标准的审查数据</li><li>选择需要解决的问题，基于上述分析的生理参数。 如果在基线传感器读数高于或低于正常值，治疗的目标是帮助客户带来，非压力的环境中正常的生理参数。 如果在应激传感器的读数高于或低于正常值，治疗的目标是帮助客户识别压力和压力下能够保持生理功能的正常水平。 在停机时间，如果传感器的读数表明缺乏复原，治疗的目标是帮助客户带来到正常水平，很快一个应激，生理参数。 任何或所有这些目标可能包括在治疗计划。 </li></ol>附录I 解释压力评估准则 （所有的“大胆”的答案显示领域要解决的） <table border="0"><tbody><tr><td colspan="3"> 肌电图（正常阅读&lt;3微伏） </td></tr><tr><td> 1。肌电图是高架在基线？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。是肌电图基线升高过去3压力？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 3。是否有肌电图恢复到基线水平在休息时间吗？ </td><td>是_ </td><td> 没有 _ </td></tr><tr><td colspan="3"> 心率（正常范围为每分钟60-80次） </td></tr><tr><td> 1。在基线心率升高？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。过去的基线心率升高3压力？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 3。在休息时间是否有心率恢复到基线水平？ </td><td>是_ </td><td> 没有 _ </td></tr><tr><td colspan="3"> 呼吸频率（正常率&lt;12，每分钟呼吸） </td></tr><tr><td> 1。呼吸率在基线升高吗？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。过去任何应激基线是呼吸率升高？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 3。是否有休息时间，呼吸率基线恢复？ </td><td>是_ </td><td> 没有 _ </td></tr><tr><td colspan="3"> 皮肤电导 </td></tr><tr><td> 1。在基线升高的皮肤电导？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。皮肤电导任何应激过去的基线升高？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 3。是否有皮肤电导基线恢复期间的休息时间呢？ </td><td>是_ </td><td> 没有 _ </td></tr><tr><td colspan="3"> 手指的温度（正常阅读&gt; 90F） </td></tr><tr><td> 1。手指温度在基线低吗？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。手指温度的降低是否过去的基线任何应激吗？ </td><td> Ÿ ES _ </td><td>没有_ </td></tr><tr><td> 3。是否有在休息时间，手指温度恢复到基线？ </td><td>是_ </td><td> 没有 _ </td></tr><tr><td colspan="3"> 心率变异性（规范按年龄而异 ） </td></tr><tr><td> 1。心率基线变异低？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 2。是否过去的基线心率变异减少任何应激？ </td><td> 是 _ </td><td>没有_ </td></tr><tr><td> 3。在休息时间是否有恢复心率变异性呢？ </td><td>是_ </td><td> 没有 _ </td></tr></tbody></table>附录I 压力评估的解释： <table border="0"><tbody><tr><td colspan="3"> 肌电图（正常阅读&lt;3微伏） </td></tr><tr><td> 1。肌电图是高架在基线？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。是肌电图基线升高过去3压力？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 3。是否有肌电图恢复期间的休息时间到基线水平吗？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 心率（正常范围为每分钟60-80次） </td></tr><tr><td> 1。在基线心率升高？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。过去的基线心率升高3压力？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 3。在休息时间是否有心率恢复到基线水平？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 呼吸频率（正常率&lt;12，每分钟呼吸） </td></tr><tr><td> 1。呼吸率在基线升高吗？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。过去任何应激基线是呼吸率升高？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 3。是否有休息时间，呼吸率基线恢复？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 皮肤电导（正常阅读&lt;5微欧姆） </td></tr><tr><td> 1。在基线升高的皮肤电导？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。皮肤电导任何应激过去的基线升高？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 3。是否有皮肤电导基线恢复期间的休息时间呢？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 手指的温度（正常阅读&gt; 90F） </td></tr><tr><td> 1。手指温度在基线低吗？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。手指温度的降低是否过去的基线任何应激吗？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 3。是否有在休息时间，手指温度恢复到基线？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 心率变异性（规范按年龄而异 ） </td></tr><tr><td> 1。心率基线变异低？ </td><td> 是_ </td><td>没有_ </td></tr><tr><td> 2。是否过去的基线心率变异减少任何应激？ </td><td> 是_ </td><td>没有_ </td></tr><tr> &lt;TD&gt; 3。在休息时间是否有恢复心率变异性呢？ </td><td> 是_ </td><td>没有_ </td></tr></tbody></table>治疗计划： 如果读数高于或低于正常值，在基线，治疗的目标是帮助客户带来在任何情况下都正常，生理参数 如果读数高于或低于正常值，在应激，治疗的目标是帮助客户识别压力和压力下能够保持正常生理功能水平 如果读数显示，在缺乏复苏的停机时间，治疗的目标是帮助客户带来到正常水平，很快一个应激，生理参数。

<ol>
	<li>Arena, J., Schwartz, M. S., Schwartz, M. S., Andrasik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychophysiological+assessment+and+biofeedback+baselines.">Psychophysiological assessment and biofeedback baselines.</a> A Practitioners Guide. , 128-158 (2005).</li><li>Schwartz, N. M., Schwartz, M. S., Schwartz, M. S., Andrasik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitons+of+biofeedback+and+applied+psychophysiology.">Definitons of biofeedback and applied psychophysiology.</a> A Practitioners Guide. , 27-42 (2005).</li></ol>

The authors have nothing to disclose.

完成appendi压力评估的心理生理提供了一个与生物反馈医生制订治疗计划所需的信息。这项评估提供有关个人的响应stereotypy，或所有压力均匀的某些生理反应，并刺激反应的特异性，或个人的独特的，以各种不同的压力的生理反应信息。有了这些信息，客户端就能够学会预测什么样的生理反应，期望在这种情况，并能够相应地修改他/她的反应。 评估之后，重要的是医生询问?...

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psychophysiological stress assessment using biofeedback 

在过去的半个世纪中，在生物反馈研究显示人的心灵在何种程度上能够影响植物神经系统的功能，以前被认为是有意识的控制之外。让人们观察自己的身体信号​​，生物反馈，使他们能够开发提高他们的生理和心理反应，如紧张，的认识，并学习如何修改这些反应。生物反馈的练习者，可以促进这个过程中，评估让人心动轻度压力事件的反应，并制定一个基于生物反馈治疗计划。医生首先在压力评估记录生理读数基线，然后提出了一些温和的压力，如认知，身体和情绪的应激，客户端。压力品种，以确定一个人的刺激反应的特异性，或在每个人的质的不同刺激的反应的差异。此影片将展示使用生物反馈治疗计划和目前的一般准则的心理生理压力评估的过程。

Watch this Scientific Journal Video about Psychophysiological Stress Assessment Using Biofeedback at JoVE.com

Psychophysiological Stress Assessment Using Biofeedback

在这个视频中，我们将描述评估人的心理生理反应，强调使用生物反馈的方法之一。我们也将目前的治疗计划的一般准则。

使用生物反馈的心理生理压力评估

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

psychophysiological-stress-assessment-using-biofeedback

Research

JoVE Journal

Biology

Psychophysiological Stress Assessment Using Biofeedback 

13.3K 次观看.  Cambridge Health Alliance. 在这个视频中，我们将描述评估人的心理生理反应，强调使用生物反馈的方法之一。我们也将目前的治疗计划的一般准则。

视频: Psychophysiological Stress Assessment Using Biofeedback

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

科研

生物学

Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion.  Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction.  Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm.  This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.

Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement.

显微剪辑闭塞大脑中动脉瘤术中流量评估

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

在临床的虚拟现实使用生物反馈：勇敢的项目

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

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 ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...