我々はFCU1遺伝子の配列を我々に提供するための博士フィリップのERBSを感謝したいと思います。この作品は、NIHの助成金AI 26649によってサポートされていました。

1。 赤痢アメーバのFCU負の選択システム<ol><li>ベクトル制御ライン（HM1 / pKT3M）と実験ライン（HM1/pKT3M-FCU1）は、前述のテクニックの1,2により生成された。シードはT - 25フラスコにストックチューブから研究対象の行とTYI - S - 33 3培地で適切な抗生物質（12μg/ mlのG418）を追加することで選択を維持する。フラスコは37℃で成長の16〜18時間後に70から80パーセントコンフルエントになるはず℃に</li><li>暖かいTYI - S - 33培地中で5 - フルオロシトシン（5 - FC、シグマ）の新鮮な50mmのストック溶液を準備します。完全に5 - FCを溶解するのに数分間、厳密に渦。 0.22μmのフィルターを通すことでストック溶液をろ過滅菌します。 </li><li> 3〜5分間氷上にフラスコを置き、室温で5分間200xgで遠心分離により細胞を収集することによって収穫のトロフォゾイト。新鮮TYI培地にペレットを再懸濁し、細胞を数えます。 </li><li>典型的な実験のデザインは、1mLの最終容量で48ウェルプレートのウェル当たり0.5 × 10 4細胞を使用して、そして、各テスト条件のための三連を繰り返す。さらに、蛍光測定を容易にするために、別々のプレートは、1時点の各系統に使用されます。 </li><li>各テスト5 - FCの濃度、三連のために、ウェルあたりの種子0.5 × 10 4細胞。 TYI培地中の抗生物質選択圧を維持する。今すぐ5 - FCのストック溶液を各ウェルにトロフォゾイトの必要量を追加します。 </li><li> 37℃で嫌気袋でプレートをインキュベート℃に</li></ol> 2。選択効率の決定<ol><li> 2 mgを/ DMSOでセルトラッカーグリーンCMFDA（Invitrogen社）のmlストック溶液を調製。ワーキングソリューションを作成するには、無血清TYI媒体の株式1000倍に希釈する。 </li><li>栄養型培養プレートから完全に培養液を除去し、150μLの無血清TYI含むCMFDAと交換してください。 37℃インキュベーションを続ける℃で1時間。 </li><li>井戸から染料溶液を除去し、Spectramax M2マイクロプレート光度計でプレートを読み取る。励起波長492nmで設定し、蛍光発光は517 nmで測定されるべきである。 </li><li>テストのトランスフェク対コントロールの細胞の蛍光値を比較します。 </li><li>各時間間隔でプレートの読み取りを続けます。各プレートに適切な正（TYIのみ）と負（25μg/ mLのハイグロマイシン）コントロールウェルを使用してください。 </li></ol> 3。代表的な結果 E.赤痢のトランスフェクタントは、コドン最適化された組換えFCU1（図1）の強い発現を示した。このプロトコルはEの選択的除去では、結果が正しく実行されている場合赤痢は、細胞を0.5mMの5 -フルオロシトシンの存在下でFCU1を表現する。コントロール細胞は、対照的に5 - FCの5 mMの濃度までで正常に成長し、72および96時間（図2）との間でコンフルエントに達するために引き続き。処理された井戸が顕微鏡で観察しているときに48時間でFCU1持つ細胞が完全に溶解されています。これらはまた、サンプル（図2 - B）の多数を含む定量的な研究で使用されている生体染色色素CMFDA、で染色したときの蛍光の減少を示す。 FCU1/5-FCシステムはE.のための効果的かつ強力な負の選択ツールです。 赤痢はトロフォゾイト。 <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="図1" /> 図1。 E.の世代負の選択マーカーを発現赤痢 HM1トランスフェク （A）FCU1組換えタンパク質は、抗c mycの抗体（上部パネル）を使用してウェスタンブロットで検出された。予想通り、42kDaバンドは、FCUのトランスフェクタントからではなく、コントロールのトランスフェベクター単独で合計ライセートに（矢印で示す）を検出することができた。下のパネルは同じブロットのローディングコントロールとして抗アクチン抗体でプローブを示しています。 （b）の定量的リアルタイムPCRの結果はlgl4コントロールに正規FCU1特定のmRNAの発現を示す。 <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="図2" /> 図2 E.の in vitro薬剤感受性の負の選択マーカーを発現赤痢のトランスフェク （a）制御トランスフェクタントと（b）FCU1表現するトランスフェクタントは、48ウェルプレートで培養の生存曲線。細胞は濃度の増加の存在下で成長させたプロドラッグ5 - フルオロシトシン、X軸に示されるように、細胞の生存率は各時間間隔でCMFDA染色により定量した。 Y軸は蛍光の単位を表し、生き残った細胞集団を直接反映しています。 FCU1表現するトランスフェクタントは、コントロールと比較して0.5mmのプロドラッグの濃度で有意な感度を示したことに注意してください。セルがCOMとして、これらの井戸で120Hの時点で観察されなかった顕微鏡下で対応するコントロールウェル（データは示さず）に見られるようにコンフルエントに成長して比べ。 Hygroは、ネガティブコントロールとして使用される25μg/ mLのハイグロマイシンを表します。

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. Microbiol. 45, 656-660 (1998).</li><li>Erbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cancer+gene+therapy+by+adenovirus-mediated+transfer+of+a+bifunctional+yeast+cytosine+deaminase/uracil+phosphoribosyltransferase+fusion+gene.">In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.</a> Cancer Res. 60, 3813-3822 (2000).</li><li>Braks, J. A. M., Franke-Fayard, B., Kroeze, H., Janse, C. J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+positive-negative+selectable+marker+system+for+use+in+reverse+genetics+in+Plasmodium.">Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.</a> Nucleic Acids Res. 34, e39-e39 (2006).</li><li>Maier, A. G., Braks, J. A. M., Waters, A. P., Cowman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+selection+using+yeast+cytosine+deaminase/uracil+phosphoribosyl+transferase+in+Plasmodium+falciparum+for+targeted+gene+deletion+by+double+crossover+recombination.">Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination.</a> Mol. Biochem. Parasitol. 150, 118-121 (2006).</li></ol>

このビデオの制作は、この記事で使用する試薬の一部を生成するシグマアルドリッチ株式会社後援した。

赤痢アメーバの分子生物学的ツールの大幅な進歩があったが4の遺伝子を削除したり、混乱させるために利用できる現在の方法はありません。遺伝子特異的発現のノックダウンは、ヘアピンRNA 5の使用によって達成低分子干渉RNA 6と細菌のdsRNA 7を発現されている。しかし、それぞれの標的遺伝子の中に効果的なこれらの方法は、高価な化学物質の使用、抗生物質と時間の経過とともに発生復帰の高い発生率に起因する一定の検証の必要性に対する継続的な選択（アリシアリンフォード、私信）8を含むいくつかの制限があります。 DNA複製9の起源のための厳密な順序の要件が明らかにないとして、任意のプラスミドは、 赤痢アメーバに複製することができ、ので、一度トランスフェクション、それは通常、プラスミドを治すことは困難です。これは、組換えと遺伝子ノックアウト実験で無傷プラスミドの活用可能性を制限する。負の選択マーカーは、それらが組換え亜集団を排除するために使用できるようにメソッドを遺伝子ターゲティングの重要なコンポーネントです。私たちは正常にコドン赤痢アメーバでFCU1遺伝子を最適化し、負の選択マーカーとしての可能性をテストを表明している。この遺伝子の融合は、ヒト腫瘍細胞の自殺遺伝子治療のために使用され、RNAとDNA合成10の阻害をもたらす有毒な川下製品へのプロドラッグ5 - FCを代謝されています。 FCU1は最近マラリア 、別の原生動物寄生虫における負の選択マーカーとして使用されています。FCU1を表現マラリアberghei細胞は 5 - FCとのin vitroおよび in vivo治療におけるプロドラッグへの約1000倍感度が高くなることが判明したのは、99.9％以上死滅マラリア11の赤血球段階のマウスモデルにおける組換え寄生虫に感染。E.は FCU1を発現している赤痢細胞はP.で観測された感度とは対照的にプロドラッグ5 - FC〜30倍高い感度を示したberghei。このために考えられる理由は、有毒な代謝産物と競合する増殖培地で利用可能なヌクレオチドの大規模なプールである可能性があります。 P.両方のbergheiとP.熱帯熱マラリア FCU1マーカーは、標的遺伝子破壊に使用されています。研究11,12。我々は、相同組換えを用いた赤痢アメ​​ーバのゲノムを操作することを目指しています。 FCU1/5-FCネガティブ選択系の検証は、この目標に向けた重要なステップです。

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

赤痢アメーバはアメーバ症の原因物質であり、世界人口の10％まで感染します。遺伝子発現のアップとダウンレギュレーションを有効にしている分子技術は、安定的に維持プラスミドのトランスフェクションに依存しています。これらは、赤痢アメーバの病原因子についての理解を増加している一方で、逆遺伝学の実験を可能にするゲノムに外来DNAを統合する能力は、、この寄生虫の研究で大きな利点となります。この微生物によって提示される課題は、相同組換えのイベントとトランスフェトロフォゾイトからエピソーム、プラスミドDNAを治すために困難のために選択することができないことなどがあります。外因性DNA、善意のゲノム統合イベントが発生したトロフォゾイトの同定における主要な問題の高バックグラウンドで後で結果。我々は、酵母のシトシンデアミナーゼとウラシルプロドラッグ5 - フルオロシトシン（5 - FC）とホスホトランスフェラーゼキメラ（FCU1）と選択のトランスジェニック発現に基づいて、負の選択シス​​テムの開発について報告する。 FCU1酵素は毒性の5 -フルオロウラシル、5 -フルオロウリジン-5&#39; -モノリン酸。E.に非毒性の5 - FCを変換するFCU1を表現赤痢ラインはコントロール株と比較してプロドラッグに対してより敏感に30倍であることがわかった。

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

私達は中の負の選択シス​​テムの開発について報告する<em&gt; E.赤痢</em&gt;トランスジェニックキメラタンパク質の発現（FCU1）およびプロドラッグ5 - フルオロシトシンと選択に基づいて。 FCU1タンパク質は、酵母のシトシンデアミナーゼおよびウラシルホスホリボシルトランスフェラーゼの融合です。 FCU1の発現は増加をもたらした<em&gt; E.赤痢</em5 - フルオロに向かって&gt;感度。

のための負の選択マーカーの開発赤痢アメーバ</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

development-of-a-negative-selectable-marker-for-entamoeba-histolytica

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.4K ビュー.  University of Virginia Health System. 私達は中の負の選択シス​​テムの開発について報告する E.赤痢トランスジェニックキメラタンパク質の発現（FCU1）およびプロドラッグ5 - フルオロシトシンと選択に基づいて。 FCU1タンパク質は、酵母のシトシンデアミナーゼおよびウラシルホスホリボシルトランスフェラーゼの融合です。 FCU1の発現は増加をもたらした E.赤痢

ビデオ: Development of a Negative Selectable Marker for Entamoeba histolytica

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal.
Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55&deg;C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application. 
Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.

Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.

家畜の感染症アウトブレイクの制御のためのBiocontained枝肉コンポスト

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

免疫・感染学

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

赤痢アメーバのDNAとtRNAメチルトランスフェラーゼDnmt2（Ehmeth）酵素を用いたin vitro tRNAのメチル化アッセイに

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

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 ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

ラベルフリーバイオセンサー技術を活用乳癌におけるヒト白血球抗原バイオマーカーの検出

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Biocontainmentにおけるウイルスサンプルの陰性染色のためのカプセルの利用

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

のための負の選択マーカーの開発赤痢アメーバ

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