この作品は、NIHの助成金1R03AI090465によって賄われています。

1。データの取得<ol><li>軌跡データはハンドヘルドGPSユニット、GPS対応スマートフォントラッキングアプリケーションだけでなく、A-GPS（GPSを支援）など、我々の研究で使用されている1つ、商業子トラッカー装置などの装置を使用して収集することができます。 </li><li>軌跡データは、通常の時間緯度経度レコードの条件で保存されます。所望の時間間隔は、アプリケーションのニーズに基づいて設定する必要があります。多くの場合、最も頻繁な間隔は、時空間活動の研究のために望まれている。 </li><li>コンマで区切られた値にデータを変換したり、。それぞれのレコードIDごとに個別の列を持つcsvファイル、緯度、経度、時刻、。その後、一般的に使用される地理情報システムへ。csvファイル（GIS）のファイル形式（ すなわち 、ESRIシェープファイル8）に変換します。 </li><li>建物ポリゴンと軌道アナライザによる研究領域の境界の別のシェープファイルにロードします。 3Dディに対して適切に建物の &quot;押し出し&quot;を設定スプレイと空間と時間を表すz次元を表すx、yの次元と時空キューブ6,9を表示するには、適切に&quot;押し出し&quot;と境界層の&quot;透明度&quot;を設定します。 </li></ol> 2。前処理<ol><li>二つのオプションは、前処理うるさい生の軌道データに使用できます。一つは、前処理メニューのドロップダウンリストから選択することができます。 </li><li> &#39;対話&#39;が選択されている場合は、3次元軌跡の2D投影を簡単に表示および選択のために作成されます。空間的にも時間的に生の軌跡を調べるために3D表示を操作します。形状、速度および/またはトラックセグメントのトポロジに基づいて、データのエラーを特定します。非現実的な高速または突然の方向変化を伴う通常のトラックポイント（頂点）のエラーを意味する。選択して、元の軌道からそれらを削除します。選択して、3D軌道またはその2D投影のどちらかからそれらを削除します。 </li><li>先端のとがったSHAとトラックポイントのクラスタPES（ 図1）空間的にも時間的に最も長い期間、おそらくGPS信号が弱い屋内位置によって引き起こされるエラーを意味する。これらの点のグループが選択された場合は、プログラムが選択されているポイントの時空重心を計算し、重心を通過する軌道を調整することができます。 </li><li> &#39;自動&#39;は前処理メニューから選択された場合、あるいは、異常な高速性とポイントの突然の転換を決定し、入力と出力の場所としてだけでなく、経験的なパラメータを設定します。プログラムがロードされた軌道データを検索し、視覚的なエラー検出の手法を模倣したアルゴリズムに基づいて自動的に実行されます。 </li></ol> 3。軌道セグメンテーション＆アクティビティスペースの特性<ol><li>軌道セグメンテーションは、建物の層を必要とするので、建物形状ファイルがロードされていることを確認してください。 </li><li>機能を起動するには、ツールバーのセグメンテーションツールをクリックします。入力と出力を設定し、基準層として建物形状ファイルの場所を指定。セグメント化された軌道にラベルを付けるために建物名を使用します。アルゴリズムは、速度、時間などのトラックポイントだけでなく、建物との関係で空間トポロジーとして設定するか、デフォルトの基準に基づいて、室内のセグメントを識別します。 </li><li>セグメント化された軌道にロードすると、それに総活性半径、一定期間における半径、屋内対屋外で費やされる合計時間の比率、など、1つの活動空間を特徴づけるために選択したサマリー属性を計算する活動空間集約ツールをクリックします。 </li><li>属性は、定量的なモデル化の目的で使用する場合は、スプレッドシートにエクスポートすることができます。 </li></ol> 4。密度表面マッピング<ol><li>密度表面は時間次元と空間での活動の密度が崩壊を示しています。 3つのオプションは、密度表面マッピング]メニューのドロップダウンリストから利用できます。 </li><li> &quot;トラックポイントの密度 &#39;オプションが選択されている場合、入力および出力情報を含むダイアログボックスに記入して3Dや2Dのいずれかで表示するように選択する。軌道データからすべての頂点がポイントのカーネル密度を計算するために使用される図2は密度面を示す。 </li><li> &quot;トラックパス密度&quot;を選んだ場合には、アルゴリズムが計算し、個々の走行経路（ 図3）の密度が表示されます。 </li><li> &quot;再サンプリングポイントの密度 &#39;オプションが選択されている場合は、アルゴリズムの再サンプル点の密度が時間内に均等に広げ、設定された時間間隔とマップを使用して軌道データ。このオプションは、様々な物理的条件やセグメント化された軌道の下にあるデバイスの感度の違いにより、不規則な時間間隔でトラッキングポイントを集めるトラッキングデバイス用に設計されています図4は、セグメント化された軌道の2Dおよび3D密度面を示しています。 </li><li> &#39;時空焦点&#39;が選択されている場合上記のオプションのny、時間的な焦点10は 、異なる期間での活動パターンを調べるために行うことができる。たとえば、日中に様々なタイミングで活動密度面は時間を越えて、ホットスポット（ 図5）の識別を容易にするために視覚化することができる。 </li></ol> 5。密度ボリューム推定とボリュームレンダリング<ol><li>密度のボリューム可視化は軌道の可視化のように時空キューブの概念を使用します。このような可視化の中核は、ボクセル空間11への分解である。視覚密度·ボリュームへの我々のアプローチは、最初のボクセルと交差時空トラックの数を数えることによって、個々のボクセルの密度の体積を推定する。一つは、このステップの密度のボリューム可視化メニューの下に &quot;密度体積計算&quot;をクリックすることもできます。 </li><li>同じ3つのオプションは、密度表面を可視化するために密度のボリューム可視化のために用意されています。 </li><li>インタラクティブなボリュームレンダリング12のための3Dボリュームデータの可視化インタフェースを起動するには[次へ]をクリックして&quot;ボリュームレンダリング&quot;。各軸に沿った分割数を設定することで、一つは、異なるスケールでクラスタを調べるかもしれません。 Z値の倍率は、より良い視覚化のための垂直誇張を設定するために使用されています。建物などの基準層も同様に可視化を支援するためにロードすることができます。ボリュームレンダリングの結果を対話的密度からカラーへのマッピングを制御伝達関数を操作することによって調整することができます。 （ 図6）。 </li></ol> 6。他の探索的データ解析（EDA）とビジュアライゼーション<ol><li>手順は、Google Earthに表示されるアニメーションシリーズを作成することが可能です。 &quot;その他&quot;の下では、この手順にアクセスする &quot;EDAのKMLへのエクスポート&quot;をクリックします。それは軌道のインタラクティブなアニメーションにGoogle Earthで開くKML 13ファイルを作成します。 </li><li>一つは、ラーベする軌道をたどることがGoogle Earthのタイムラインに沿ってスクロールして時間内にlは環境。 </li><li>手順は、 &quot;接続の分析 &#39;を通じて興味のある場所同士のつながりを視覚化することが可能です。例えば、大学キャンパス内の異なる建物間の接続は、学生（ 図7）によって収集されたセグメント化された軌道データから導出されます。 </li><li>派生した接続に基づいて、そのようなほとんどの発信または着信トラフィックと最もトラフィックの多い場所をつなぐハブとそれらの建物のようにホットスポットを識別することができる。 </li></ol>

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

特別な利害関係は宣言されません。

我々は、インターフェースを開発するために、ArcGISのアドインのメカニズムを使用していました。すべての対話的な操作を使用して実装されたC + +です。すべて自動処理と分析機能がPythonを使って開発されました。 エラーが原因で建物や頻繁な屋内停留所に隣接する大規模なことができるように歩行者が収集したAGPSデータ、またはGPSデータは、前処理に特有の課題を提?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> WorldTracker GPRS </td><td>世界のトラッキング</td><td></td><td></td></tr><tr><td>分析を実行するためのパソコン</td><td></td><td></td><td></td></tr><tr><td> ArcGISソフトウェア</td><td> ESRI </td><td></td><td></td></tr><tr><td>軌道アナライザ拡張</td><td></td><td></td><td></td></tr></tbody></table>

trajectory data analyses for pedestrian space-time activity study

これはよく空間的および時間的な次元における人間の運動は病気伝送1-3直接的な影響力を持っていることが認識されている。感染症は、通常、それらの重なり活動空間における感染と感受性個体間の接触を介して広がっています。したがって、毎日のモビリティアクティビティ情報は、感染の危険因子への曝露を測定するための指標として使用することができる。しかし、このように大きな難題とミクロスケールでの感染症伝播の研究が不足している理由は、詳細な個人のモビリティデータの不足から生じる。以前はしばしば被験者が積極的に時間と空間での活動を記録するために必要な時間空間日記技術、に依存していた交通、観光研究の詳細な時空間活動データインチこれは非常に参加者からの参加者とのコラボレーションを求めている大幅にデータ4の品質に影響を与える。 現代のGPSやモバイル通信などの技術が可能軌跡データの自動収集を行っている。収集されたデータは、しかし、既存の機器の精度によって制限され、人間の時空間活動をモデル化するための理想的ではありません。人間行動研究のためのデータの効率的な処理のための容易に利用可能なツールもありません。私たちはここでのメソッドのスイートと軌跡データの前処理、時空間解析のための統合されたArcGISのデスクトップベースのビジュアルなインターフェイスを提示。我々は、特にそのような感染症の伝送モデリングなど、公衆衛生学に有用である可能性のあるエラーの豊富な歩行者の軌跡データと、人間の時空間活動をモデル化するために使用されるかもしれないか、このような処理の例を示します。 提示の手順では、前処理、軌道セグメンテーション、活動空間の特性、密度推定と可視化、および他のいくつかの予備的な解析手法を含む。プリprocessingがうるさい生の軌道データのクリーニングです。私たちは、対話型のビジュアル前処理インタフェースと同様に自動モジュールをご紹介します。軌道セグメンテーション5は前処理された時空のトラックから、屋内と屋外の部品の識別を必要とする。繰り返しになりますが、インタラクティブな視覚的セグメンテーションと自動セグメンテーションの両方がサポートされています。セグメント化された時空のトラックはその後派生するために分析されるような活動半径等密度推定と可視化など、1つの活動領域の特性は、ホットスポットとの相互作用をモデル化するために軌道に大量のデータを調べるために使用されます。我々は、密度表面マッピング6および密度ボリュームレンダリング7の両方を示しています。我々はまた、Google Earthのアニメーションをサポートし、接続の分析など、他の探索的データ解析（EDA）と視覚化ツールのカップルが含まれています。分析のスイートだけでなく、この論文で提示ビジュアルメソッドはsの任意の軌道データに適用してもよいペースタイム活性研究。

軌跡データは、2010年春にキーン大学（ニュージャージー州、米国）からの学部学生をボランティアにより収集した。目的はなかった人と比較して（医師によって診断や自己診断）がインフルエンザをひいた生徒の行動パターンを研究することであった。本稿で提案手法や手順を説明するために、我々は代表的な結果を生成するために郊外キャンパスエリア内で収集軌道を取った。キャンパス...

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

Trajectory Data Analyses for Pedestrian Space-time Activity Study

時空間処理方法のスイートは、モデリング·歩行者の時空間活動の目的のため、GPSデバイスを使用して収集されたものと人間の軌道データを分析するために提示されています。

歩行者空間·時間·アクティビティ·研究のための軌道データ解析

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

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

Research

JoVE Journal

Biology

13.4K ビュー.  Kean University. 時空間処理方法のスイートは、モデリング·歩行者の時空間活動の目的のため、GPSデバイスを使用して収集されたものと人間の軌道データを分析するために提示されています。

ビデオ: Trajectory Data Analyses for Pedestrian Space-time Activity Study

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

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

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

トカゲの運動の速度と方向の視覚感度のテスト

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

生物学

Computer-Generated Animal Model Stimuli

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

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

コンピュータによって生成された動物モデルの刺激

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

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

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

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

切断者のためのOsseointegratedインテリジェントインプラント設計システムの生体電気分析

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

の単離と精製ショウジョウバエ</em磁気ビーズソート別>末梢ニューロン

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

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.

プロテオーム解析のためのマウスの硝子体と網膜の内臓摘出

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

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

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

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

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

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

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

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

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

免疫組織化学および免疫蛍光分析のための腸組織の調製のための改良されたスイス・ローリングテクニック

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

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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

体外相撲 E3 リガーゼ活性を研究する sumo 化アッセイ

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

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

電気生理学測定の電気中立運送者の活動を研究への応用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

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.

-オミクス分析と原発細胞培養のためのコウモリの組織採取

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