我々は、原稿の批判的な読みのために博士はヴェスパーのFeマリーラモスに感謝したいと思います。この作業は、研究資源のためのナショナルセンター（P20 RR0115635-6、C06 RR17417-01とG20RR024001）と国立神経疾患研究所およびストローク（2R01 NS052609）によってサポートされていました。

1。機器の準備<ol><li> Misonix 3000または37の温度を一定に保つためにサーモエレクトロンNeslab EX-7水槽（ニューイントン、ニューハンプシャー州）に接続Misonix 4000ソニケーター（ファーミングデール、ニューヨーク州）℃を使用するサーモサイエンティフィック（ウォルサム、マサチューセッツ州）から入手したドーム型のキャップ付き200μlの薄壁PCRチューブストリップのサンプルを超音波洗浄します。 </li><li>真新しいソニケーターは連続運転9の&quot;慣らし&quot;期間を必要とします。 2ヶ月のブレークイン期間は10に設定されて振幅レベルを持つ40秒の超音波処理バーストと10分インキュベーションサイクルで構成されています。一つはチタンプレートホーン（ 図1、パネルB）の表面全体に浸食を確認する必要があります。循環水は、チタンマイクロホーンの浸食による白濁を見ていきます。毎日この水を交換してください。 </li><li> PrP Scの増幅は、マイクロプレートホーンを通して変化します。最高のPrP Scの増幅効率が内に取得することができるマイクロホーンの中心から5cmの半径（ 図1、パネルCを参照してください）。 </li><li> PMCAの一周は144サイクルで構成されています。各サイクルは、5秒の超音波処理し、10分間のインキュベーションで構成されています。これは大体PMCAのラウンドごとに24時間に負わせるでしょう。 </li><li>超音波破砕機のコントロールパネルに表示されて超音波処理の電力出力は、超音波破砕機のラックと水浴から循環水の温度に存在するPCRチューブの数によって異なります。水は37℃で平衡化されていることを確認し、超音波処理装置のチューブラック（ 図1、パネルC）の30％以上を記入しない。マイクロプレートホーンを通してPCRチューブを広げた。 PCRチューブストリップ間の空の空間の少なくとも1つの行を持っている（ 図1、パネルC）ストリップあたり一緒に接続されていない4つ以上のチューブを許す。 </li><li>超音波処理の強度は、成功したプリオン株の増幅のために重要です。超音波処理しながら、パワー出力を持つように振幅を調整155〜170ワットの間の範囲。私たちの研究室では、電力レベルは、それぞれMisonix 3000およびMisonix 4000の6と7に設定されています。 </li><li>硬水の汚れを蓄積しないように水浴中で蒸留水を使用します。毎週この水を交換してください。インキュベーションおよび超音波処理サイクル中に、凝縮水は、マイクロプレートホーンのふたの内側に発生します。 図1に示すように、結露を避けるために、パネルA、超音波破砕機の筐体ボックスの上部に小さな水槽の熱·アタッチ·パッド</li></ol> 2。サンプルの準備<ol><li>動物を含むすべての手順は、クレイトン大学施設内動物のケアと使用委員会によって承認され、実験動物の管理と使用に関する指針に準拠した。任意の組織採取する前に、動物を麻酔とtranscardially氷冷リン酸を用いて灌流しなければなりませんが、脳から血液を除去するとの阻害を回避するために、pH7.4の5mMのEDTAとの緩衝生理食塩水血液7によるPMCA反応。無菌技術を用いて急速に動物組織を採取し、それぞれのプリオン株のための専用の解剖ツールを持っている。切開した後、1.5 mlのマイクロチューブに組織を置き、フラッシュがドライアイスの上に凍結し、-80℃で保管して℃を均質化するまで。 </li><li> PMCA基板（PrP Cの ）溶液を調製し、Tenbroeck組織グラインダーを用いて氷冷変換バッファで10％重量/体積（w / v）溶液（パート3を参照）に感染ハムスター脳（国連BH）ホモジナイズする。ホモジナイズしながら、溶液の過剰な泡立ちを避けるために、グラインダーの内側に空気を入れないように注意してください。 30秒、アリコートを1.5ml微量遠心チューブの上清を500×gで遠心分離することによって解決策を明らかにする。 -80℃で保存さらなる使用まで。 </li><li> PMCAシード（PrP Scの）溶液を調製し、氷冷ウォートを10％（w / v）の溶液中に感染性物質（脳、脾臓、リンパ節、他の組織または汚染された土壌）をホモジナイズrとアリコート0.6ミリリットルマイクロチューブ内の溶液。 -80℃で保存さらなる使用まで。 </li></ol> 3。変換バッファの準備<ol><li> 50mlのメスフラスコ（結果はトリトンX-100の1％の最終濃度である）（;シュタインハイム、ドイツのSigma-Aldrich社）でのTriton X-100の0.5093グラムの重量を量る。 </li><li> 1コンプリートプロテアーゼインヒビタータブレット（;マンハイム、ドイツロシュ·ダイアグノスティックス社）を追加します。 </li><li> EDTAの0.0931グラム（これはEDTAの5mMの最終濃度になります）（;フィリップスバーグ、ニュージャージー州マリンクロット·ベイカー社）を追加します。 </li><li> 1N NaOHでpHを7.4に調整してください。 </li><li>ダルベッコのリン酸50mlに音量を調節するには（;マナッサスメディアテック株式会社）（DPBS）緩衝生理食塩水。 </li><li>真空ろ過装置と0.45μmのニトロセルロース膜を通して溶液をフィルタリングします。変換バッファが一箇月以内に4℃で保存することができます。 </li></ol> 4。 PMCAを使用したプリオン株の増幅<ol><li>PCRチューブに1:20の比率（ すなわちミックス PMCAシードの5μlとPMCA基板の95μl）をPMCAで基板にPMCAシードを希釈します。 -80℃で15μlのアリコート°unsonicated制御のためのCを削除して保存すること。このunsonicatedコントロールはウエスタンブロット（WB）の分析に続いてプロテイナーゼK（PK）消化によりPrP Scの増幅を定量化するために使用されます。追加unsonicated制御はPMCA反応の持続時間のために37℃で第二15μlのアリコートを配置することによって生成することができます。 3回繰り返しの最小値は統計上の目的のために必要です。 </li><li>主題図1に示すように、PMCAの1ラウンドにサンプルの残り（70μl）を、パネルA.シェイクチューブPCRチューブのドーム型キャップの結露を避けるために、すべての5時間。 </li><li>サンプルのチューブがhomog確保するために、任意の手順の後に開く前に（溶液はドーム形キャップに行っていないことに注意して）スピンダウンし、軽くボルテックスして下さいeneityと潜在的な汚染を避ける。 </li><li>短い潜伏期間株（ すなわち 、HY、TME、HaCWD、または263K）のシリアルPMCA（sPMCA）では、新鮮な未感染の95μLにラウンド1からの超音波処理したサンプルの5μlを転送することにより、1:20の割合で超音波処理した材料を希釈脳ホモジネート（ 図2、パネルB）。 </li><li>潜伏期間が長い株（ すなわち DY TME、139H、22CH、22AH、またはME7H）のsPMCAについては、感染していない新鮮な50μlのにラウンド1からの超音波処理したサンプル50μlを転送することにより、1:1の割合で超音波処理した材料を希釈脳ホモジネート（ 図2、パネルB）。 </li><li>以前に希釈混合超音波処理（ステップ4.4または4.5）から215μlのアリコートを取り出して、-80℃で、それらのいずれかを格納し、37℃と他の℃（PMCAラウンド2用unsonicated制御ソリューション）。 PMCAラウンド2への残党を施す。 </li><li>この手順は、無限に繰り返すことができます。私たちの研究室では、我々ひずみ特性は変わらずで、PMCA 15ラウンドまで行ってきた。 </li><li> PKはサンプルを消化。典型的なWBのために、サンプルを5μl（この溶液は、40μg/ mlの最終濃度のPKを持つことになります）にPKを80μg/ mlの5μlを混合し、37℃で1時間インキュベートする。ゲルローディング緩衝液10μlを追加します。 10分間この溶液を沸騰。解決策は冷えるとゲルに10μlをロードしましょう​​。 </li><li>増幅の成功を計算するには、どちらかにそれらを比較することによって増幅されたPrP Scの量を推定するために、PK-消化サンプル、unsonicatedコントロール20,21 図2、パネルCまたは組換えPK-消化されていない既知の量に上のWB分析を実行プリオン。 </li></ol> 5。クロスコンタミネーションの回避重要なステップは、クロスコンタミネーション及びプロテアーゼ耐性製品の de novo形成に起因する偽陽性の発生を最小限に抑えるように注意する必要があります。 </P&gt; <ol><li>各プリオン株のためのサンプル収集のための解剖ツール専用セット（ すなわち鉗子、ピンセット、はさみ）を使用します。 </li><li> PMCA基板として使用されるために感染していない脳を均質化する前に、1％の漂白剤でピペットを拭いてください。 10分間1％の漂白剤で超音波処理装置のPCRチューブラック、組織ホモジナイザー、及びPCRチューブの収納ラックを浸して、蒸留水で十分に洗い流してください。 </li><li>新しいサンプルをホモジナイズするたびに新鮮なVersi-ドライラボ浸すと作業領域をカバーする、新しいPMCA実験が準備されるとき、またはsPMCAのラウンドの間に小分け。 </li><li>新しい手袋に新しいサンプルが（sPMCAラウンド間のアリコートを転送する場合は特に）に処理されるたびに使用してください。 </li><li>各PMCAサイクルの短い超音波処理のバーストを使用しています。 10分間のインキュベーションを組み合わせる5秒の超音波処理のサイクルは 、de novo の PrP Sc 1,20,21,23,24を生成せずにプリオンを増幅するのに十分である。他の研究室が示したように、増加超音波処理時間は、超音波の振幅を増加させ、PMCAのサイクル数を拡張することはde novoの PrP Scの形成2の確率を増加させる。 </li></ol>

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感染性プリオンタンパク質を増幅するという課題は、長い潜伏期間および in vivo実験での経費です。 PMCA技術は感染性プリオンエージェントを増幅するための費用効果的な手段です。いくつかの研究室では、正確に試験管 7、9、12、14、19,24 のプリオン株を増幅するPMCAの能力を確認しています。 プリオン病は、種間で送信することができ...

<table border="1"><tbody><tr><td>試薬/機器</td><td>メーカー</td><td>猫。数</td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeckティッシュグラインダー</td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7ウォーターバス</td><td>サーモエレクトロン</td><td> Neslab EX-7 </td></tr><tr><td> 0.2ミリリットルのPCRチューブストリップ</td><td>サーモサイエンティフィック</td><td> AB-0451 </td></tr><tr><td>トリトンX-100 </td><td>シグマアルドリッチ</td><td> T9284-100ML </td></tr><tr><td>コンプリートプロテアーゼインヒビター</td><td>ロッシュ</td><td> 11 697 498 001 </td></tr><tr><td> EDTAを</td><td> JTベーカー</td><td> 4040から00 </td></tr><tr><td> DPBS </td><td>マリンクロット·ベイカーメディアテック</td><td> 21から031-CV </td></tr><tr><td> Versi-ドライラボSoakers </td><td>フィッシャー·サイエンティフィック</td><td> 14 206 28 </td></tr><tr><td> REPTIサームヒーター</td><td>動物園メッド·ラボラトリーズ社</td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

プリオンは、動物とヒト9,18で必然的に致命的な伝達性 ​​海綿状脳症（TSE）を引き起こす感染性病原体である。プリオンタンパク質は、2つの異なるアイソフォームは、非感染性宿主でエンコードされたタンパク質（PrP C）および感染質（PrP Sc）は、PrP Cの8の異常に折り畳まれたアイソフォームを持っています。 プリオン剤での作業の課題の1つは、ホスト13接種後の臨床徴候の開発の前に長い潜伏期間があります。これは、伝統的に長く、高価な動物のバイオアッセイ研究を命じた。さらに、PrP Scの生化学的および生物物理学的特性はあまり彼らの異常なコンフォメーションと凝集状態に起因する特徴がある。 PrP Sc は 試験管 14 内 の PrP ScへのPrP Cの種子変換することができます。 PMCAはアドバを取るのin vitro手法ですのPrP CとPrP Scの種子19微量の過剰な量を含んでいるシステムから、加速度的に、PrP Scのを大量に生成するために超音波処理およびインキュベーションサイクルを使用してこの能力のntage。この手法は、効果的にプリオン株の干渉をエミュレートするためには、PrP CからPrP Scの変換の種および株特異性を再現するために、感染した組織、体液、および環境試料6,7,16からPrP Scの非常に低いレベルを増幅することが証明されています23。 一貫性のある結果を生成し、それらの結果を定量化、汚染を最小限にするための提言を含め、本稿では詳細PMCAプロトコルを、。我々はまた、感染性プリオン株、プリオン株の干渉、及び環境中のプリオンの検出の発生と特性化など、いくつかのPMCAアプリケーションを、議論する。

タンパク質のミスフォールディングサイクリック増幅（PMCA）はin vitroで 7 におけるPrP Scは、12、14、19、24を増幅するために使用されます。 図3に示すように、成功したPrP Scの増幅が PK耐性プリオンタンパク質（ハムスター由来のプリオン株の19および30kDaの間の移行）のウエスタンブロットのバンド強度の増加によって示されている<str...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

タンパク質のミスフォールディングサイクリック増幅（PMCA）はプリオン変換とひずみと種の壁の研究のためのin vitroアッセイである。また、プリオン検出アッセイとして使用することができます。

プリオンのタンパク質のミスフォールディングサイクリック増幅

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K ビュー.  University of Nebraska at Lincoln. タンパク質のミスフォールディングサイクリック増幅（PMCA）はプリオン変換とひずみと種の壁の研究のためのin vitroアッセイである。また、プリオン検出アッセイとして使用することができます。

ビデオ: Protein Misfolding Cyclic Amplification of Prions

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

免疫・感染学

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

の種特異的検出のためのLAMP法（LAMP）アッセイ<em&gt;アイメリア</em&gt;鶏に感染する

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

の検出のための凍結乾燥LAMP法試薬の開発<em&gt; Q熱リケッチア</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

解離性個体の親和性の浄化 (BiCAP) によって多蛋白質シグナリング複合体

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

自動行動分析の大規模なc. の elegans集団による視野が広い追跡プラットフォーム

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

世代、増幅、および組換え呼吸器合胞体ウイルスの滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

チラピア湖ウイルスの特異的かつ迅速な検出のための逆転写ループ媒介等熱増幅(RT-LAMP)アッセイ

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.