我々はCPRIT（RP100119）とウェルチ財団（I-1665）からの資金支援を認める。

プロトコル全体は約4日かかります。 1。のsiRNAとレポータートランスフェクションのための細胞の調製我々はここで例示の目的のためにゲノムワイドなsiRNAライブラリからsiRNAの小さなセット（単一遺伝子を標的とする4倍のsiRNAから成る各プールで、96ウェルフォーマットで384の異なるsiRNAのプール·アレイ）​​を使用したsiRNAライブラリーを尋問するためのプロトコルを紹介します。この特定の研究のために、我々はHCT116逸脱WntシグナルとKrasのシグナルを示す細胞、p53活性を活用します。関心のある他の細胞プロセスに問い合わせを目的とした研究において、適切な細胞株は同定され、同様の方法で評価されなければならない。 <ol><li>トリプシン処理ソリューション/プレートを1mlを用いてトリプシン処理によって収穫細胞は、2％のFEを含むダルベッコ改変イーグル培地（DMEM）10mlで中和した後、PBSで80〜90％コンフルエントHCT116細胞を5×10 cm 2とプレートを洗うタルウシ血清（FBS）および1％ペニシリン/ストレプトマイシン/プレート。 </li><li> DMEM / 2％FBS / 1％ペニシリン/ストレプトマイシンを10ml、50 mlコニカルチューブに細胞懸濁液を移し、5分間〜130×gで遠心分離し、上清を除去し、再懸濁細胞ペレット。 </li><li>セルカウンターで細胞数をカウントした後、10 5細胞/ mlの細胞溶液を行い、次のステップを150ミリリットルの細胞懸濁液を維持する。マルチドロップ自動液体ディスペンサー（以下、マルチドロップ）を用いて96ウェル細胞培養プレートの各ウェルのプレート10 4細胞（100μl）を。細胞懸濁液は、めっき12×96ウェルプレートで、この場合には十分であるべきである。 </li><li>トランスフェクション混合物を準備しながら、5％CO 2で37℃で細胞とプレート°インキュベーターでCをインキュベートする。 </li></ol> 2。レポーターコンストラクトストック溶液を調製<ol><li>分離し、高品質な記者は、標準的なミディ​​プレップキット（NucleoBondエクストラミディが生産で使用してDNAを構築とクロンテック）は各レポーターのために1 mg / mlの最終濃度にDNAを希釈。 </li><li>記者のp53-FL30μlの、8X TCF-RL、Elk1-Gal4の30μlの、6μlのUAS-CLとでDNAミックスを達成するためのH 2 O 4μlの30μlのを組み合わせることにより、原液を構築100μlのを準備記者の最終1:1:1:0.2比率。このDNAミックス原液の最終DNA濃度は、0.3 mg / mlのです。 </li></ol> 3。 siRNAを/ DNAトランスフェミックスの準備プロトコルのこの部分では、12×96ウェルプレートをトランスフェクトするために十分であるsiRNAを/ DNAトランスフェクションミックスを準備します。 siRNAの4×96ウェルプレートのこのテストライブラリのために、我々は三重にそれぞれのsiRNAプールをトランスフェクトされます。我々が使用するトランスフェクション試薬は、のEffectene（キアゲン）と呼ばれています。我々の手では、これは培養細胞へのsiRNAやDNAの同時配信のために働くだけ試薬である。 <ol><li>リポーターコンストラクト株式ゾルの80μlの希釈3μgの/ mlの最終濃度でDNA溶液を達成するために、EC緩衝液（キットのEffectene）8mlのでution。一般的には、すぐに使用するために十分なDNA溶液ミックスを調製する。 </li><li> 96 /ウェルプレートの各ウェルに、このDNA溶液を20μlのプレート。 96ウェルプレートの数は、siRNAプールがテストされますどのように多くに依存します。たとえば、この場合には、我々は384の異なるsiRNAのプールを評価するために4×96ウェルプレートが必要になります。 </li><li>テストするsiRNAは、5μMのストック濃度になっているはず。ていない場合は、レプリカプレートし、各ライブラリに関連付けられた推奨される希釈液を使用して、この濃度に希釈することができる。 </li><li> BIOMEK自動液体ハンドラーで希釈したDNAストックを含むPCRプレートの対応するウェルに各5μMのsiRNAストック2μlのを転送します。調査の規模に応じて、マルチチャネルピペットで十分である。 </li><li>今、私たちは、DNA / siRNAのPLAの各ウェルにエンハンサー溶液（のEffecteneキット）を1μlを追加しますTE。これは、再び自動液体ハンドラまたはマルチチャンネルピペッターのいずれかを達成することができます。 </li><li>その後、各ウェルにのEffecteneトランスフェクション試薬の3μLを加え、さらに10分間室温でインキュベートエンハンサー反応は5分間室温で発生することができます。 </li><li>トランスフェクション複合体形成を停止するには、PCRプレートの各ウェルに10μlのDMEM / 2％FBS / 1％ペニシリン/ストレプトマイシンを追加します。 </li><li>細胞（細胞インキュベーターから取得）を含む96ウェルプレートの各ウェルにトランスフェクション混合物10μlを移す。各トランスフェクションは、トリプリケートで実施されるように、同一の組合せは、細胞を含む2つの付加的な96ウェルプレートに適用される。 </li><li> 36時間、5％CO 2を含む加湿環境下で37℃で添加し、トランスフェクション混合物で細胞をインキュベートする。 </li></ol> 4。細胞サンプルからのルシフェラーゼ活性を決定 36時間後、ルシフェラーゼ活性は、測定のための準備が整いましたトランスフェクションされた細胞である。エンドポイントごとの実験に基づいて決定されるべきである。我々の研究では、36時間のインキュベーション時間は、以前に結果決定を意味するために十分に堅牢な信号が得られていた。本研究では、複数の記者（培養培地中に分泌一つであり、細胞質で発現している2つの他の人）が内蔵されたように、我々は、培養培地と同様、細胞溶解物の両方からの測定値を取得します。 <ol><li>培養培地中のCL活性の検出： <ol><li>培地20μlのは、白色不透明な96ウェルプレート（どちらBIOMEK液体ハンドラまたはマルチチャンネルピペッターを使用して）でレプリカメッキです。 </li><li>各ウェルにCLアッセイ緩​​衝液20μlを（システムのターゲット）を追加します。 </li><li>各ウェルにウミホタルルシフェリン基質（システムをターゲット）を10μlを加え、ルミノメーター（例えばBMGによって生成Pherastarプレートリーダー）を使用して、CL活性を検出。 </li></ol></li><li> FLとRL活性の検出： <ol><li> Cを溶解し第培地を除去することによってエルボ。これは、逆さまにプレートを回して、シンクに培地を投げることによって、急速に行うことができます。残りの培地を穏やかにペーパータオル上にプレート逆さまをタップして削除することができます。プラットフォームロッカーにマルチドロップと場所を用いて細胞の各ウェルに1Xパッシブ溶解バッファー（Promega社）を30μl加え、室温で5分間程度の速度を設定します（ベルコは、これらが行う一つの会社です。） </li><li>ルミノメーターを用いたマルチドロップし、直ちに測定FLアクティビティを使用してルシフェラーゼアッセイ試薬II（プロメガデュアルルシフェラーゼキットの一部）の20μlのを追加します。次に、ストップ＆FL活動をクエンチとRL活性の測定が可能になりますグロ試薬（Promega）を追加します。注：拡張信号の長さを（例えばプロメガからデュアルグロルシフェラーゼキットなど）を許可基板ミックスを使用している場合を除き、フラッシュキットの使用は、基質添加時の迅速な測定（5分以内）が必要です。 </li></ol></li></ol>

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OKとLL多重ルシフェラーゼ技術に関連する特許出願中で著者がある。

初めてのハイスループットスクリーンを楽しま人のために便利にオンラインリソースを提供ルシフェラーゼベースのレポーターシステムのいくつかの御用達（例えば、プロメガ、ターゲットシステム、New England Biolabs社、及びサーモフィッシャーなど）があります。また、遺伝子の発見と方法のRNAiベースのスクリーニング結果は、他の-オミクスベースのデータセットを統合することができる?...

細胞生物学的プロセスの多様な配列を監視するためのルシフェラーゼに基づくレポーターの様々な市販されている。これらのDNAの大部分は、特定の細胞の刺激や摂動によって発現するように誘導されているルシフェラーゼタンパク質をコードする構築。最も堅牢な転写基づい記者は生理学的に関連する転写イベント1,2の報告では、その信頼性のために十分に確立された合成エンハンサーエレメントまたは遺伝子プロモーター領域の制御下にルシフェラーゼ酵素の発現を配置。ルシフェラーゼタンパク質の発現を駆動するためGAL4-UASを利用するトランス - ルシフェラーゼレポーターシステムも存在する。 GAL4は、酵母転写活性化因子であり、UASは、Gal4のは、特にそれ3の下流に配置された遺伝子配列の転写を活性化するために結合するエンハンサーである。ルシフェラーゼこれらのタイプのシステムは、典型的には、2つのDNAプラスミドに支持されている - オンはregulatoに融合GAL4タンパク質をコードするリュー目的のタンパク質の部分と、第二は、1つまたは複数のUAS配列の制御下に置かれたルシフェラーゼタンパク質をコードする。したがって、セルラルシフェラーゼシグナルは目的のタンパク質の活性レベルを反映する。ルシフェラーゼベースのレポーターの別の一般的な用途は、目的のタンパク質がルシフェラーゼ酵素4に融合させたタンパク質の安定性を監視するためのものです。一つはよく尋問されたため、任意のレポーターの特異性を仮定することはできませんように関係なく、レポーターのタイプの、買い主の危険負担は、ここで注目される。したがって、デューデリジェンスは、任意の研究戦略における新たなレポーター構築の組み込みが必要になります。 ルシフェラーゼ酵素読み出しの選択は、その発現を制御するDNAエレメントの信頼性と同じくらい重要になることがあります。ホタルルシフェラーゼ（FL）は、市販の構成要素の中で最も一般的に使用される酵素であるのに対し、（場合のようにATPを必要としない新しいルシフェラーゼレポーターシステムの出現FLベースの反応）またはより強いシグナルを発する組み込むより安定した酵素は、この研究プラットフォームの効率と信頼性を向上させることを約束します。その化学研究でルシフェラーゼレポーターの選択に関する重要な注意事項は、虚偽の報告を生じさせる可能性があり、化学阻害にFLの感受性である。我々の経験では、そのようなウミや基質としてセレンテラジンを利用ガウルシフェラーゼ（それぞれRLとGL、）のような他の酵素が少なく容易に小分子5によって阻害される。 多重化ルシフェラーゼリードアウトのための最も一般的な形式は、主に単一のサンプルから順次に酵素活​​性を測定する機能を簡単に使用できるキットの利用可能性を与えFLとRLの使用を伴う。ほとんどのキットにおいて、サンプルは、最初に同時にseconを得セレンテラジンを用いて堆積される消光用試薬が続くFL活性のレベルを明らかにするルシフェリンにさらされているDARY RL信号。例えばウミホタルルシフェラーゼ（CL）のようなさらに別の基板GL、またはその用途として分泌され得る他のルシフェラーゼを添加して、ルシフェラーゼを用いた高コンテンツデータを生成する可能性がより多くの数が浮上している。この技術を用いたゲノムワイドRNAiスクリーニングが6ここで見つけることができた例。これらの技術的進歩は、順番に、クロストーク7を制限するために、ルシフェラーゼ酵素の各タイプをクエンチするために特定のメカニズムの開発に拍車をかけた。私たちの手で、私たちはそのようなGLやCLなどの分泌ルシフェラーゼと、 &quot;エッジ効果&quot;（高力価の培養プレートのエッジに関連する細胞活動の不可解な傾向）の大きい周波数に注意してください。それらは、細胞生存率をadulteratingずに信号サンプリングを可能にするように、その一方で分泌ルシフェラーゼ、運動アッセイに有用である。 ここで紹介する私たちの研究では、我々は同時に3癌関連のアクティビティを監視します細胞プロセス：p53を、Krasの、及びWntシグナル伝達経路。 、、我々の研究に組み込まれた記者はクロンテックからPP53-TA-リュックFLレポーター（以下のp53-FL記者と呼ぶ）8、Elk1-GAL4/UAS-CLレポーター系（アジレント以後エルク-1レポーター）であるおよびT細胞因子（またはのTCF）9-11として知られるWnt経路転写調節因子によって認識され、複数の合成エンハンサー要素を取り入れ8X TCF RLレポーター。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

RNA干渉（RNAi）を使用して、遺伝子機能のゲノムスケールの尋問は、化学的に扱いやすい癌細胞の脆弱性の迅速な同定のための途方もない可能性を秘めています。この技術の可能性を制限することで、迅速に結果の表現型の基本メカニズムを描くため、分子標的治療戦略の開発を知らせることができないことである。私たちは、ここで重要な癌細胞関連のプロセスを監視できるように多重化されたレポーターシステムを用いた標的RNAiによる遺伝子によって誘導される細胞の表現型を解体するためのメソッドの概要を示します。このハイコンテントスクリーニング方法は汎用性があり、容易に大規模な分子ライブラリの他のタイプのスクリーニングに適合させることができる。

大規模なゲノム配列の取り組み12-14を使用して、様々な癌の変異風景をマッピングにおける進歩にもかかわらず、我々はまだ場所に介入戦略にこれらの観察を翻訳するための体系的なアプローチを持っていません。結腸直腸癌（CRC）は、3つの細胞プロセスのコンポーネントに影響することが予測されている変異 - / Wntシグナルβ-カテニン、p53の、そしてKrasのシグナリングは - 彼らは?...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

細胞プロセスのシステム·レベルの理解を達成することは、現代の細胞生物学の目標です。我々はここでゲノム規模のRNAiライブラリーを用いた遺伝子の機能を調べるために、様々な細胞機能のエンドポイントのルシフェラーゼレポーターを多重化するための戦略について説明します。

培養細胞における癌関連シグナル伝達を問い合わせるための多重ルシフェラーゼベースのスクリーニング·プラットフォーム

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Research

JoVE Journal

Medicine

11.0K ビュー.  UT Southwestern Medical Center. 細胞プロセスのシステム·レベルの理解を達成することは、現代の細胞生物学の目標です。我々はここでゲノム規模のRNAiライブラリーを用いた遺伝子の機能を調べるために、様々な細胞機能のエンドポイントのルシフェラーゼレポーターを多重化するための戦略について説明します。

ビデオ: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

ローカルGyrificationのインデックスを計算するステップバイステップのチュートリアル：MR画像からの皮質折りたたみを測定する方法

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

医学

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

神経変性疾患におけるハイコンテントスクリーニング

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

小型げっ歯類における膀胱内圧測定の使用：膀胱Chemosensationの研究

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

ハイコンテンツ<em&gt;インビトロ</em&gt;膵島β細胞の複製ディスカバリー・プラットフォーム

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

トール様受容体シグナル伝達の阻害剤に対する食作用性免疫細胞における生物活性ナノ粒子のスクリーニング

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板輸血で微粒子のスクリーニング法のルーチン

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

ヒドラ、心血管分析および診断で臨床医を支援するコンピュータ ベースのプラットフォーム

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

体外培養肺胞上皮細胞におけるハロゲン化ガスの濃度を制御する方法

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...