我々はマイケルFeissとファージの作成と精製に関するガイダンスジャンシッピーに感謝しています。我々は、細胞認識ソフトウェア、Schnitzcellを提供するためのマイケルElowitzに感謝します。 、ウェルチ財団（助成Q-1759）とヒューマン·フロンティア·サイエンス：ゴールディングラボの仕事は国立衛生研究所（R01GM082837）、国立科学財団（生きている細胞の物理学研究センター082265、PFC）からの補助金によってサポートされていますプログラム（RGY 2008分の70）。

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

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菌株、ファージ及びプラスミド： ひずみLE392はsupFです。それは（詳細については表1を参照）は、ファージゲノム中SAM7突然変異を抑制するために選ばれました。溶菌経路を選択した感染細胞が意志としてこのように、誘導され溶原は、最終的に、ファージ粒子を溶解し、解放します。溶原性細胞は30℃で栽培されている℃のファージゲノム中857アレルCI温度感受性の存在に起因する。熱誘導後、GPD-EYFPおよび野生型GPDは、λLZ1のゲノムおよびプラスミドpPlate * Dのそれぞれから同時発現される。その結果、新たに作成されたλファージLZ2のキャプシドはGPD-EYFPおよびGPDタンパク質の混合物を含んでいます。このモザイクファージは、構造的に安定しており、個々のファージ5の検出を可能にするために十分に蛍光性である。 PP RE - mCherryは溶原pathwaの選択を検出するために使用されるプラスミドレポーターですyである。プロモーターP REは溶原性1,11の確立時にCIIによって活性化される。 PP RE - mCherry 5は mCherry 12でGFPを置き換えることにより、PE-GFP 11から派生したものです。詳細については、当社の以前の仕事5を参照してください。 成長条件パラメータ： 溶原誘導（第1節）の間に、180 rpmで軽度の揺れは良いウイルス収量13を与える。グルコース代謝があるので避けてください成長培地中のグルコースの使用は酸性代謝産物を生成し、成熟したラムダ粒子が酸性pH 13で不安定である。 MgSO 4の加算はファージカプ3を安定させることを目的としている。野生型CI（代わりにCI 857）を運ぶファージは、溶原菌は、DNA損傷剤のマイトマイシンC 3を使用して誘導することができる。ステップ1.3では、37℃でインキュベーション℃は通常90分を超えてはいけません。それは、USEFです<html> 30分ごとにOD 600により細胞密度を確認してUL。良いライセートは、OD 600が約0.2以下に低下し、残りのOD 600が細胞破片の結果です。新しく作成されたファージは、細胞の破片に自分のDNAを注入するために始めることができるから時間が経ちすぎインキュベートすると、下のファージの収率で発生する可能性があります。ステップ2.11と2.13で見えるファージバンド（少なくとも1×10 11ファージ粒子）を取得するには、ステップ1.2で少なくとも500 mlの培養液を栽培しています。ステップ5.1および5.2の増殖培地中に0.2％マルトースの添加は子羊、ラムダファージの吸着3,14に対する受容体の発現を誘導することを目的としている。 mCherry -ステップ5.2の代わりに、100倍1000倍に希釈し、レポータープラスミドのPP REからmCherryのバックグラウンドレベルを減らすことを目的としています。 、35をトリガファージDNA注入のためのステップ5.5°Cでは温度感受性cI857対立の誘発を避けるために選択されます。 ファージ精製： jove_content &quot;&gt;ファージの精製工程（2.11〜ステップ2.1）は、他の精製プロトコル5に置き換えられますが、CsCl平衡勾配（ステップ2.12と2.13）を介して最終的に超遠心分離は不可避であることができます。スイングバケットローターはステップ2.10と2.12で必要とされる鋭い目に見えるファージバンドを確保する。純粋ファージ株式の取得には一週間ほどかかる場合がありますので、中間段階の間に何かがうまくいかないことを確認するための方法に沿ってファージ力価を確認する必要があります。 ファージの取り扱い： 第2節の全精製手順の間に、それはファージは、ファージ頭部からファージ尾部を剪断避けるために穏やかにライセートを処理するために重要です。第5節で細胞感染（例えば、5.7を通じて5.5ステップ）の間に、それはまた、感染細胞からファージ粒子のせん断を避けることが重要です。のIE、ファージのDNAを注入した後、感染細胞から剪断される場合、結果は &quot;暗い&quot;感染症であることに注意してくださいクション結果が実験で観察されたが、感染ファージはしませんされます。ファージまたはファージ/細胞混合物を取り扱うときは、必ずこのような問題の発生を防ぐために、我々は広いピペットチップを使用しています。 DAPIのテスト： DAPI（4節）とファージストックを染色すると、ファージストックの純度を確認するには、迅速かつ効率的な方法です。また、時間をかけて既存のファージストックの分解の可能性をテストするために使用することができます。純粋な株式については、蛍光顕微鏡下でYFPおよびDAPI信号の共局在はほぼ100％であるべきである。我々は通常のYFPスポットの1％未満では、これらの粒子が正常にウイルスDNAをパッケージ化しなかったことを示していたり​​、既に他の場所で彼らのDNAを注入したDAPIを（ウイルスゲノムなしキャプシドを表す）は、含まれていないことを確認します。 DAPIのスポットの1％未満はYFP（非蛍光性のファージに相当）が含まれていません。この条件が満たされない場合、2.14を通じて2.12はoに繰り返されるために必要な手順再び精製するrder。長期的なライブセルイメージングがここに必要としないため、撮像パラメータに関しては、ステップ4.3の顕微鏡のセットアップでは、第5節でほど重要ではありません。 1の願いはただ一つのファージ粒子の蛍光強度を校正する場合は、第5節と同様に顕微鏡の設定を保存しておくことは有用である。 PBS-アガローススラブは非常にきれいで、またはDAPI染料が使用されても過言ではない場合には、ファージDNAに対応するいくつかのDAPIスポットは &quot;ハロ&quot;で囲まれていてもよい。少なすぎるDAPIの染料が使用されている場合は、DAPIのチャネ​​ルからの信号は非常に弱いかもしれません。 顕微鏡システム： 第6節のイメージングのために、私たちは100倍対物レンズ（プランのFluo、開口数1.40、油浸）と標準フィルターセット（ニコン）と、商業倒立落射蛍光顕微鏡（エクリプスTE2000-E、ニコン）を使用します。蛍光光源は、光強度の制御とアーク灯である。次の機能は、コンピュータ制御されています：x、y、zのPOsition、明視野と蛍光シャッター、及び蛍光フィルター選択。オートフォーカス機能が必要です。そうしないと、フォーカスが容易にタイムラプスムービー（通常は4時間長い）の間に流れ出すことがあります。それが並列で複数の感染イベントを追跡することが可能として、各時点で（x、y）は、複数のポジションを獲得する能力は、便利です。我々は一般的に100感染イベントに追従し、それぞれの映画の中で8段階のポジションを獲得。我々が使うカメラは16ビット（Cascade512、Photometrics）のダイナミックレンジを持つ16×16μmのピクセルのカメラと512×512冷却CCDである。買収は、MetaMorphソフトウェア（Molecular Devices）を使用して実行されます。顕微鏡は、恒温室に置かれるべきである、或いは、顕微鏡ステージは、温度制御室で囲む必要があります。 画像取り込み： ライブセルイメージングのために、それは漂白とフォーにつながる可能性があり、試料の不必要な被ばくを避けるために重要ですtotoxicity。したがって、それは最初に漂白過大または細胞増殖を阻害するに至らない間に蛍光を検出することができ、最適な露光量を見つけるためにあなたのシステムを特徴づけるために最善の方法です。良い蛍光画像を得るためには、励起光強度、露光時間とカメラゲインと遊ぶ。ステップ6.2から6.3で、10分のフレーム間隔は、光の露出を最小限に抑えることを目的として選択されます。各フレームでは、単一の合焦画像は、位相コントラスト（細胞認識のために）および蛍光チャネル（細胞の運命を決定するため）が必要である。最初の時点では、しかし、YFPのチャネルを介して、複数のz位置の画像は、細胞表面上のすべての感染したファージをキャプチャする必要があります。初期フレームにおけるYFPの露光時間はまた後で時間枠でタイムラプスムービーで使用しているよりも高くする必要があるかもしれません。 画像解析： 非常に慎重にステップ7.1における細胞表面の周りファージ粒子を数える。として上で述べたように、我々は、ステップ6.2におけるYFPのチャネルを介してZ-スタックのシリーズを取る。しかし、これはまだカウントに挑戦一部の蛍光ファージ粒子アウトフォーカスを離れることができる。最初の時間枠内のセルの長さがMetamorphソフトウェアを使用して測定されています。セル長はまた、ImageJのか、他のソフトウェアツールによって測定することができる。さらに、自動化された家庭MATLAB組み込みプログラムは、そのような細胞系譜に沿って時間をかけて蛍光変化などの情報を取得する際に非常に役立つことがあります。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>クロロホルム</td><td>フィッシャー·サイエンティフィック</td><td> C298-500 </td></tr><tr><td> NaClを</td><td>フィッシャー·サイエンティフィック</td><td> S271-3 </td></tr><tr><td> DNase Iを</td><td>シグマ</td><td> D4527-10KU </td></tr><tr><td>アーゼ</td><td>シグマ</td><td> R4642〜10mgの</td></tr><tr><td> PEG8000 </td><td>フィッシャー·サイエンティフィック</td><td> BP233-1 </td></tr><tr><td> SMバッファー</td><td> Teknova </td><td> S0249 </td></tr><tr><td> NZYM </td><td> Teknova </td><td> N2062 </td></tr><tr><td>セシウム</td><td>シグマ</td><td> C3011-250G </td></tr><tr><td>注射器</td><td>ベクトン·ディッキンソン</td><td> 309585 </td></tr><tr><td>針</td><td>ベクトン·ディッキンソン</td><td> 305176 </td></tr><tr><td>透析カセット</td><td>サーモサイエンティフィック</td><td> 66333 </td></tr><tr><td>顕微鏡スライド</td><td>コー​​ニング</td><td> 2947-75x50 </td></tr><tr><td>アガロース</td><td>フィッシャー·サイエンティフィック</td><td> BP160-100 </td></tr><tr><td> SW40Ti超明確な管</td><td>ベックマン·コールター</td><td> 344060 </td></tr><tr><td> SW60Ti超明確な管</td><td>ベックマン·コールター</td><td> 344062 </td></tr><tr><td> SW40Tiローター</td><td>ベックマン·コールター</td><td> 331302 </td></tr><tr><td> SW60Tiローター</td><td>ベックマン·コールター</td><td> 335649 </td></tr><tr><td>屈折計</td><td>フィッシャー·サイエンティフィック</td><td> 13から947 </td></tr><tr><td>落射蛍光顕微鏡</td><td>ニコン</td><td>エクリプスTE2000-E </td></tr></tbody></table> 表2試薬と機器。

following cell-fate in e. coli after infection by phage lambda

バクテリオファージ（ファージ）ラムダと細菌大腸菌を含むシステム大腸菌は長い細胞運命決定の1,2のためのパラダイムを務めている。ファージの数によって、細胞の同時感染後、2つの経路のものが選ばれる：溶解（強毒性）または溶原（休止状態）3,4。我々は最近、蛍光個々のファージを標識するための方法を開発し、個々のファージとセル5のレベルでは、顕微鏡下でリアルタイムで感染後の決定を調べることができました。ここで、我々は以前の仕事5に記載された感染実験を行うための完全な手順について説明します。これは蛍光ファージの作成、細胞の感染、顕微鏡下でのイメージングおよびデータ分析が含まれています。蛍光ファージは、カプシドGPDタンパク質の &quot;ハイブリッド&quot;、共発現する野生型およびYFP融合バージョンです。原油ファージ溶菌液は最初GPD-EYFP（ENHの溶原菌を誘導することによって得られるプラスミドを発現する野生型GPDを宿す、黄色蛍光タンパク質）ファージをとれた。精製工程のシリーズは、その後、顕微鏡下でのDAPI標識やイメージング、続いて行われます。これは、均一性、DNAパッケージング効率、蛍光シグナル及びファージストックの構造安定性を確認するために行われます。細菌にファージの初期吸着は、その後、35℃で短時間のインキュベーションに続いて、氷の上で実行される°Cは、ウイルスDNA注入6をトリガする。ファージ/細菌混合物を、次いで、薄い栄養寒天平板の表面に移動したカバーガラスで覆われており、落射蛍光顕微鏡下で撮像される。感染後の処理は10分間隔で、4時間続きます。複数のステージの位置は約100細胞の感染は、単一の実験でトレースすることができるように追跡されます。それぞれの位置と時間の時点で、画像は位相コントラストと赤と緑の蛍光チャネルで取得されています。位相コントラスト画像は、自動化されたセルのために後で使用されている新しい蛍光ファージの生産（緑）細胞溶解、または再開細胞の増殖と分裂に続いて溶原性因子の発現（赤色）が続いています。蛍光チャンネルが感染の転帰を特徴付けるために使用されるのに対し、Lは認識。取得したタイムラプスムービーは、手動と自動の方法を組み合わせて使用​​して処理されます。各感染事象（感染ファージの例数と位置）だけでなく、感染症の転帰（溶解/溶原性）のための感染パラメータの同定におけるデータ解析結果。必要に応じて追加のパラメータを抽出することができる。

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

この記事では、バクテリオファージラムダの感染の蛍光標識されたバージョンを準備する手順を説明します<em&gt; E大腸菌の</em&gt;細菌、顕微鏡下での感染の結果に続く、感染結果の分析。

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

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Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

23.2K ビュー.  University of Illinois at Urbana-Champaign. この記事では、バクテリオファージラムダの感染の蛍光標識されたバージョンを準備する手順を説明します E大腸菌の細菌、顕微鏡下での感染の結果に続く、感染結果の分析。

ビデオ: Following Cell-fate in E. coli After Infection by Phage Lambda

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

免疫・感染学

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

全タンパク質の抽出および2次元ゲル電気泳動法のための<em&gt;バークホルデリア</em&gt;種

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

ファージディスプレイされた合成抗体ライブラリーから、スケーラブルな高スループットの選択

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

神経病原の非侵襲的モデル<em&gt;エシェリヒア·コリ</em&gt;新生ラットにおける感染

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

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

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

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

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

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

マウスにおける強制経口投与に続いてプロバイオティクス酵母の形質転換および消化管免疫組織から彼らの回復

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

密なトランスポゾン挿入ライブラリを使用して細菌活用腸内細菌系統など、大腸菌や赤痢菌の作成

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...