この研究は、医学研究評議会、カナダ自然科学工学研究評議会からの補助金、およびカナダ脳卒中ネットワークによってサポートされていました。 GAMは、医学研究のためのアルバータヘリテージ財団によってサポートされていました。

ラダーのラング歩行試験装置 。メッツとWhishaw、試験装置を歩く水平ラダーのラングは、ラングの間に1cmの最小距離（図1を参照して床を作成するために挿入できる透明なプレキシグラスと金属のラング（3 mm径）、製側壁で構成され、2003）。側壁は長さ1 m、19センチの高さ横木の高さから測定。ラダーはニュートラルスタートケージと終了時に避難所（ホームケージ）で地上30センチ上昇した。動物が訓練中に慣れていたため、装置の高度は、不安を引き起こす可能性は低いでした。路地の幅は、それが周りの回転から動物を防ぐために、動物より約1cm幅になるように、動物の大きさに調整した。 タスクの難易度は、金属のラングの位置を変えることによって変更されました。ラングの規則的なパターンは、動物はいくつかのトレーニングセッションを介してパターンを学習し、ラングの位置（図1、パターン）を予測することができました。裁判から裁判に変更された不規則なパターンは、パターン（図1、パターンB）の学習から動物を防いだ。規則的な配列の場合、ラングは、2 cmの間隔を置いていた。不規則なパターンの場合は、ラングの距離は1〜5 cmに体系的に変化した。不規則なラングパターンの五のテンプレートは、同じパターンが（結果を参照のこと）、テストの難易度を標準化し、結果の比較可能性を高めるためにすべての動物に適用されたように、使用されていました。 Videorecording カメラ（Canovision、キヤノン株式会社）はわずかに腹側の角度に配置されたので、すべての四肢の位置を同時に記録することができます。 2000秒 - シャッタースピードを500に設定されていましたvideorecordingsは、30 F /秒でフレームごとの分析を用いて分析した。 行動訓練とテストの分析動物は自分のホームケージに到達するための中立的なケージから梯子を横断するように訓練された、同腹子とホームケージは歩くために正の強化を提供するため。すべての動物は同じ方向にラダーを交差させた。それ以上の補強は、はしごを渡る動物をやる気にさせるための指定されませんでした。すべての動物は訓練され、セッションごとに5回の試験を行った。 フットフォルトのスコア前肢と後肢の配置の定性的な評価は（メッツとWhishaw、2003）前述のようにフットフォルトのスコアリングシステムを用いて行った。分析は、ビデオ録画フレーム毎の検査によってなされた。各肢の唯一の連続的段階を分析した。したがって、停止またはフットフォールトなどの歩行の中断、および中断後の最初のステップの前に最後のステップは、採点されませんでした。はしごの最後に実行した最後のステップのサイクルは、得点から除外されました。肢の配置は、ミスが発生したラング間ラングと肢突起上肢の配置の点で採点した。 ラングの足や足の配置の種類は、（図2参照）、7カテゴリースケールを用いて評価された。ラング上で足や足の配置は、それらの位置と配置精度で発生したエラーに応じて評価された。 四肢が完全にラングを逃した時に合計ミス。0ポイントが与えられていた（0）、すなわち、それを触れていない、と秋が発生しました。秋深くに - との間のラングと体の姿勢とバランス立ち手足のように定義されていたが乱された。 （1）ディープスリップを。手足が最初にラング上に配置され、その後、時体重がオフ滑って落下を引き起こした。 （2）わずかなスリップ。手足がラング上に配置されたが、ときに体重負荷をオフに滑って、しかし秋になるも、歩行サイクルを中断しなかった。この場合、動物はバランスを維持し、協調歩行を継続することができた。 （3）交換。手足はラング上に配置されたが、それは体重負荷になる前に、それはすぐに解除し、別のラング上に配置されました。 （4）問題の修正。肢一ラングを目指したが、その後最初のものを触れることなく別のラング上に配置された。手足がラング上に配置されている場合あるいは、4得点を記録したと同じラング上に存在したまますぐに再配置されました。 （5）部分の配置を。肢は前肢またはヒールや後肢のつま先の手首または数字のいずれかとラング上に配置された。 （6）正しい配置を。肢の手のひらの中央部が全重量のサポートとラング上に配置されました。 別のエラーが同時に発生したとき、得点の最も低い記録した。例えば、足が（スコア1）最初のラングに配置し、同じステップ（スコア3）で別に配置して、ラングの合間に滑って落ちていた場合、1得点を記録した。 WH動物はすべての手足を再配置するまでに発生した秋の途中で、エラーを開始するだけ手足が評価されたと他の肢のどれも得点されなかった。 5件の試験のエラースコアは、分析のために平均した。 足の位置精度の分析（エラーの数） 各交差点でのエラーの数をカウントした。エラーがフットフォルトのスコアリングシステムに基づいて決定された。エラーが0、1点または2点のスコアを受け取った各肢の配置として定義され、エラーが足スリップまたは合計ミスのいずれかの種類を表す、すなわち。エラーやステップ数の数は、個別に各肢について記録した。これらのデータから、ステップごとの誤差の平均数は5件の試験のために計算して平均した。分析された第二定量的パラメータには、はしごのタスクの全体の長さを横断するために必要な平均時間でした。時間の測定は、動物がラダーに配置された後に開始し、それの全体の長さを歩いて始めた。動物が停止に費やされた時間は測定に含まれていませんでした。 前足桁得点前足桁のスコアは、前足がラング上で、その中央部（フットフォルトのスコアリングシステムで、すなわちスコア6）で正しく置かれたときに記録された。前足の桁がラングの周りに屈曲することができるか程度は（図2）三点尺度で評価された。スコアは、前肢がラング上に垂直に配置された、すなわち、最大の重量をサポートする位置に適用した。スコアは以下のように与えられた： （0）数字はおおよそ90度の角度でクローズ。 （1）数字はおおよそ45度の角度でクローズ。 （2）数字は、完全にラングの周りに屈曲。 5つのステップの得点を平均し、分析のために使用されていました。 図の説明文<img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="図1" /> 図1は、。装置の具体的な測定と、frontolateralビューで試験装置を歩いて熟練したラダーのラングが示された。パターン、定期的なラングの配置、パターンB、不規則なラングアレンジメント：二つの異なるラングのパターンが本研究で使用されていた。不規則なラングの配置が連続する試行間で変化した。 <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="図2" /> 図。 （2）代表的な写真は、桁得点の3つのカテゴリを示す図。ときに全体重のサポートの前肢との関係で数字の角度に注意してください。

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本研究では、熟練した歩行、四肢の配置と四肢協調を評価するための新たなテストとして、タスクを歩いてラダーのラングを紹介します。タスクを歩いてラダーのラングは、熟練した歩行の定性及び定量分析を組み合わせることにより、運動機能の微妙な障害の間に差別をすることができます。ビデオの分析手順は、障害のより記述的なアカウントのために使用され、ラングを把握する際にこのホワイトペーパーで説明されているシンプルな評価スケールは、四肢の配置と桁の屈曲の誤りを明らかにすることができます。足音のエラーの数を数える定性的な解析は行われ、時間は、装置を通過する必要がありました。これらの措置は、足の位置決め精度と桁の使用の効率的かつ有効な検査を可能にする。 rotorodウォーキングのテスト、ビームの歩行、シリンダーテスト、および歩行解析、および床反力の解析（ミューア、2005）を含む歩行のテストの数があります。これらのテストのそれぞれは、行動分析のために有用フラグメント肢配置することができます。強度の数値は、しかしながら、現在の試験のプロトコルのためにあります。動物はモチベーションのための食料や水剥奪を必要としないため、最初に、テストでは、自発的な歩行状況を確認することができます。第二に、横木を越えステップ実行するステップと把握の両方の分析を可能にする、正確な足の配置と把握する必要があります。ラングの間隔が変化しているため、最も重要なのは、本試験は進行中の運動に挑戦しています。変動が進行中の歩行および/またはステッピングパターン（マクビーとピアソン、2007）のメモリを挑戦するために使用することができます。 ラダーのラング歩行テストの強みは、それが十分に船首と使用し、前脳の制御を必要とする障害をアンマスク後肢両方で微妙な慢性的な障害を明らかにするために挑戦されていることです。タスクの不規則なラングの状態では、動物はラングの位置を予測し、特定の歩行パターンを学習することができません。その結果、各ステップは、足の配置、ストライドの長さ、および体重の分布での調整が必要になります。通常の動物が少ない試行内で不規則なラングのパターンに調整することができますが、この能力は、モータシステムの病変を持つ動物に限られている。不規則なラングの配列はまた、動物が変化するラングの距離に、通常の四肢協調を適応させることが必要です。四肢協調を適合させるために、動物はすぐにあらゆる肢の配置誤差を補正するために、自分の体重のサポートを制御する必要があります。片側病変後、動物は、部分的に重量をサポートするため無傷の手足を使って、配置ミスを補う。そのため、ラダーのラング歩行テストは、そのままサイドに足の位置の誤差を明らかにすることによって、補正を測定することができます。 、ファーら、リエック- Burchardtら、2004;。。ラング歩行テストは、脳卒中のラットモデル（エメリックとKartje、2004などの運動系への成人と新生児の病変後の慢性的な運動の赤字、に敏感であることが示されている2006;農夫ら、2007）、パーキンソン病（メッツとWhishaw、2002;。Farajiとメッツ、2007）、および脊髄損傷（Z&#39;Graggenら、1998;。Merklerら、2000）。。また、ラダーのラング徒歩タスクは、そのような軽度のストレス（メッツ）と食事でさえ変化などの生理的変数（スミスとメッツ、2005）により誘発される微細運動性能の変化を検出します。ラットモデルに加えて、タスクは、マウス（ファーら、2006）で熟練した歩行を研究するのにも便利です。ラング徒歩タスクが動物は体重のかかる手順を実行できることが必要として重度の脊髄病変は、（メッツら、2000）テストされている場合は、このテストを使用すると、使用する必要がある注意。 。Miklyaevaら、1994;。Kleimら、1998;。Chuさんとのモータシステムの病変を持つ動物は、熟練した運動（Whishawら、1997a、B、1998年に傷害誘導性の赤字を補うためにできることを実質的な証拠があるジョーンズ、2000）。それにもかかわらず、重度の障害が依然として存在していてもよい。例えば、ドーパミン作動性または赤核の病変を有する動物は、滑らかな表面や狭いビームに歩行能力を回復するが、床反力の対策は（ミューアとWhishaw、1999、2000）、その障害が慢性的であることを明らかにする。それも微妙な残りの運動障害と補償の調整を明らかにするので、ラング歩行テストが原因で脳や脊髄損傷、および治療法の利益のために機能の喪失と回復を評価するための貴重なツールです。

the ladder rung walking task: a scoring system and its practical application.

/脳卒中、脊髄損傷、および他の神経変性疾患の動物モデルの開発の進捗状況は、運動機能の異なる側面を詳しく説明すると、運動能力の微妙な損失を決定するために高感度のテストを必要とします。テストの有効性と分解能を向上させるために、テストでは、運動機能の定性的および定量的な措置を許可し、回復期間中のパフォーマンスの変化に敏感でなければなりません。本研究では、同時に前肢と後肢機能の両方を測定するためにラットで熟練した歩行を評価するための新しいタスクを説明します。動物は、ラングの間隔が可変であり、定期的に変更されている水平方向のラダーに沿って歩くことが必要です。ラングの間隔の変化は、ラングの絶対および相対位置を学習から動物を防ぐなどの学習を通じて、障害を補うために動物の能力を最小限に抑えることができます。さらに、ラング間の間隔を変更すると、テストが長期試験で繰り返し使用することができます。メソッドは、協調、ステップ、置くこと四肢を含む前部と後肢のパフォーマンスの両方の定量的および定性的な説明は、両方に記載されている。また、代償的戦略の使用は失策または他の肢の紛失への対応の代償手順で示されます。

Watch this Scientific Journal Video about The Ladder Rung Walking Task: A Scoring System and its Practical Application. at JoVE.com

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

タスクを歩いてラダーのラングは、熟練した歩行を評価し、前肢と後肢、配置ステップ、および間四肢協調の両方を測定するための新しいテストです。

タスクを歩くラダーラング：スコアリングシステムとその実用化。

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

the-ladder-rung-walking-task-scoring-system-its-practical

Nanyang Technological University

Research

JoVE Journal

Biology

25.9K ビュー.  University of Lethbridge. タスクを歩いてラダーのラングは、熟練した歩行を評価し、前肢と後肢、配置ステップ、および間四肢協調の両方を測定するための新しいテストです。

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

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

誘導および慢性-再発実験的自己免疫性脳脊髄炎の臨床スコアリング

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

生物学

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

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.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

ラットの歩行の質的分析のための方法としてRotorodの使用

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

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic analyses of these traits. Tomato Analyzer (TA) is a software program that measures 37 attributes related to two-dimensional shape in a semi-automatic and reproducible manner1,2. Many of these attributes, such as angles at the distal and proximal ends of the fruit and areas of indentation, are difficult to quantify manually. The attributes are organized in ten categories within the software: Basic Measurement, Fruit Shape Index, Blockiness, Homogeneity, Proximal Fruit End Shape, Distal Fruit End Shape, Asymmetry, Internal Eccentricity, Latitudinal Section and Morphometrics. The last category requires neither prior knowledge nor predetermined notions of the shape attributes, so morphometric analysis offers an unbiased option that may be better adapted to high-throughput analyses than attribute analysis. TA also offers the Color Test application that was designed to collect color measurements from scanned images and allow scanning devices to be calibrated using color standards3. 
TA provides several options to export and analyze shape attribute, morphometric, and color data. The data may be exported to an excel file in batch mode (more than 100 images at one time) or exported as individual images. The user can choose between output that displays the average for each attribute for the objects in each image (including standard deviation), or an output that displays the attribute values for each object on the image. TA has been a valuable and effective tool for indentifying and confirming tomato fruit shape Quantitative Trait Loci (QTL), as well as performing in-depth analyses of the effect of key fruit shape genes on plant morphology. Also, TA can be used to objectively classify fruit into various shape categories. Lastly, fruit shape and color traits in other plant species as well as other plant organs such as leaves and seeds can be evaluated with TA.

Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

トマトアナライザ：2次元オブジェクトからの正確で詳細な形態学的および測色データを収集するために有用なソフトウェアアプリケーション

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

の可視化線虫（Caenorhabditis elegans）</em親油性生体染色色素、DIIを使用する>キューティクルの構造

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Nonexpertsための系統学の実用ガイド

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3次元組織学ボリュームとマウス乳腺研究への応用再建

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...