由加拿大卫生研究院，加拿大中风网络，和阿尔伯塔省文物基金会的医学研究机构的拨款支持。

Rotorod步行仪 rotorod是一个旋转的圆柱体，直径4厘米，高出地面35厘米，由透明有机玻璃封闭固定。 rotorod缸覆盖质感的橡胶涂层，这有利于牵引。仪器的地板所用的泡沫层，以防止伤害动物下跌时。小型电动马达提供的电源打开通过橡胶带rotorod。 rotorod是轮换一次，每5秒录像和运动学一个高速的数码摄像机，用于拍摄动物，使用了1000秒的快门速度。而动物rotorod行走前，侧和后方的意见被摄制。两手臂尼康，信息产业部冷光源和光纤灯设置在录制过程中被用来提供足够的照明。磁带进行了分析帧一帧，使用了索尼DSR 20 DV CAM的甲板。使用图像采集卡和Macintosh计算机的视频帧被抓获。 测试过程和分析为了评估步行6 - OHDA和对照组大鼠的运动之间的差异，从视频记录手术后的天15 - 30（任何病变的恢复可能会预计将基本完成后），由两个独立的研究盲目的实验条件下的观察员。 6 - OHDA黑质纹状体病变引起的大脑右半球，造成持久的损伤在左侧前后肢。 A级规模开发率老鼠的姿态和前肢跨步动作（Whishaw等，2003）。五，加强组件被评为： <ol><li> 姿势 ，姿势的评级是由研究鼓大鼠的位置; </li><li> 升降机和发布 ，加强循环，爪子是从鼓解除部分; </li><li> 携带 ，部分肢体以上的鼓举行大鼠前移，加强循环; </li><li> 提前 ，延长肢体的恢复与鼓接触达到的部分; </li><li> 安置 ，其中的爪子放在鼓。 </li></ol>下列标准被用来定义一个正常​​的运动： <ul><li> 姿势：大鼠的身体是在鼓头的导向。 </li><li> 电梯和释放：肩部，肘部和手腕弯曲的爪子提高从感光鼓表面。 </li><li> 卡里：爪子举行的感光鼓表面上方的数字是松散的弯曲，身体中线为中心的数字提示。进展：肢体延长的数字，从一个semiflexed位置移动到一个扩展和开放的立场。 </li><li> 安置：肢体是延长的肩，肘，和手腕和数字都开到的地方足跖向前和感光鼓表面上两位数的2位于约在老鼠的中心（第二位从中线），身体。 </li></ul>每个动作被评为规模3点：1 = /运动就像一个对照组大鼠，0.5 = /运动出现轻度异常，0 = /运动显然是不正常的（Whishaw等，2003）。然后用此评定量表评估五个加强运动，从各组大鼠术后天1，3，5，7，9，和15。五项措施分数平均每天每个大鼠，结果天作为一个重复测量方差分析。 走在6 - OHDA和对照组大鼠rotorod概述运动评定量表是适用于术后定性运动分析测试会话。 6 - OHDA病变组测试会议期间，收到了显着较低的分数比对照组，F（1,8）= 33.2，P &lt;0.001，但考试当天没有显着的效果，也没有显着的集团测试日的相互作用。加强分数，然后分别与作为一个变量的前肢（6 - OHDA大鼠对侧病灶与病灶同侧的前肢前肢为对照组，与左右爪子爪子）的分析。是前肢，F（1,40）= 15.8，P &lt;0.01有显着疗效，并有一个显着的交互作用，F（1,40）= 15.0，P &lt;0.01前肢集团。后续t -检验表明，收到的6 - OHDA组对侧病变前肢比同侧病变前肢或对照组大鼠，没有差异的前肢的得分显着降低。为了确定运动的组成部分穷人分数由6 - OHDA组获得，姿势的措施作出了贡献加强循环的6 - OHDA组同侧和对侧前肢的各个组成部分进行了比较，对照组大鼠中获得的值。 6 - OHDA组不同的姿势和对侧肢体加强组件，但没有同侧肢体的组成部分（P &lt;0.05）。 在控制和6 - OHDA大鼠姿势的定性和描述性的差异在对照组大鼠，头部保持在一个水平位置，以及配套的前肢和后肢跨越鼓，形成了支撑拱。 6 - OHDA大鼠通常走到头受损前肢定位紧密联系起来（面向观众）和更高的感光​​鼓表面上发生较对照组大鼠同侧肢体或四肢（为对照组大鼠的视频评分“1”的姿势，和“0”分6 - OHDA病变动物）。通常情况下，异常的姿势可以看出，从老鼠的头部的相对位置一目了然。 前肢加强在控制序列，加强前肢抬起弯曲的肩膀，肘部和手腕。由于肢体被解除，是松散的数字弯曲，肢体，远高于感光鼓表面解除。当患肢是先进的，它是在肩部，肘部和手腕延长，因为它是放在数字是扩展和开放。在对照组相比，DA耗尽大鼠对侧肢体病变的所有组件的幅度衰减。肢体释放鼓后，解除少，是先进的，在较短距离比是控制前肢。此外，肢体解除时，数字是弯曲的多，当患肢放置，扩展和开放的数字少于一个对照组大鼠的。一旦达到鼓顶部控制前肢抬起，手腕弯曲抬起的爪子和前瞻性，延长肢体的进步和手腕。相比之下，6 - OHDA大鼠对侧病变前肢进行进一步尾端鼓的运动，从而释放后依然延续手腕。屈曲手腕，似乎是被动的，发生鼓的运动落后的数字，时提出，前肢的爪子和前臂不升空的感光鼓表面清楚。一个对照组大鼠的前肢以及向前延伸的数字，随着肢体的进步打开。由于肢体上，它是放在一个琶音运动，与数字接触表面的鼓第一5，随后先后由4位数字，3和2。相比之下，6 - OHDA大鼠肢体解除和扩展，数字延长，他们几乎同时接触了“巴掌”鼓表面。视频显示在控制动物的前肢加强，取得了为“1”，并在6 - OHDA病变动物，主要为“0”所取得的前肢加强运动运动。 步进后肢在6 - OHDA大鼠对侧病变的后肢也有损伤。由于控制后肢向后移动，它似乎对转动的滚筒推，因为它这样做，脚尖向外旋转。由于肢体向前移动，脚向外摆动。 6 - 羟基多巴胺大鼠后肢直接向后移动，磁鼓旋转，如果有的话，变成略微向内。由于肢体向前移动，脚下是进行直接转发。对照组大鼠外摆，因为它是先进的，脚趾举行高于脚踝的脚，脚趾延长。在对照组大鼠的位置，脚趾5点分布在第一和重量鼓表面的脚趾和掌心。相比之下，6 - 羟基多巴胺大鼠脚趾弯曲期间携带和安置是没有琶音运动。对照组大鼠的脚趾都公布在扭转琶音模式，5趾解除和脚趾1最后解除。这一运动是缺少在6 - OHDA病变大鼠踝关节保持其弯曲的姿势进行运动期间。再次提前6 - OHDA大鼠运动减弱，肢体少exorotated，解除少，和先进低于对照组大鼠。作为对照组大鼠置于他们的脚趾，他们扩大和开放，这样，脚趾5点横向相对于大鼠的身体。 5趾接触大鼠支持脚趾提示其重量由剩余的脚趾先跟着鼓。 6 - 羟基多巴胺大鼠，脚趾有点弯曲的脚是结转，脚趾到脚踝的水平相对较低，有脚趾开幕时脚放在鼓上。步幅也较对照组短。为了量化CONT之间的差异ROL和6 - OHDA后肢运动，升降机及释放的组件，携带，进步，和额定位置上规模3点，前肢。措施上手术后20天的视频记录，以及各组大鼠的额定三个步骤的周期。有显着的组间差异，F（1,8）= 47.6，P &lt;0.001。后续的组件t -检验表明，对照组大鼠的所有组件收到显着较高的分数比6 - OHDA大鼠的组成部分。视频显示在控制动物的后肢加紧动作，分别为“1”取得。后肢加强在视频显示，在6 - OHDA的变动，主要是堪称一个“0”的得分。

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本研究的目的是研究是否可以用于定性检查的姿势，前肢加强和后肢踩着在中枢神经系统损伤大鼠rotorod灭鼠运动技能测试。单方面DA枯竭和对照组大鼠进行了分析，录制的视频前，外侧和后侧的意见。一个姿势和前肢动作的评定量表表明，加强运动，手术后长期受损。肢体运动的研究表明，而DA耗尽的老鼠可以使用支持病变对侧肢体，他们收到的转移重量极少使用。这项研究的结果表明rotorod任务，除了提供定量?...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

rotorod测试，在一个转鼓上行走的动物，被广泛用于评估在啮齿类实验动物的运动状态。性能测量的持续时间，动物保持鼓，转鼓转速的功能。在这里，我们报告的任务，提供了丰富的步行运动的定性方面的信息来源。因为运动是在一个固定的位置进行的，他们可以很容易地用高速视频记录方法研究。目前的研究进行了检查rotorod潜力揭示半帕金森模拟到右侧黑质纹状体束注射6 - 羟多巴胺（6 - OHDA）制作大鼠，步行运动的质的变化，消耗黑质纹状体多巴胺（DA ）。翌日，手术开始，然后定期在未来的两个月中，老鼠是从正面，横向，和后部的看法拍摄，因为他们走进rotorod。帧帧回放视频记录的行为进行了分析。加强行为评定量表表明，半帕金森大鼠长期在他们的姿势和使用的肢体对侧的DA消耗受损。不仅显示姿势和运动异常，对侧肢体，他们参加发起和持续推进比同侧肢体。这些研究结果不仅揭示继发单方面的DA消耗新的赤字，但也表明，rotorod可以提供一个强大的工具，对运动的定性分析。

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

rotorod测试是用来评估半侧帕金森模拟大鼠的行走运动的电机状态。

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

use-rotorod-as-method-for-qualitative-analysis-walking

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12.9K 次观看.  University of Lethbridge. rotorod测试是用来评估半侧帕金森模拟大鼠的行走运动的电机状态。

视频: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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走在大鼠作为定性分析方法使用Rotorod