この研究は、公衆衛生サービス助成金AI - 34431（DAHに）アレルギーと国立感染症研究所からとVA医学研究基金（DAHに）によっても支持された。

1。スライドガラスへのレプトスピラの固定<ol><li>レプトスピラinterrogansはクラスBSL2病原体です。生細胞での作業には、手袋、実験室、コートと無菌フードのピペッティング操作などの適切な取り扱いが必要です。 </li><li> 30 ° Cに到達するまで半ばまで約6日間の後期対数増殖期（5 × 10 8細胞/ mlに5 × 10 7の密度）。で、1％ウサギ血清を補充した、EMJH medium11でレプトスピラを拡大</li><li>室温で7分間〜2000 × gで遠心分離によって培養を回収する。 </li><li>吸引により上清を除去し、穏やかにリン酸塩でペレットを再懸濁さは5 × 10 8細胞/ mlの最終濃度にのMgCl 2、pH7.2生理食塩水（PBS）-5 mMのバッファ付き。 </li><li>二ウェルチャンバースライドガラスの各ウェルに細胞懸濁液1 mlを加え、30℃でインキュベート℃を80分間、細胞が付着できるようにする。 </li><li>慎重に吸引して未結合の細胞を含む液体を取り除く。 </li><li> PBS - 5 mMのMgCl 2の2％パラホルムアルデヒドを1ml /ウェルを添加することによりガラススライドに、残りの無傷の細菌を修正。 30℃で40分間インキュベート℃にこれらのスライドは、表面に露出したタンパク質の評価になります。 </li><li>サブ表面タンパク質を評価するコントロールのスライドの場合は、/、100％の氷冷メタノール1mlで固定することにより外膜を透過性。 20分間-20℃でインキュベートする。メタノールは、いくつかの方法で機能する：それは、外膜をpermeabilizes、タンパク質をdenaturates、およびガラススライドに細胞を修正しています。 </li><li>吸引してエージェントを修正削除する。 </li></ol> 2。特異的抗体で標識<ol><li> /ウェルのブロッキングバッファー（Difco社レプトスピラ濃縮EMJH）1 mlを添加することにより、非特異的結合をブロックする。 90分間30℃でインキュベートする。 </li><li>特異抗体（ここで免疫ウサギの血清またはマウスモノクローナル抗体、ポリクローナルウサギOmpL54 10の特定の血清、FlaA1 12、およびOmpL1 13）と、抗体を含まない免疫前ウサギ血清やマウスの禁欲的な液を希釈する（ネガティブコントロールとして利用するとき）にバッファをブロック。各抗体の希釈は、細胞中の抗体価、抗原抗体反応やタンパク質の豊富に応じて経験的に決定する必要があります。通常の範囲は、1:50〜1:600​​である。 </li><li>吸引でブロッキングバッファーを取り除き、希釈した一次抗体を1 ml /ウェルを追加。 </li><li> 1時間、30℃でインキュベートする。 </li><li>吸引して溶液を取り除き、PBS（1ミリリットル/ウェル）を各ウエル3回洗浄する。 </li></ol> 3。レプトスピラの可視化<ol><li>を1ml /ウェルのアレクサ希釈® 488標識二次抗体（ヤギ抗ウサギIgGまたはヤギ抗マウスIgGのいずれか）1:2000と蛍光核酸染色、4&#39;6 -ジアミジノ-2 - フェニルインドール二塩酸塩を（追加するDAPIは）をブロッキングバッファーで0.25μg/ mlの最終濃度に希釈した。このステップでは、それぞれ、抗体の検出結合と抗体の結合に関係なく、すべてのスピロヘータの存在を保証する。 </li><li> ° C 45分間30℃のスライドをインキュベートする。 </li><li>吸引によって液体を除去し、蒸留水（1ml /ウェル）で一度PBSで2回ウェルを洗浄して。 </li><li>チャンバーとスライドガラスと約10分間空気乾燥から粘着ストリップを外します。 </li><li>ゴールド耐フェードマウンティング培地（スライドあたり2 × 20μl）を、24​​ × 50 mmのカバースリップを褪色追加。 </li><li>暗所で室温で一晩インキュベートする。このステップでは、治すためにマウンティング培地（硬化）するために必要です。 </li><li>マニキュアでカバーガラスをシール。 </li><li> DAPI用シアン/青検出フィルタとAlexa Fluor 488のための緑の検出フィルタを用いて蛍光顕微鏡による染色を視覚化する。サンプルの正確な表現がなされていることを確認するには、各サンプル全体チャンバーの面積を評価することを確認してください。 </li><li>イメージング各サンプルの代表的なフィールドでデータを記録します。イメージングのAlexa Fluor 488の蛍光が、すべてのサンプルに対して同一の露光時間を使用する場合。一貫性のある露光時間を使用すると、テストの抗原に対するコントロールと結果のより正確な比較ができるようになります。 </li></ol> 4。代表的な結果： 表面および表面蛋白質に対する抗体を用いたレプトスピラinterrogans細胞表面IFA結果の例​​を図2に示されています。実質的な蛍光が、表面（図2A）公開されている目的の蛋白質のために無傷レプトスピラのサンプルで検出されると、テストは陽性とみなされます。通常、正のテスト（ここで、OmpL54表面露出）で、ほぼ同じ蛍光強度はそのまま、膜透過した細胞（図2A）の両方で観察されるそれは、地下に対する抗体を用いて外膜の完全性のためのネガティブコントロールを含めることが不可欠です。 、好ましくはペリプラズムタンパク質（例えばレプトスピラFlaA1、図2B）。データは外膜が無傷の杜日残っていることを示しているときにのみngの実験で、サブ表面蛋白質に対するすなわち抗体は、インタクトな細胞（図2のように透過処理したサンプルと比較した場合、蛍光または非常に弱い蛍光がない。2B）に反応しない、それは目的のタンパク質（s）があると結論することができます表面が露出。追加の陰性対照として免疫前血清（ポリクローナル抗体の場合）を用いた実験を含めることは、明確なデータが不可欠であり、無傷または膜透過した細胞（図2A、2C）に実質的な蛍光が得られないはず。染色DAPIは陰性の結果が（ないのAlexa Fluor ® 488蛍光）が観察されているときにレプトスピラを可視化する必要があります。ヨウ化プロピジウムで対比すると、DAPIに受け入れ可能な代案です。 明るい蛍光が等しい豊富の他の抗原に対する相対的な単一の抗原ではなく、より少ない表面露出エピトープと認識されている複数の表面露出抗原エピトープが原因である可能性があるため、このメソッドは、定性的ではなく定量的である。したがって、蛍光強度は、そのような別のタンパク質間の無傷対透過性細胞と蛍光ではない強度と同じ抗体の反応性を比較するために使用することができます。この区別は大幅に少ない蛍光を透過性細胞（図2C）に比べて無傷の細胞で観察されている場所、あいまいな結果が得られた場合に重要です。そのような結果はどちらのタンパク質は、（完全な細胞のいくつかの非特異​​的なバックグラウンド染色を持つ）サブ表面の場所にあることを示し、抗体は複数の可能性のある優先的にメタノールの透過性やタンパク質の後に公開されている非ネイティブエピトープへの結合です。局在部位は、別のスピロヘータ、 ボレリアブルグドルフェリ 14のERPとELPタンパク質について説明した。どちらの場合でも、このようなあいまいなIFAの結果は、目的のタンパク質を10表面に露出されているかどうかを判断するためにそのような表面ビオチン化または表面のタンパク質分解のよ ​​うな代替的なアプローチが必要です。 <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="図1"/> レプトスピラinterrogansスピロヘータの表面免疫蛍光アッセイのための図1。フローチャート。最初に、細胞懸濁液の1mlは、2つのウェルチャンバースライドの各ウェルに添加し（各実験のための少なくとも二つのスライド）と付着するレプトスピラインキュベートする。次に、レプトスピラはそのまま外膜（OM）と透過性OMの試料1ミリリットル/よく100％冷メタノールを持つサンプルを/ウェルの2％パラホルムアルデヒド（PFA）の1mlを添加することによりチャンバースライドに固定されています。次に、レプトスピラは、30の地​​下タンパク質（一次抗体の）のためのコントロール抗体で90分間Cと一緒に表面に露出したタンパク質を認識する抗体とインキュベートされています。その後、一次抗体/ウェルのAlexa Fluor ® 488標識二次抗体を1mlとのインキュベーションによって検出されます。最後に、細胞は蛍光顕微鏡により可視化。 <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="図2"/> レプトスピラタンパク質の図2。表面免疫蛍光分析。レプトスピラinterrogansスピロヘータは免疫と免疫前血清でプローブした、と否定的な結果は、スピロヘータの存在を実証するためにDAPI対比染色と対になっていた。緑色蛍光フィルターは、細胞のDNAへのDAPIが結合を検出するために露出タンパク質と青/シアン蛍光フィルターを表面に結合のAlexa Fluor ® 488標識二次抗体を検出するために使用されていました。 A）レプトスピラは、表面露出タンパク質、OmpL54 10を認識する抗体でプローブ。 B）細胞は、細胞周辺地下タンパク質、FlaA1（ネガティブコントロール）に対する抗体でプローブ。 C）細胞は、レプトスピラポーリン、OmpL1に対する抗体でプローブ。レプトスピラのウサギの血清の結合は、のAlexa Fluor ® 488ヤギ抗ウサギIgG抗体の断片を検出した。すべての画像は4秒長時間露光後に撮影されています。

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利害の衝突は宣言されません。

私達の表面IFA技術は、様々なborrelial 15、16、レプトスピラ12、17のタンパク質の表面の露出を示すために使用されているものと似ています。ここで説明する表面IFAの方法の詳細は、スライドガラスに付着しレプトスピラ細胞の傾向を利用しながら外膜の完全性を維持する目的で、細胞の操作を最小限に抑えるように設計されています。方法は、外膜安定化バッファー（PBS 5 mMのM...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>二つウェルチャンバースライドガラス</td><td>ラボテック</td><td> 177380 </td><td></td></tr><tr><td>ウサギ血清</td><td>ロックランドのImmunochemicals </td><td> D209 - 00から0100 </td><td></td></tr><tr><td> レプトスピラ濃縮EMJH </td><td> BD Difco社</td><td> 279510 </td><td></td></tr><tr><td>のAlexa Fluor ® 488ヤギ抗ウサギIgG（H + L） </td><td> Invitrogen社/ Molecular Probes社</td><td> - 11034 </td><td></td></tr><tr><td>のAlexa Fluor ® 488ヤギ抗マウスIgG（H + L） </td><td> Invitrogen社/ Molecular Probes社</td><td> - 11029 </td><td></td></tr><tr><td> 4&#39;6 -ジアミジノ-2 - フェニルインドール二塩酸塩（DAPI） </td><td> Invitrogen社/ Molecular Probes社</td><td> D1306 </td><td></td></tr><tr><td>ゴールドアンチフェードマウンティングメディウムを延長</td><td> Invitrogen社/ Molecular Probes社</td><td> P36930 </td><td>使用前に室温に平衡化</td></tr><tr><td> Fisherfinestプレミアムカバーガラス</td><td>フィッシャー</td><td> 12-544-14 </td><td></td></tr><tr><td>蛍光顕微鏡</td><td>ツァイス</td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

細菌の表面タンパク質は、宿主細胞と直接接触し、環境1からの栄養素の摂取に関与している。このような理由から、細胞内局在は、細菌タンパク質の機能的役割についての洞察を提供することができます。細菌タンパク質の表面の局在は、病原性のメカニズムに関与病原因子の同定に向けた重要なステップです。 レプトスピラ膜2-5を分画するための方法はそのようなリポタンパク質対貫通OMPSとして外膜タンパク質（OMPS）、特定のクラスに対して選択的であるため、誤分類する可能性があります。この可能性は、構造的な違いと、それらがどのように外膜に関連付けられているためです。リポタンパク質はN末端 ​​脂質部分（3つの脂肪酸）と脂質二重層のリン脂質6,7の間に疎水性相互作用を介して膜に関連付けられています。対照的に、膜貫通OMPSは通常、樽状の構造8,9に配置されたβ-シートの両親媒性で脂質二重層に組み込まれています。さらに、外膜のタンパク質の存在は、必ずしも蛋白質またはそのドメインが表面に露出していることを保証するものではありません。スピロヘータ外側の膜は壊れやすくなるため、サブ表面のタンパク質の細胞とインクルージョンの穏やかな操作を含む方法を必要とすることが知られている外膜の完全性を評価するために制御します。 ここで、我々が直接そのままレプトスピラ上のタンパク質の表面の露出を評価するために蛍光抗体法（IFA）法を提案する。この方法は、抗原特異的抗体によるレプトスピラ表面タンパク質の認識に基づいています。ここで、OmpL54 10の特異的な抗体は、ネイティブ、表面露出エピトープにaftero結合detetctedされています。無傷対透過性細胞への抗体の反応性の比較は、細胞分布の評価を可能にし、蛋白質は、レプトスピラ表面上に選択的に存在するかどうか。外膜の完全性、好ましくは、ペリプラズムに位置する1つまたは複数の地下タンパク質、抗体を用いて評価すべきである。 表面IFA法は、特異的抗体が利用可能な任意のレプトスピラタンパク質の表面の露出を分析するために使用することができます。法の有用性と限界の両方が採用さ抗体がネイティブエピトープに結合することができるかどうかによって異なります。抗体はしばしば組換えタンパク質、天然のエピトープに対して提起されているので、表面に露出したタンパク質が認識されない場合があります。それにもかかわらず、表面IFA法は無傷細菌の表面の成分を研究するための貴重なツールです。この方法は、レプトスピラだけでなく、他のスピロヘータとグラム陰性菌に対してだけでなく、適用することができます。 OMPSの表面露出に関する強力な結論のために、いくつかの細胞の局在の方法を含む包括的なアプローチは、10をお勧めします。

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

レプトスピラタンパク質の表面露出を評価するために効率的な方法が記載されている。方法は、特にレプトスピラ細胞の脆弱な外膜の崩壊を回避するために設計されています。この手法にはいくつかのネガティブコントロールの雇用は、外膜と抗体反応の特異性の整合性を評価する必要があります。

レプトスピラ表面に露出したタンパク質の免疫蛍光アッセイ

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

Research

JoVE Journal

Immunology and Infection

15.7K ビュー.  University of California, Los Angeles. レプトスピラタンパク質の表面露出を評価するために効率的な方法が記載されている。方法は、特にレプトスピラ細胞の脆弱な外膜の崩壊を回避するために設計されています。この手法にはいくつかのネガティブコントロールの雇用は、外膜と抗体反応の特異性の整合性を評価する必要があります。

ビデオ: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

TCRとそれに関連するシグナル伝達タンパク質のリアルタイムの同時イメージングのための全反射顕微鏡法

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

免疫・感染学

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

マウスノロウイルスのプラークアッセイ

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

蛍光タグ付きワクシニアウイルスタンパク質の効率的な生成のための方法論

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

ファージフェノミクス：小説のウイルスタンパク質を特徴づけるために、生理的アプローチ

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

アレナウイルス - 細胞付着の測定のための高感度アッセイ

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量的マイクロ中和アッセイの最適化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

組換えタンパク質の部位特異的糖体外のシステインのチオール基を対象と

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Methods Article

レプトスピラ表面に露出したタンパク質の免疫蛍光アッセイ