1。 Nicotiana benthamianaの植物からウイルス接種とVLPの精製<ol><li> ポテトウイルス X（PVX）ベース運ぶベクタープラスミドから頂いた、T7-RNA転写産物を生成MRFV野生型（WT）およびCys変異コートタンパク質（CP）遺伝子12、AmbionのT7-mMessage mMachineキットを使用しています。 </li><li>各T7転写反応には、Nの2の完全展開葉に接種10μlの反応と、25で光（25,000-30,000ルクス）で16時間、60％の湿度で、温室内で10日間インキュベートbenthamianaの植物を20℃と暗8時間℃の</li><li>ハーベストンN. benthamianaのウイルスに感染した葉は10日後に接種（DPI）は、氷の上で植物組織（50グラム）と場所を量る。 </li><li> 0.5Mのクエン酸ナトリウム緩衝液（pH 5.5）（植物組織1グラムあたり緩衝液2mlを使用）の冷却溶液を100mlの存在下でブレンダーで植物材料をホモジナイズする。 </li><li> cheeseclの3つの層を通ってホモジネートをフィルタリングOTH、30分間27000×gで遠心分離することにより明らかにする。冷やしたメスシリンダーに上清を移し、体積を測定した後、冷却した無菌のビーカーに移す。ペレットを廃棄します。 </li><li> 、10％ポリエチレングリコール8000（PEG 8000）を添加することにより上清を処理し、4℃で15分間撹拌し、4℃でさらに45分間インキュベート℃のウイルス様粒子（VLP）-PEGのマトリックスを沈殿させる。 </li><li> 4℃で20分間27000×gで遠心し、上清を除去します。 </li><li> pH5.5のTriton X-100を2％含む、0.05Mクエン酸ナトリウム緩衝液8mlを加えることによってペレットを処理します。渦巻遠心管はよくペレットを解放します。チルド滅菌ビーカーにペレットとバッファを転送します。同じ緩衝液2mlで直ちにチューブをすすぎ、ビーカーに加える。ペレットは（一般的に1時間）に溶解するまで4℃で懸濁液をかき混ぜる。 </li><li> 27000×gで5分間遠心分離することにより明らかにし、ペレットを廃棄し、process 4℃で2時間14万xgで遠心分離により上清℃、 </li><li> 1ミリリットルの0.05Mクエン酸ナトリウム緩衝液（pH 5.5）でペレットを再懸濁し、0.05Mのクエン酸ナトリウム緩衝液（pH 5.5）で行われた百分の10から40のショ糖勾配の上に懸濁液を配置します。 </li><li> 4℃14万xgで3時間勾配を遠心</li><li>遠心管の上に光を当て、管の上部から4 cmの光散乱上部のバンドを集めて、4℃で0.05Mのクエン酸ナトリウム緩衝液（pH 5.5）、14万×gで3時間遠心機で薄める℃の</li><li> 6ヶ月まで4℃で滅菌水、店舗の0.5mlにペレットを再懸濁します。 </li><li> Bradfordタンパク質アッセイを行うことにより、VLPの濃度を定量する。 VLPの収量は植物組織の1〜3μg/ gである。 </li></ol> 2。 VLPのフルオレセイン-5 - マレイミド標識反応<ol><li> 50mMリン酸ナトリウムphosphatにおけるフルオレセイン-5 - マレイミド（427.37グラム/モル）の1mM溶液を調製電子緩衝液（pH7.0）、1 mMのEDTA。ボルテックスを用いて混和し完全に溶解させていることを確認します。アルミ箔を使用して光からチューブを保護します。 </li><li>ステップ1とミックスで生産のVLPにフルオレセイン-5 - マレイミドを追加します。スルフヒドリル基のモル量のフルオレセイン-5 - マレイミドの25倍モル過剰に使用します。一般的に、1μg/μLのとフルオレセイン-5 - マレイミド溶液60μlの濃度でVLPの30μlを使用すると、許容可能な結果を​​生成します。 </li><li> 4℃の室温で、または一晩で2時間反応をインキュベート</li><li> 50mMの最終濃度までジチオスレイトール（DTT）を加えて反応を終了します。 </li><li>削除する非反応は室温で1500×gで2分間脱塩スピンカラム（サーモサイエンティフィック社は、ロックフォードIL）と遠心分離を用いてフルオレセイン。 </li><li> SDS-PAGE分析では、各反応液10μlに2×SDS-PAGEのLaemmliサンプルバッファー10μlを加える。 4℃で光から保護標識タンパク質を格納するまでの1ヶ月間、Cまたは最大3ヶ月まで-20°Cで単回使用アリコートインチ</li></ol> 3。ビオチン定款のラベリング反応と定量<ol><li>水からのリン酸緩衝食塩水（PBS）、ステップ1で作製したVLPのpH7.0に緩衝液交換を実行するために脱塩スピンカラム（サーモサイエンティフィック社は、ロックフォード、IL）を使用します。 </li><li>ピペッティングによりマレイミド-PEG2-ビオチンとミックスを無計量でのPBS 190μlを添加してマレイミド-PEG2-ビオチンの20mMストック溶液を調製します。 </li><li> VLPおよびミックスにマレイミド-PEG2-ビオチンを追加します。 VLPのソリューション≤2 mg / mlのための試薬の10倍モル過剰に使用します。 </li><li> 4時間室温で反応をインキュベートする。 </li><li>マレイミド-PEG2-ビオチンは、室温で1500×gで2分間脱塩スピンカラム（サーモサイエンティフィック社は、ロックフォードIL）と遠心分離法を用いて非反応削除します。 </li><li>ピアースビオチン定量キット（サーモサイエンティフィック）とFOを使用したVLPのモル当たりビオチンのモル数を決定するメーカーの指示に従ってllowing。 </li></ol> 4。 Cysは2-VLPおよびFのペプチドの架橋<ol><li> （2-VLPはCys）は、ステップ2で、架橋のために利用可能なシステイン残基をもたらしVLPは、次のステップで使用されます。ジメチルスルホキシドの680μlのNHS-PEG4-マレイミド架橋剤10mgを溶解させることにより、新鮮な架橋原液（28.63 mM）を準備します。 </li><li> 0.1 mMの濃度で、結合緩衝液50μlの17（F）-アミノ酸長のペプチド（LLPNMOKDKEACAKAPL）（1mM EDTAを1×PBS緩衝液、pH7.2）に溶解する。 </li><li> 1mMの最終濃度（ペプチド溶液の0.1mMのために、10倍モル過剰である）で溶解したペプチド（タンパク質-NH2）50μlに架橋剤の4μlを添加する。 </li><li> 4℃で2時間反応をインキュベート</li><li> 1500 xgで遠心分離により、結合緩衝液（1mM EDTAを1×PBS、pH 7.2）で平衡化した脱塩カラム（サーモサイエンティフィック社は、ロックフォード、IL）を用いて架橋されたペプチドを精製する室温で1分間。 </li><li> VLPは（タンパク質-SH）と反応し、VLPおよびペプチドの間の分子量の差に関与チオールと活性アミンの数によって決定されるモル比で架橋されたペプチド（タンパク質-NH2）を組み合わせた反応混合物を設定します。 </li><li> 4℃で2時間室温で反応混合物をインキュベート</li><li>ウェスタンブロット分析は、Laemmli緩衝液、10分間沸騰させ、結果として得られると共役製品1:1に混ぜて10〜20％Tris-グリシンゲル（Invitrogen）プレキャストのSDS-PAGEに進み、上にエレクトロ続くニトロセルロース膜（Invitrogen社）。 1×PBS緩衝液（pH7.2）、0.05％のTween-20（PBS-Tween）でかつ適切なホスファターゼ標識二次抗体、ペプチド特異的抗体を用いて、5％スキムミルク粉末及びプローブを含有する膜をブロックします。 </li></ol>

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ここで紹介する方法は、植物生産VLPの表面上だけでなく、他のタンパク質複合体上に存在する反応性のシステインの非常に高感度で迅速な分析を可能にします。マレイミドは安定なチオエーテル結合を形成する遊離スルフヒドリル含有分子と反応チオールに特異的な試薬である。この方法は、他のアミノ酸との相互作用に関与していないスルフヒドリル基と反応するマレイミドの特異性を利?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>薄肉、超クリアチューブ</td><td>ベックマン</td><td> 344059 </td><td></td></tr><tr><td> mMESSAGE mMACHINE T7キット</td><td>ライフTecnologies </td><td> AM1344M </td><td></td></tr><tr><td>フルオレセイン-5 - マレイミド</td><td>サーモサイエンティフィックライフテクノロジーズ</td><td> 46130 F150 </td><td> 46130は、F150との順序の代用外です</td></tr><tr><td>ピアースビオチンの定量キット</td><td>サーモサイエンティフィック</td><td> 28005 </td><td></td></tr><tr><td> EZ-Linkのマレイミド-PEG2-ビオチン、無計量フォーマット</td><td>サーモサイエンティフィック</td><td> 21901 </td><td></td></tr><tr><td> SM（PEG）のn個の架橋剤</td><td>サーモサイエンティフィック</td><td> 22107 </td><td></td></tr><tr><td> 10から20パーセントトリス - グリシンゲル</td><td>インビトロジェン</td><td> EC61352 </td><td></td></tr><tr><td> Laemmli緩衝液</td><td>バイオ·ラッド</td><td> 1610737 </td><td></td></tr><tr><td>トリスグリシンSDSランニングバッファー</td><td>インビトロジェン</td><td> LC2675 </td><td></td></tr><tr><td>トリスグリシン転送バッファ</td><td>インビトロジェン</td><td> LC3675 </td><td></td></tr><tr><td>ニトロセルロース膜フィルターペーパーサンドイッチ</td><td>インビトロジェン</td><td> LC2001 </td><td></td></tr><tr><td>ウサギIgGに対する抗体を精製ホスファターゼ標識アフィニティ</td><td> Kirkegaardとペリーラボラトリーズ</td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIPホスファターゼ基質</td><td> Kirkegaardとペリーラボラトリーズ</td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

ウイルスの特性と物理化学的および物理的特性を模倣して、利用することは、世界の最も緊急の課題のいくつかの解決策を提供する可能性を秘めています。薄手の範囲およびそれらの魅力的な特性と相まってウイルスの種類は潜在的にウイルスベースの技術でのアプリケーション用に無限の機会を与える。ウイルスは温度とpH幅広い条件下で離散形状や大きさ、対称性の特異性、多原子価、安定した特性を有する粒子に自己組織化する能力を持っています。驚くことではないが、プロパティのような顕著な範囲で、ウイルスが生体材料9、ワクチン14、15、電子材料、化学ツール、および分子の電子コンテナ4、5、10、11、16、18、12で使用するために提案されている。 ナノテクノロジーでウイルスを利用するためには、それらが新たな機能を付与するために彼らの自然なフォームから変更する必要があります。この挑戦的なPRocessはウイルスゲノムの遺伝子改変を含むいくつかのメカニズムを介して行われ、化学的にウイルス粒子の反応性基を8に外国又は所望の分子を結合することができます。ウイルスを変更する機能は、主にウイルスの物理化学的および物理的特性に依存します。また、遺伝的または物理化学的修飾は、悪影響ウイルスネイティブ構造とウイルス機能に影響を与えずに実行する必要があります。 トウモロコシrayadoフィノウイルス （MRFV）コートタンパク質は、タンパク質-タンパク質によって安定化されて安定しており、空のVLPを生産する大腸菌で自己集合相互作用とそのウイルスベースの技術アプリケーション8で使用することができます。タバコ工場で生産VLPはペプチドの様々な共有結合13に表示することが可能な足場として検討した。ここで、我々は、1）の手順について説明しMODIFIできますウイルスキャプシドの溶剤アクセスシステインの判別陽イオンと、2）修正されたカプシドにペプチドをバイオコンジュゲート。ネイティブまたはmutationally -挿入されたアミノ酸残基および標準カップリング技術を使用することにより、多種多様な材料は、 ブロムモザイクウイルス 3、 カーネーション斑紋ウイルス 12、 ササゲ退緑斑紋ウイルス 6、 タバコモザイクなどの植物ウイルスの表面に表示されているウイルス 17、 カブ黄斑モザイクウイルス 1、MRFV 13。

Nの変異MRFVコートタンパク質（CP）遺伝子の一過性発現PVXベースのベクター生産VLPにおけるbenthamianaの植物は、 図1に記載されている。修正MRFVコートタンパク質遺伝子をPCRで増幅された後、PVX-basedベクター、pP2C2S 2、（D. Baulcombeの贈り物、セインズベリー研究所、ノーリッチ、イギリス）で重複サブゲノムCPのプロモーターの転写制御下に置かれた。 に...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

のシステイン残基のチオール基の溶媒アクセス可能性を分析する手法<em&gt;トウモロコシrayadoフィノウイルス</emペプチド架橋反応が続く&gt;（MRFV）ウイルス様粒子（VLP）が記述されている。メソッドは、特定の反応のためのターゲットに設定することはできないVLPの表面上の複数の化学物質グループの可用性を活用しています。

上のシステイン残基の溶媒アクセシビリティの分析<em&gt;トウモロコシrayadoフィノウイルス</emで生産&gt;ウイルス様粒子<em&gt; Nicotiana benthamianaの</emVLPは、ペプチドの&gt;植物と架橋

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

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Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K ビュー.  Agricultural Research Service, United States Department of Agriculture. のシステイン残基のチオール基の溶媒アクセス可能性を分析する手法トウモロコシrayadoフィノウイルス（MRFV）ウイルス様粒子（VLP）が記述されている。メソッドは、特定の反応のためのターゲットに設定することはできないVLPの表面上の複数の化学物質グループの可用性を活用しています。

ビデオ: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

&#39;シングルウイルスゲノミクス&#39;を使用してシングルビリオンの単離とゲノム解析

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

免疫・感染学

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

プロデュース上皮損傷の解析<em&gt;赤痢アメーバ</em&gt;感染症

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronicミニゲノムを含むウイルス様粒子とバイオセーフティレベル2の条件の下ではエボラウイルスのライフサイクルのモデル化

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

新しいウイルスのゲノムを発見する感染葉からの RNA の解析とウイルス粒子粗抽出 DNA の結合解析

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

FcγR 架橋への応答におけるマクロファージの活性酸素産生の流れフローサイト メーターによる測定

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...