We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

USGS国立野生生物保健センターで行わ動物の作業は実験動物福祉ガイドラインのNIHオフィスに合わせて、制度的動物のケアと使用委員会プロトコル＃のEP080716で行った。狩猟採取した動物からの組織は、ウィスコンシン州、または天然資源のミシガン部門からの贈り物だった。 1.溶液の調製注：以下に記載されているソリューションは、下記の第2でCERアッセイ基質の製造のために必要とされる。より高濃度のストック溶液からこれらの溶液の各々を準備し;ストック溶液の推奨濃度は、リストされた各それぞれの化学名の後の括弧内に与えられます。使用するまで4℃で基板準備や店舗の際に新鮮なすべてのソリューションを準備します。 <ol><li> 100mMの秒：溶解緩衝液は、10mMトリス、pH7.5の（1 Mトリス、推奨pH7.5のストック溶液）で以下の溶液を調製する憎悪塩化ナトリウム（NaCl、1 Mのストック溶液）、10mMのエチレンジアミン四酢酸（EDTA、500mMのEDTA、pH8.0のストック溶液）、0.5％NP-40（10％ストック溶液）、0.5％デオキシコール酸（DOC、10％ストック溶液）。 </li><li>変換バッファは、1リン酸緩衝生理食塩水（PBS、10、pH7.4のストック溶液）、0.05％のドデシル硫酸ナトリウム（SDS、10％のストック溶液）及び0.5％のTriton X-100（10％ストック溶液）の溶液を調製する。 </li><li>カオトロピックソリューションでは、1×PBS（10倍原液）における塩酸グアニジン（GdnHCl、8 Mストック）の2つの3 M溶液を調製する。濃塩酸（HCl）を用いて、3.5±0.05の解決策の1のpHを調整する。第2の溶液のpHが7.4±0.05のままであり、必要に応じて、濃HClまたは水酸化ナトリウム（NaOH）を用いて調整することを確認する。 </li></ol> 2. CERアッセイ基質ペアを準備<ol><li>興味の種から健康的、非TSE感染脳組織を入手し、10％の重量あたりのボリュームを準備ダウンス、ビーズミル、又は乳鉢及び乳棒均質化：次のいずれかの方法を使用して、溶解緩衝液中（w / v）のホモジネート。さらに、シリンジおよび針均質に供する基板を吸引し、27 G注射針を介して容易に排出することができるまで増加ゲージの針を使用して得られたホモジネートを絞り込む。 <ol><li>齧歯類および他の小型哺乳類から調製基板について、脳全体（複数可）を均質化する。大型哺乳類から調製した基板について、OBEX（脳幹）ホモジネートを準備するための組織を使用しています。脳ホモジネートの量を選択する場合、ステップ2.6において、タンパク質沈殿のために使用される遠心分離器の容量の50％以下を準備していない。 </li><li>取得された脳組織の品質は疑問がある場合は、SDS-PAGEおよび免疫ブロット16 17基板の前に準備することによってのPrP Cのレベルを評価する。 </li></ol></li><li>独立したラベルプラスチック製コニカルチューブ内の2つの同等のボリュームに脳ホモジネートを分割。 （GdnHClを追加脳ホモジネートへ1体積比：1の各チューブにpHが3.5または7.4のいずれかで1 P​​BS中の3 Mソリューション）。 </li><li> 2の溶液のpH値を確認してください。濃HClを使用して0.05​​±pHにpHを3.5基質のpHを調整します。他の基板のpHは7.4±0.05のままで確認し、必要に応じて、必要に応じてHClまたはNaOHでpHを調整する。酸または塩基の賢明な添加によってpH値をオーバーシュート避ける。 </li><li> 5時間室温（RT）で転倒ミキサーで解決策を回転させます。 </li><li>各コニカルチューブ内の基板ソリューションへのメタノールの4つのボリュームを追加することによって、タンパク質を沈殿させ、ボルテックスよく混ぜ、そして16〜18時間、-20℃でインキュベートする。 </li><li> 4℃で30分間、13000×gで遠心分離により堆積物試料。慎重にデカントし、上清を廃棄し、メタノールはペレットから蒸発させる。チューブの内側にしがみついメタノールを吸収を助けるために綿棒アプリケーターを使用してください。オーバー-DRへのペレットを許可しないyおよびメタノールが見えなくされたら、次のステップに進んでいない。 </li><li>変換バッファにおけるタンパク質ペレットを再懸濁する。ステップ2.2において、各チューブに分注し、脳ホモジネートの出発材料の量に等しい変換バッファの量を使用する。 注：これは、pHが3.5と7.4で処理した基板の製剤の両方からのタンパク質ペレットを再懸濁するために使用する変換バッファは、（洗剤の低レベルと生理的pHを含む）上記のステップ1.2で定義されて解決することが重要です。 </li><li> cuphorn音波処理で簡単に超音波処理基質溶液（30％最大電力〜10秒）、0.5へのアリコート - ラベル1.5ミリリットルマイクロ遠心チューブ中で1.0mlのボリューム。各基板のアリコートに品質管理イムノブロット（ステップ2.9）を実行します。 CERアッセイ（第4節）を実行する準備ができるまで-80℃で、他のアリコートを保管してください。 </li><li>使用前CER基板対（ 図2）上の品質管理を行う。これらのステップは、CER基板が提供します：することを保証する変換の研究で、電子最適な結果を得る。 <ol><li>二枚の基板中のPrP Cの匹敵する量を確認してください。 SDS-PAGE 16およびイムノ17によって各基板の10-25μlの中のPrPレベルを比較。 pHは7.4と3.5の基質中のタンパク質レベルが互いに約10％以内で​​あれば、基板対はCER研究での使用に適している。 注：PrPのイムノブロットは、大規模なPrP Cから劣化の証拠を示すべきではありません。 PrPの免疫反応性は、主&gt; 20kDaのである必要があります。この分子量よりも小さいバンドは分解産物であることができる。バンドパターンは、基板対の間でほぼ同等でなければなりません。 </li><li>両方の基質中のPrP Cの PK敏感であるべきであるように、100μg/ mlの最終濃度でPKと、各基板10〜25μLを治療し、サーモシェーカーで1時間、37℃で1,000rpmで試料を消化する。 SDS-PAGE 16およびイムノ17で残りのPrPの信号を評価する。PrPのRESを持つ基板は、CERの研究に使用するために適切ではありません。 </li></ol></li></ol> 3.東証エージェントの種子を準備<ol><li>興味の種からTSE感染脳組織を入手し、上記のステップ2.1に記載されている方法を使用して、1×PBS pH7.4の中で10％（w / v）のホモジネートを準備します。 NOTE：種子はまた、中枢神経系外の他のTSE感染組織から製造することができ、しかし、TSE感染末梢組織に由来する種子による脳由来の基質の変換効率はまだ実験的に公開された文献において評価されていない。 </li><li> 100へのホモジネート（S）結果の分量 - 直前まで、-80℃で0.5 mlマイクロチューブ内の300μlの体積およびストアはCERアッセイを実行する。 </li></ol> 4. CERアッセイ注：CERアッセイは、ある種から（「シード」で提供される）のPrP TSEの比較的少量の添加を含む部分的にpHが3.5または7.4のどちらかで変性された別の種からのPrP C（非感染脳「基板」で提供される）の相対的過剰に。拡張振盪インキュベーション期間の後、各反応中のPrP TSEによるPrP Cからのテンプレート駆動型の変換は、PrPの解像度の残りのPK消化および免疫ブロット検出後の濃度測定信号によって評価される。以前にpHは3.5 対 7.4で変性基質中のPrP RESレベルを比較することは、種の壁の尺度を提供する。このアッセイ法の実験の概要を図1に提供される。 <ol><li>ゆっくりと非感染CER基板のペア（アッセイは、以前にpHが3.5と7.4の両方で変性基質の等容量を必要とする）と氷のベッド（約1時間の解凍時間）の上にそれらを配置することによってTSE感染シード·ソリューションを解凍。 </li><li>完全に解凍されると、吸引した後、各貫通基質溶液を追い出す27 Gの注射針を数回に再均質化する。簡単に説明すると約30％最大パワーで10秒間各TSE感染シードソリューションを超音波処理。変換反応の準備中に氷の上にすべてのソリューションを保管してください。 </li><li>ラベルTSEエージェントあたり5個々の低結合、薄壁PCRチューブがテストされている。 </li><li> pHは7.4で調製（a）はCER基板およびpH 3.5で調製（b）はCER基板の95μlに10％のTSE感染シード溶液5μlを添加することにより、これらのチューブ内変換反応を準備します。 <ol><li>各実験サンプルについては、以前にpHは3.5と7.4の両方で変性CER基板対における平行反応を準備します。 </li><li>経験的決定を通じて感染していない脳の基板へのTSE因子の種の比率を最適化します。共通シード：採用基板比は100μlの総反応容量を維持し、1：99μL、2：98μLと5:95μlのがあります。ほとんどのアプリケーションでは、5：95μlのシード基板の体積比が適当であると目に提示されるものであるプロトコルである。 </li></ol></li><li>次のようにTSE因子ごとに3つのコントロールサンプルがテストされているの準備：（a）の入力のPrP RESのコントロールとして95μlの変換バッファに10％のTSE感染シード溶液5μlを追加します。非特異的、非テンプレートの変換のための対照として（b）のpHは3.5、及び（c）は、pH 7.4で調製95μlの基質を5μlの変換バッファを追加する。 </li><li>簡単に言えばよく、種と基板を混合するためにその閉じたPCRチューブに各サンプルをボルテックス。 PCRチューブの蓋の中に閉じ込め、反応混合物の任意のボリュームを削除するには、低速ベンチトップミニ遠心機で瞬間的なパルスで従ってください。 </li><li> PCRチューブを受け入れるために装備ベンチトップサーモシェーカーに個別に標識PCRチューブで負荷サンプル。 24時間、37℃で1000rpmでサンプルを振る。 </li><li>以下（w / v）のおよびPKを、100μg/ mlの最終濃度2％の最終濃度までサルコシルを追加し、振盪。ダイジェストサンプルサーモシェーカーで1時間、37℃で1,000rpmで少なくとも。 NOTE：PrP CからのPK消化のサンプル変換後のエイズへのサルコシルを添加する。播種していないサンプル（ステップ4.5）において偽陽性シグナルは事実上サルコシルを添加することによって除去される。 </li><li> 5分間95℃にSDS-PAGE試料緩衝液、熱のサンプルを追加して、17をイムノブロッティング、続いてSDS-PAGE 16により各サンプルの残りのPrP resとを解決する。 注：前のイムノブロッティングサンプルバッファーの添加と加熱後、サンプルをすぐに処理してもよいし、サンプルは-20℃で保存するか、または-80℃である。ここに提示されたすべてのデータは、ノベックスNuPAGEゲル及び転送システムを使用して生成された。 CERアッセイからの個々のサンプルがサンプル·バッファと、製造者の指示に従って還元剤で処理した。サンプルは、その後95℃で5分間加熱し、各サンプルの30μlを、12％ビス - トリスプレキャストゲルにロードした。 </li><li>対策として、変換効率比（CER）を計算種の壁の。デンシトメ17によるPrPのRESの測定レベルと、pHが3.5試料のそれによってpHが7.4の試料の総密度を分割。パーセントを生産する100で掛けます。 </li><li>与えられたTSE因子を有する種の平均CER±標準偏差を生成するために、独立した実験の反復からのデータをコンパイルします。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+sheep+and+goats+with+transmissible+mink+encephalopathy+virus.">Experimental infection of sheep and goats with transmissible mink encephalopathy virus.</a> Can J Vet Res. 51 (1), 135-144 (1987).</li><li>Rubenstein, R., 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=Immune+surveillance+and+antigen+conformation+determines+humoral+immune+response+to+the+prion+protein+immunogen.">Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.</a> J Neurovirol. 5 (4), 401-413 (1999).</li><li>Castilla, J., 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=Crossing+the+species+barrier+by+PrP(Sc)+replication+in+vitro+generates+unique+infectious+prions.">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134 (5), 757-768 (2008).</li><li>Kubista, M., 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+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27, 95-125 (2006).</li><li>Atarashi, R., Sano, K., Satoh, K., Nishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quaking-induced+conversion:+a+highly+sensitive+assay+for+prion+detection.">Real-time quaking-induced conversion: a highly sensitive assay for prion detection.</a> Prion. 5 (3), 150-153 (2011).</li><li>Ladner-Keay, C. L., Griffith, B. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaking+Alone+Induces+De+Novo+Conversion+of+Recombinant+Prion+Proteins+to+beta-Sheet+Rich+Oligomers+and+Fibrils.">Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to beta-Sheet Rich Oligomers and Fibrils.</a> PLoS One. 9 (6), e98753 (2014).</li><li>Mathiason, C. 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=Susceptibility+of+domestic+cats+to+chronic+wasting+disease.">Susceptibility of domestic cats to chronic wasting disease.</a> J Virol. 87 (4), 1947-1956 (2013).</li><li>Seelig, D. M., 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=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., 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=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

このプロトコルが正常に完了するために、注意が基板準備（ステップ2.1.2）と、変換反応（ステップ4.4.2）用の比率を基板へのシードに使用する非感染脳組織中のPrP Cレベルに支払われるべきである。我々の経験では、脳は、限りのPrP Cは、免疫ブロット法（ 図4）によって存在するように、かなりの期間、死後後CER基板として使用するために抽出することができる...

伝達性海綿状脳症（TSE、プリオン病）は、動物およびヒトの様々な影響を与える長時間のインキュベーション期間が致命的な神経変性疾患の一群である。 TSEの推定上の病原体は、感染、疾患関連したPrP（PrP Cから）の正常な細胞形態のテンプレート駆動変換によって自己増殖可能な宿主プリオンタンパク質（PrP）のミスフォールド異性体から構成されている感染したホスト1の中枢神経系組織内に蓄積フォーム（PrPのTSE）。感染哺乳類のプリオンは、一般的に種間の伝送イベント2を制限する「東証種の壁」の概念に上昇を与えている種特異的な方法、ホスト·ツー·ホストから送信する。プリオン種の壁の生物学的決定要因はよく理解されていない。感染性プリオンTSEとホストのPrP C cとのアミノ酸配列類似性強く変換が行わ3-5かかりますが 、 インビボ 6,7 で観察されたすべてのプリオン伝達事象を説明するには不十分なままであるかどうかに影響を与える。 したがって、TSE種の障壁の特性は、主に動物チャレンジ試験に頼っている：別のプリオンに所定の種からのナイーブ動物を曝露し、伝送効率の指標として、疾患の発症および発病率を生じるインキュベーション時間を測定する。異種の種由来のPrP Cを発現するマウスはまた、試験8のこのタイプのために使用される。トランスジェニックマウスの生産と長引くプリオンバイオアッセイと同様に、動物使用の倫理的配慮に関連するコストは、東証種の壁の実験的調査の障害となっている。 TSEはヒト種の壁の評価は、ヒトPrP Cを発現するように遺伝子操作したマウスに依存しています。これらのマウスは、tに人間のTSEや変更に屈するように長い潜伏期間を必要と彼の迅速な疾患発症9用のヒトPrP C分子 。これらのマウスにおける非ヒトのTSEの潜伏期間は、困難な負の結果の解釈を行う通常のマウスの寿命を越えて延びることができる。非ヒト霊長類は、ヒト種の壁を研究するためのプロキシとして使用されてきたが、これらの研究は、ヒト宿主に進むように、他の動物実験の種類、非ヒト霊長類と同様の課題を正確疾患を再現しない可能性をはらんでいる。 動物のバイオアッセイは、東証への種の感受性を測定するための「ゴールドスタンダード」方式のままであるが、障害物、コストとこれらの生きた動物研究の倫理は、代替案調査を強要している。 PrPのTSEによって播種プロテイナーゼK（PK）耐性状態（PrPの解像度 ）にホストのPrP Cの転換を評価することに基づいて、in vitroアッセイの数は、開発され、TSE種を調査するために使用されているECIES障壁10-12。 インビトロアッセイの例は、無細胞転換アッセイ、タンパク質サイクリック増幅（PMCA）のミスフォールディング、および変換効率率（CER）アッセイ10-14を含む。これらのアッセイのいずれも、自然感染した後、種の壁に関与するアカウント末梢要因を考慮しないが、すべてのTSEのための潜在的に感受性のホストを識別することが有用であり得る。 ここでは、正常な脳ホモジネート由来の2つの変性したPrP C基板をベンチトッププリオン転換反応に使用されるCERアッセイのためのプロトコルは、（ 図1）13 を提示する。 pH7.4で変性した基板中のPrP Cは、種の壁14の非存在下での反応中に播種したPrP TSEによってPrPの解像度に変換することができる。対照的に、pHが3.5で他の基板中のPrP Cの変性は、インキュベーション後のPrP 解像度に変換することができ任意の種からのPrP TSEと、変換のためのコントロールとして機能します。 pH3.5の基板とpH7.4の基板の相対中のPrP resはへのPrP Cの転換の比率は種の壁の尺度を提供する。我々は、CERアッセイは、種々のTSEに実験用マウスの既知の種の壁を予測することが見出されており、慢性消耗病（CWD）にオオツノヒツジ、および他のTSE 14を含む多くの哺乳動物種の種の壁を予測するための努力においてアッセイを使用している15。便利なこの方法論を見つける東証種の壁またはPrP Cから-to-PrPの解像度の変換の評価の迅速なスクリーニングを可能なツールに興味を持って研究者。

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

CERアッセイの使用の成功は、変換反応に使用される基板のペアの品質に大きく依存する。この理由のために、CER基板対を調製する手順に従い、品質管理イムノブロット（ 図2）を実行すべきである。両基板の場合、アッセイ免疫ブロット法によって10〜25μL。 PrP Cからは、各基板において容易に検出可能であるべきとのPrP Cレベルは、2つの間でほぼ同じで?...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

と伝達性海綿状脳症種の障壁を評価する<em&gt;インビトロ</em&gt;プリオンタンパク質変換アッセイ

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K ビュー.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

ビデオ: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

正常なヒト脳内の可溶性および不溶性のPrPオリゴマーの分離

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

膵神経可塑性をシミュレートする。<em&gt;体外</em&gt;デュアルニューロンの可塑性アッセイ

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生児低酸素性虚血性脳症の子豚モデル

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

ラットにおける羊膜内リポ多糖で出生前低酸素性虚血を使用した未熟児のモデル化脳症

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

抗がん剤の特異性を評価します<em&gt;インビトロ</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...