私は、技術支援のためのオルガ·デ·ソウザ·シルバを承認したいと思います。この作品は、スイス国立科学財団（SNSF）グラント31003A_127029によってサポートされていました。

TransFLP法（ 図1）は、3つの異なるアプローチが例示される：I）Vの病原性決定因子をコードする2つの隣接した遺伝子の欠失コレラ菌 （ 例えば 、ΔctxAB）、病原性島のⅡ）除去（ 例えば 、ΔVPI-1）、およびその後にそのを可能にする目的遺伝子の上流にT7 RNAポリメラーゼ依存性のプロモーター配列のIII）の統合それぞれのひずみ背景に人工的な発現（ 例えば、T7-tfoX）（ 図2）。 1。キチンフレークの調製<ol><li>標準1.5mlのプラスチックチューブでキチンフレークの重量50から80ミリグラム。これは大量に調製することができます。キチンフレークは、Sigma（カタログ番号C9213）から市販されている。 </li><li>チューブの蓋が開いたままのフレークをオートクレーブ。 </li><li>オートクレーブを冷却した後、直ちに蓋を閉じます。 </li><li>ストアオートクレーブ処理キチンフレーク室温で。 </li></ol> 2。オプション：Vの人工変換FLPリコンビナーゼをコードするプラスミドでコレラ <ol><li>エレクトロ準備V.標準的な方法11を用いて 菌の細胞。 -80℃でコンピテント細胞のアリコート℃で保存</li><li>エレクトロにプラスミドpBR-FLP 5のレギュラーミニ準備中1-2μlを加えV.コレラ菌は、細胞およびエレクトロポレーションキュベット（0.2 cm商品のギャップ幅）に混合物を移す。 </li><li> 1.6 kVでパルスを印加します。 </li><li> 0.9ミリリットルのSOC培地を加え、穏やかに、標準の14 mlチューブに細胞を移す。 30℃で2.5から3時間にわたり非移動インキュベート℃に</li><li>プレートを100μl、30℃で一晩100μg/ mlのアンピシリンとインキュベートを含むLBプレート上に300μlの。 </li><li>将来TransFLP実験のグリセロールストックのような単一のクローンや店舗を精製する。 </li></ol> 3。オリゴヌクレオチドの設計およびポリメラーゼ連鎖反応<ol><li>少なくとも6つのオリゴヌクレオチドは、目的とするDNA領域（複数可）と同様にPCRによりFRT-挟まれた抗生物質カセット（ 図3）を増幅する必要があります。また、挿入されたPCR断片外オリゴヌクレオチドプライミングのペアを含めることをお勧めします。これは、統合とFRT-挟ま抗生物質耐性カセット（ 例えば 図3〜図5に&#39;CHKアップ&#39;と&#39;CHKダウン&#39;プライマーとして描かれている）の正確な切除をチェックすることができます。 </li><li>オリゴヌクレオチド＃2、＃3、＃4、＃5の注意を払って設計を行ってください。彼らは十分に鋳型DNAに（ 例えば 〜28 bp）をアニールすることができるはずです。テンプレートDNA、プライマー＃2と＃5などpROD17 12及びプライマー＃3と＃4のPBR-FRT-KAN-FRT（本研究）（としてFRT-カンイFRT含有プラスミドDNAのゲノムDNA（gDNA）です図3A）。 </li><li> （それぞれ、＃2 /＃3、＃4 /＃5の間にそれらの5 &#39;末端の塩基対広範図3Aを許可＃5＃2プライマーを設計</stronグラム]&gt; [挿入図）。 PCR法の2つのラウンドが続きます。 </li><li> PCRの第1ラウンドとして3独立したPCR反応を行う。最初のものは、オリゴヌクレオチド＃1と＃2を用いて遺伝子/ DNA関心領域の上流領域を増幅する。 PCR法は、細胞内での相同組換えを可能にするために、長さが少なくとも500bpの断片をもたらすべきである。短い断片（約250 bp）は8で十分できますが、推奨されていません。 </li><li>並列にFRT-挟ま抗生物質カセットを増幅する第2のPCRを行う。例はここで提供されるために私達は主にAPH（館 R）を使用しました。この反応（ 図3A）のためのオリゴヌクレオチド＃3と＃4を使用します。 </li><li>付随して、PCR法（第三のサンプル）によって下流のDNA領域を増幅する。 PCR断片はまた、長さが少なくとも500bpである必要があります。テンプレートおよびオリゴヌクレオチド＃5と＃6（ 図3A）にgDNAを用いてPCRを行う。 </li><li>すべての3つのPCR産物の精製後断片は、PCRの第2ラウンドを行う。テンプレートとして、最初のラウンドで得られたすべての3つの断片の等量混合物を使用しています。増幅は、オリゴヌクレオチド＃1と＃6によって触媒される。得られたPCR断片（ 図3B）は、自然形質転換実験（ 図1）でDNAを変換として機能します。 </li></ol> 4。キチン誘起自然形質転換<ol><li>成長V. 30℃における富栄養培地で好気的にコレラ菌は、細胞は、約0.5の600nmでの光学密度に達するまで。 </li><li>遠心分離により菌体を回収します。 DASW 2容量の中に細胞を再懸濁する前に、一度定義する人工培地（DASW 6）でペレットを洗浄します。無菌キチンフレーク（ポイント1で説明）の50〜80 mgの培養液1 mlを加える。文化への細菌港プラスミドpBR-FLP（オプション2点）、ADDアンピシリン（50μg/ ml）の場合。運動せずに16から24時間30℃でインキュベートする。 </li><li> G＆追加eであり、第2回目のPCR由来断片（ポイント3で説明）は200 ng。広くキチン表面から細菌を切り離さずに慎重に混ぜる。運動なしで24時間30℃でインキュベートする。 </li><li>渦≥30秒広く文化。選択LB培地プレート（ここで提供される例については、 例えばカナマイシン、ゲンタマイシン含有LBプレート）上に100から300μlを広げた。細菌は他の抗生物質に付随してアンピシリンを追加することにより、PBR-FLPプラスミドを抱いている場合、ダブル選択プレートを使用しています。 16から24時間30℃でプレートをインキュベートするか、コロニーが見えるようになるまで。 </li><li>選択プレートから単一の形質転換体を単離する。このような形質転換体は、ダブルクロスオーバーイベントによるPCR断片によってオリジナルの染色体座を交換しました。抗生物質カセットを取り外す必要はありません場合にはこれらの株は直接さらなる実験のために使用することができます。 </li></ol> 5。 FLPによる選択カセットの除去（複数可）組換え<ol><li>自然形質転換試験の前に行われていない場合、ポイント2で説明したように人為的にPBR-FLPプラスミドを使って形質転換体を変換する。 </li><li> 16から24時間37℃でアンピシリンを含むLB寒天プレート上で細菌を培養。オプション：40に2〜3時間の間に温度を変更℃のプラスミドpBR-FLPからFLPの表現として、より高い温度での5,10 -デ抑制され、約後に新鮮なプレートへの転送細菌。インキュベーションの8時間。 </li><li>抗生物質含有および抗生物質を含まない寒天プレート上で並行してクローンをrestreakingによって、抗生物質感受性（ 図1カナマイシンにはここを実証）のテスト。シングル感受性コロニーを分離します。あなたはさらにTransFLPを使用して他の削除/挿入した株を変更する場合はグリセロールストックとしてアンピシリン耐性クローンをフリーズします。 </li></ol> 6。プラスミドキュア<ol><li>好気的条件下で一晩培養すると30℃どんな抗生物質を添加することなくリッチな培地を使用しています。 </li><li>オプション：新鮮な抗生物質を含まない培地で一晩培養した培養液の1:100希釈することにより、3から6時間、新鮮な培養する。 </li><li>板やスジ平野LB寒天プレート（S）で文化の希釈（s）および30℃にて8月16日時間のためのC（コロニーが見えるようになるまで）。 </li><li>抗生物質含有および抗生物質を含まない寒天プレート（ 図1）上で並行してクローンをrestreakingによるクローンのアンピシリン感受性をテストします。グリセロールストックとしてアンピシリン感受性株をフリーズします。 </li></ol>

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特別な利害関係は宣言されません。

TransFLP方法は、上述の他の場所と5は広く、我々の研究室で使われてきました。実現可能である遺伝子操作の種類は、とりわけ以下のとおり、単一の遺伝子や遺伝子群の欠失、ゲノムの島々の欠失（ 例えば 、VPI-1）は、目的の遺伝子の上流配列の挿入（ 例えば 、プロモーター配列）と挿入を特定の遺伝子（ 例えば 、符号化アフィニティータグ）の末尾のシーケ?...

<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>キチンフレーク</td><td>シグマ</td><td> C9213 </td><td>必要なオートクレーブ</td></tr><tr><td>オプション：Micropulser場合（抗生物質カセットはPBR-FLP上にエンコードFlpリコンビナーゼによって切除されるべきである） </td><td>バイオラッド</td><td> 165-2100 </td><td>または同等のエレクトロポ</td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

いくつかの方法は1-3細菌の染色体を操作することが可能です。これらのプロトコルの大部分は、条件付きで複製プラスミドの挿入（1,2 PIR依存または温度感受性レプリコンを宿すなど ）に依存しています。これらのプラスミドは、相同性媒介組換えに基づく細菌染色体に組み込まれています。このような挿入変異体はしばしば直接実験の設定に使用されています。あるいはまた、その損失が続くプラスミド切除の選択はしばしばsacB遺伝子4でエンコードされたカウンターで選択レバンスクラーゼの酵素に依存しているグラム陰性細菌に、これを行うことができます。切除は、事前挿入遺伝子型または染色体と改変された遺伝子のプラスミドにコードされたコピーの間の交流の結果を復元することができます。この手法の欠点は、時間がかかるということです。プラスミドは、最初にクローンする必要があり、それは Vへの水平転送を必要とするコレラ菌 （最大n<html> otably E.との交配によって大腸菌ドナー株）または後者の人工変換、およびプラスミドの切除は、ランダムであり、どちらかの初期遺伝子型を復元するか、正の選択が発揮されていない場合、必要な変更を作成することができます。ここでは、Vの迅速な操作のための方法を提示するコレラ菌染色体（S）5（ 図1）。このTransFLP法はこの生物6、などVのビブリオ属の他の代表の天然能力の最近発見されたキチン質を介した誘導に基づくシャリ 7。自然な能力は、PCRによって生成されたDNA断片を含む自由なDNAの取り込みを可能にします。一度取り上げ、染色体とDNAの再結合は、相同領域8に隣接するの250から500 bpの最小値の存在を与えられた。これらの隣接領域の中間の選択マーカーを含めることは頻繁に発生した形質転換体を容易に検出することができます。 tは&quot;&gt;このメソッドはコレラ菌の異なる遺伝子操作のために使用され、潜在的にも他の天然有能なバクテリアすることができます我々は、以前に公開された単一遺伝子欠失の研究とに加えて、この方法によって達成することができるものに3小説の例を提供アフィニティータグ配列5。キチン誘発自然変換6の初期のプロトコルに関しては、いくつかの最適化のステップはこのTransFLPプロトコルに組み込まれているのに加え、これらは他の市販のキチンフレーク8、PCRの寄付によってカニ殻断片の交換中に含める由来の材料9の変換など、DNA、およびFLP組換え標的部位の付加（FRT）5。FRT部位は、FLPリコンビナーゼ10によって媒介される選択マーカーの部位特異的切除を可能にします。

3例の代表的な結果を6〜図4に示されている。隣接する遺伝子ctxAとctxBを削除することを目的と最初のアプローチ。彼らは一緒にVの主要な病原因子をエンコードするコレラ菌 、コレラ毒素。我々は、上記のようにTransFLPメソッドのオリゴヌクレオチドを設計し、 図3を参照に記載の方法。親株は、ctxABの元のDNA遺伝子座のFRT-挟ま...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

のゲノムを変更するための簡単​​な方法<em&gt; V。コレラ</em&gt;記載されている。これらの変更は、単一の遺伝子、遺伝子クラスターおよびゲノムの島だけでなく、短い配列（例えばプロモーターエレメントまたはアフィニティータグ配列）の統合の削除があります。方法は自然形質転換およびFLP組換えに基づいています。

TransFLP - 遺伝的に変更する方法<em&gt;コレラ菌</em&gt;自然形質転換およびFLP組換えに基づき

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Research

JoVE Journal

Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K ビュー.  Ecole Polytechnique Fédérale de Lausanne (EPFL). のゲノムを変更するための簡単​​な方法 V。コレラ記載されている。これらの変更は、単一の遺伝子、遺伝子クラスターおよびゲノムの島だけでなく、短い配列（例えばプロモーターエレメントまたはアフィニティータグ配列）の統合の削除があります。方法は自然形質転換およびFLP組換えに基づいています。

ビデオ: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

の植民地化 Euprymna scolopesイカでビブリオフィシャリ</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

免疫・感染学

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

細胞内の抗リーシュマニアに対するスクリーニングのための寄生虫のRescue and形質転換試験<em&gt;ドノバンリーシュマニア</emTHP1ヒト急性単球性白血病細胞株における&gt;無鞭毛

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

の臨床応用<em&gt;眠れる森の美女</em遺伝的に末梢及び臍帯血由来のT細胞を変更する&gt;と人工抗原提示細胞

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

グラスサポート平面脂質二重層上のナチュラルキラー細胞免疫シナプスの超解像イメージング

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

哺乳動物宿主細胞に分泌された細菌毒素の影響を研究するために、透過膜を挿入ベースの感染システムの実現

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

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 ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

天然化合物ライブラリーの探査に適した抗バイオフィルムアッセイのプラットフォーム

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

バイオタイプの維持と差別化に使用される実験技術<em&gt;コレラ（Vibrio cholerae）</em&gt;臨床分離株

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...