我々は、有用な議論をブリッタヘーン、桂花日、ハリスSoiferとリサシェーラーに感謝。 CHO - EEとCHO - gp160細胞：この作品は、NIHのエイズ研究とレファレンス試薬プログラム、エイズの部、NIAID、NIHによって得られた健康AI29329と、以下の試薬をJJRに授与HL07470の国立研究所からの補助金によって支えられてライン、pNL4 - 3リュックベクトル、HIV - 1 DAIDSからBALのgp120、NIAID。

<ol>
	<li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Robertson, D. L., Joyce, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+in+vitro+of+an+RNA+enzyme+that+specifically+cleaves+single-stranded+DNA.">Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.</a> Nature. 344, 467-468 (1990).</li><li>Fitzwater, T., Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SELEX+primer.">A SELEX primer.</a> Methods Enzymol. 267, 275-301 (1996).</li><li>Weiss, C. D., White, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+stable+Chinese+hamster+ovary+cells+expressing+wild-type,+secreted,+and+glycosylphosphatidylinositol-anchored+human+immunodeficiency+virus+type+1+envelope+glycoprotein.">Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.</a> J Virol. 67, 7060-706 (1993).</li><li>Vodicka, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indicator+cell+lines+for+detection+of+primary+strains+of+human+and+simian+immunodeficiency+viruses.">Indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses.</a> Virology. 233, 193-198> (1997).</li><li>Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection,+characterization+and+application+of+new+RNA+HIV+gp+120+aptamers+for+facile+delivery+of+Dicer+substrate+siRNAs+into+HIV+infected+cells.">Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells.</a> Nucleic Acids Res. , (2009).</li><li>Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+biology+of+aptamers.">The chemical biology of aptamers.</a> Angew Chem Int Ed Engl. 48, 2672-2689 (2009).</li><li>Famulok, M., Hartig, J. S., Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+aptamers+and+aptazymes+in+biotechnology,+diagnostics,+and+therapy.">Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy.</a> Chem Rev. 107, 3715-3743 (2007).</li><li>Chu, T. C., Twu, K. Y., Ellington, A. D., Levy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+mediated+siRNA+delivery.">Aptamer mediated siRNA delivery.</a> Nucleic Acids Res. 34, e73-e73 (2006).</li><li>McNamara, J. O., 2nd, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+delivery+of+siRNAs+with+aptamer-siRNA+chimeras.">Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.</a> Nat Biotechnol. 24, 1005-1015 (2006).</li><li>Dassie, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+administration+of+optimized+aptamer-siRNA+chimeras+promotes+regression+of+PSMA-expressing+tumors.">Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors.</a> Nat Biotechnol. 27, 839-849 (2009).</li><li>Zhou, J., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+cell-internalizing+aptamers.">The therapeutic potential of cell-internalizing aptamers.</a> Curr Top Med Chem. 9, 1144-1157 (2009).</li><li>Zhou, J., Li, H., Li, S., Zaia, J., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dual+inhibitory+function+aptamer-siRNA+delivery+system+for+HIV-1+therapy.">Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy.</a> Mol Ther. 16, 1481-1489 (2008).</li></ol>

J. RとJZは、&quot;HIV - 1治療のための細胞型に特異的なアプタマー - siRNAのデリバリーシステム&quot;で特許申請中を持っている。 USPTOは、No<strong&gt; 12/328994</strong&gt;、発行日：2009年6月11日。

アプタマーは、in vitroで標的分子に8〜タイト結合、それによって高度に特異的な提供、具体的かつ安定的な三次元形状を想定して核酸を進化させています。低ナノモル結合親和性とそのターゲットへのアプタマーの絶妙な特異性がin vivoイメージング、および治療​​9 で 、それらの診断のための多目的なツールとして利用できます。標的siRNA導入のための...

<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> MF -ミリポアメンブレンフィルタ</td><td>ミリポア</td><td> HAWP01300 </td><td>孔径0.45μmの</td></tr><tr><td> Swinnexフィルターホルダー</td><td>ミリポア</td><td> SX0001300 </td><td> 13mmの直径</td></tr><tr><td> QIAquickゲル抽出キット</td><td> QIAGEN </td><td> 28706 </td><td> DNAの精製</td></tr><tr><td>マイクロコンYM - 30カラム</td><td>ミリポア</td><td> 42410 </td><td> RNAの濃度</td></tr><tr><td>バイオスピン30カラム</td><td> Bio - Rad社</td><td> 732-6250 </td><td> RNA精製</td></tr><tr><td> TaqのPCRのDNAポリメラーゼ</td><td>シグマアルドリッチ</td><td> D1806 </td><td></td></tr><tr><td> ThermoScript RT - PCRシステム</td><td>インビトロジェン</td><td> 11146-024 </td><td></td></tr><tr><td> T7転写キットをDuraScribe </td><td>シャーロット</td><td> DS010925 </td><td></td></tr><tr><td> PCR用のdNTP </td><td>ロッシュ</td><td> 1 581 295 </td><td></td></tr><tr><td>リボ核酸、大腸菌からの転送</td><td>シグマアルドリッチ</td><td> R1753 </td><td> tRNAの競合</td></tr><tr><td> HIV - 1 Baの- Lのgp120タンパク質</td><td>エイズの研究およびリファレンス試薬プログラム</td><td> 4961 </td><td>標的タンパク質</td></tr><tr><td>サイレンサーsiRNAの標識キット - Cy3標識</td><td>アンビオン</td><td> 1632 </td><td></td></tr><tr><td>酸5月1日クロロホルム/フェノール溶液（pH 4.5） </td><td>アンビオン</td><td> AM9720 </td><td></td></tr><tr><td>クロロホルム/イソプロパノール1分の24のソリューション</td><td>シグマ</td><td> C0549 </td><td></td></tr><tr><td>ウシ腸ホスファターゼ（CIP） </td><td>ニューイングランドBioLab </td><td> M0290L </td><td></td></tr><tr><td> T4ポリヌクレオチドキナーゼ</td><td>ニューイングランドBioLab </td><td> M0201L </td><td></td></tr><tr><td>グリコーゲン</td><td>ロッシュ</td><td> 10 901 393 001 </td><td> RNA沈殿</td></tr><tr><td> γ- 32 P - ATP </td><td> MPバイオメディカル</td><td> 013502002 </td><td> Radiactivity </td></tr><tr><td> 40パーセントAccuGel夜07時01分</td><td>ナショナル診断</td><td> EC - 850 </td><td></td></tr><tr><td> 10xTBE </td><td>ナショナル診断</td><td> EC - 860 </td><td></td></tr><tr><td> N、N、N、N&#39; -テトラメチルエチレンジアミン（TMEMD） </td><td>シグマアルドリッチ</td><td> T9281 </td><td></td></tr><tr><td>過硫酸アンモニウム（APS） </td><td>シグマアルドリッチ</td><td> A3678 </td><td></td></tr><tr><td> L - methioineスルホキシ</td><td>シグマアルドリッチ</td><td> M5379 - 250ミリグラム</td><td></td></tr><tr><td> RPMIメディア1640 </td><td>インビトロジェン</td><td> 11835-030 </td><td></td></tr><tr><td>重炭酸ナトリウム溶液、7.5％w / vの</td><td>インビトロジェン</td><td> 25080-094 </td><td></td></tr><tr><td>最小必須培地（MEM）（10） </td><td>インビトロジェン</td><td> 11430-030 </td><td></td></tr><tr><td> MEM非必須アミノ酸（100） </td><td>インビトロジェン</td><td> 11140-050 </td><td></td></tr><tr><td>をpCR 2.1とTAクローニングキット</td><td>インビトロジェン</td><td> K2040 - 01 </td><td></td></tr></tbody></table>

development of cell-type specific anti-hiv gp120 aptamers for sirna delivery

HIVによる感染の世界的流行は、抗レトロウイルス薬の新しいクラスのための緊急の必要性を作成しています。相補的RNA転写産物の発現を阻害する低分子干渉（SI）RNAの強力な能力は、HIVを含む疾患の様々な治療薬の新しいクラスとして悪用されています。多くのこれまでの報告では、新たなRNAiベースanti-HIV/AIDS治療戦略は、かなりの期待を持っていることが示されているが、成功した治療への応用およびsiRNAの臨床翻訳の鍵の障害は、効率的な配信です。特に、RNAiベースの治療薬の安全性と有効性を検討し、それは特定の細胞集団または組織に標的細胞内のsiRNA導入のアプローチを開発することが極めて望ましい。 HIV - 1のgp120タンパク質、HIV - 1の表面上の糖タンパク質のエンベロープは、CD4細胞へのウイルスの侵入に重要な役割を果たしている。 HIV - 1のエントリをトリガし、細胞融合を開始することをgp120とCD4の相互作用は、薬物発見のための臨床的に関連する抗ウイルス戦略として検証されています。 ここで、我々はまず、2&#39; - F改変抗HIV gp120のRNAアプタマーの選択と識別を議論する。従来のニトロセルロースフィルターSELEX法を用いて、ナノモル親和性を持ついくつかの新しいアプタマーは、50のランダムなNT RNAライブラリーから単離された。が正常に高い親和性で結合した種を得るためには、選択のストリンジェンシーは、慎重に条件を調整することによって制御される。選択されたアプタマーは、特異的に結合することができ、急速にHIV - 1エンベロープタンパク質を発現する細胞に取り込まれる。さらに、単独でアプタマーはHIV - 1感染を中和することができる。最高のアプタマー- 1に基づき、我々はまた、アプタマーやsiRNAの両方の部分が強力な抗HIV活性を持つ新規な二重の阻害機能抗gp120のアプタマー - siRNAのキメラを作成してください。さらに、我々はHIV - 1感染細胞へのsiRNAの細胞型特異的送達のためのgp120アプタマー - siRNAのキメラを利用する。キメラこの二重の機能は、抑制するHIV - 1感染の様々な核酸治療薬を（アプタマーとsiRNA）を組み合わせた高活性抗レトロウイルス療法（HAART）を失敗した患者のためのアプタマー - siRNAのキメラの魅力的な治療候補者を作るための大きな可能性を示しています。

Watch this Scientific Journal Video about Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery at JoVE.com

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

ナノモル親和性HIV - 1BA - Lのgp120に対するいくつかの2&#39; -フルオロRNAアプタマーは、RNAのライブラリーからで隔離されています<em&gt; in vitroで</em&gt; SELEX手順。新しいデュアル阻害機能抗gp120のアプタマー - siRNAのキメラが作成され、全身の抗HIV治療のためにかなりの期待を示しています。

siRNA導入のための細胞型に特異的な抗HIV gp120のアプタマーの開発

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

development-cell-type-specific-anti-hiv-gp120-aptamers-for-sirna

Research

JoVE Journal

Immunology and Infection

19.4K ビュー.  Beckman Research Institute of City of Hope. ナノモル親和性HIV - 1BA - Lのgp120に対するいくつかの2' -フルオロRNAアプタマーは、RNAのライブラリーからで隔離されています in vitroで SELEX手順。新しいデュアル阻害機能抗gp120のアプタマー - siRNAのキメラが作成され、全身の抗HIV治療のためにかなりの期待を示しています。

ビデオ: Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

Development of a Negative Selectable Marker for Entamoeba histolytica

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 up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

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 ...

免疫・感染学

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

ペプチドエピトープ特異的T細胞のMHCテトラマーベースの濃縮

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their 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, ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

腸間膜リンパダクトカニューレを挿入ラットモデル：腸のリンパ医薬品輸送の評価への応用

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用<em&gt; Q熱リケッチア</em&gt;のsiRNAサイレンシング中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

の検出のためのより高感度で特異的な発色性寒天培地の開発<em&gt;腸炎ビブリオ</em&gt;その他<em&gt;ビブリオ</em&gt;種

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

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 ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

「Acufection」と肌の研究への応用により、経済的なDNAデリバリーシステムの開発

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

ジカ特定診断エピトープの探索

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

バイオパニングから T7 バクテリオファージの量的な PCR

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...