作者は感謝してマルタ カラタユ アロヨ (FWO ポスドク ・ フェローシップ 12N2815N) フランダース研究財団から財政支援を認めます。エマ ・ ヘルナンデス サナブリアはフランダースの技術革新と起業家精神 (Agentschap フォール Innovatie ドア Wetenschap en テク、IWT) でサポートされている研究員。

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+normal+bacterial+flora+of+the+oral+cavity.">Defining the normal bacterial flora of the oral cavity.</a> Journal of Clinical Microbiology. 43, 5721-5732 (2005).</li><li>Marsh, P. D., Head, D. A., Devine, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+plaque+as+a+biofilm+and+a+microbial+community-Implications+for+treatment.">Dental plaque as a biofilm and a microbial community-Implications for treatment.</a> Journal of Oral Biosciences. 57, 185-191 (2015).</li><li>Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C., Kent, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+complexes+in+subgingival+plaque.">Microbial complexes in subgingival plaque.</a> Journal of Clinical Periodontology. 25, 134-144 (1998).</li><li>Haffajee, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subgingival+microbiota+in+healthy,+well-maintained+elder+and+periodontitis+subjects.">Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects.</a> Journal of Clinical Periodontology. 25, 346-353 (1998).</li><li>Venema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+metabolites+produced+by+the+colonic+microbiota+as+drivers+for+immunomodulation+in+the+host.">Microbial metabolites produced by the colonic microbiota as drivers for immunomodulation in the host.</a> The FASEB Journal. 27, (2013).</li><li>Marzorati, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HMI+(TM)+module:+A+new+tool+to+study+the+Host-Microbiota+Interaction+in+the+human+gastrointestinal+tract+in+vitro.">The HMI (TM) module: A new tool to study the Host-Microbiota Interaction in the human gastrointestinal tract in vitro.</a> BMC Microbiology. 14, 133 (2014).</li><li>Tanzer, J. M., Kurasz, A. B., Clive, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+displacement+of+mutans+streptococci+and+inhibition+of+tooth-decay+by+Streptococcus-Salivarius+Tove-R.">Competitive displacement of mutans streptococci and inhibition of tooth-decay by Streptococcus-Salivarius Tove-R.</a> Infection and Immunity. 48, 44-50 (1985).</li><li>Slomka, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+stimulation+of+commensal+oral+bacteria+suppresses+pathogens:+the+prebiotic+concept.">Nutritional stimulation of commensal oral bacteria suppresses pathogens: the prebiotic concept.</a> Journal of Clinical Periodontology. 44, 344-352 (2017).</li><li>Hernandez-Sanabria, E., et al. <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+increased+respiratory+activity+of+selected+oral+bacteria+may+explain+competitive+and+collaborative+interactions+in+the+oral+microbiome.">In vitro increased respiratory activity of selected oral bacteria may explain competitive and collaborative interactions in the oral microbiome.</a> Frontiers in Cellular and Infection Microbiology. 7, 235 (2017).</li><li>Alvarez, G., Gonzalez, M., Isabal, S., Blanc, V., Leon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+quantify+live+and+dead+cells+in+multi-species+oral+biofilm+by+real-time+PCR+with+propidium+monoazide.">Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide.</a> AMB Express. 3, (2013).</li><li>Ehsani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+evenness+determines+diversity+and+cell+density+dynamics+in+synthetic+microbial+ecosystems.">Initial evenness determines diversity and cell density dynamics in synthetic microbial ecosystems.</a> Scientific Reports. 8, 340 (2018).</li><li>Hernandez-Sanabria, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+particular+bacterial+PCR-denaturing+gradient+gel+electrophoresis+patterns+with+bovine+ruminal+fermentation+parameters+and+feed+efficiency+traits.">Correlation of particular bacterial PCR-denaturing gradient gel electrophoresis patterns with bovine ruminal fermentation parameters and feed efficiency traits.</a> Applied and Environmental Microbiology. 76, 6338-6350 (2010).</li><li>Masters, J. R., Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+medium+and+passaging+cell+lines.">Changing medium and passaging cell lines.</a> Nature Protocols. 2, 2276-2284 (2007).</li><li>Calatayud, M., Velez, D., Devesa, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+inorganic+arsenic+in+intestinal+epithelial+cell+lines.">Metabolism of inorganic arsenic in intestinal epithelial cell lines.</a> Chemical Research in Toxicology. 25, 2402-2411 (2012).</li><li>Minekus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardised+static+in+vitro+digestion+method+suitable+for+food+-+an+international+consensus.">A standardised static in vitro digestion method suitable for food - an international consensus.</a> Food &amp; Function. 5, 1113-1124 (2014).</li><li>Berney, M., Hammes, F., Bosshard, F., Weilenmann, H. U., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+interpretation+of+bacterial+viability+by+using+the+LIVE/DEAD+BacLight+kit+in+combination+with+flow+cytometry.">Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry.</a> Applied and Environmental Microbiology. 73, 3283-3290 (2007).</li><li>Props, R., Monsieurs, P., Mysara, M., Clement, L., Boon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+biodiversity+of+microbial+communities+by+flow+cytometry.">Measuring the biodiversity of microbial communities by flow cytometry.</a> Methods in Ecology and Evolution. 7, 1376-1385 (2016).</li><li>Yamashita, Y., Takeshita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oral+microbiome+and+human+health.">The oral microbiome and human health.</a> Journal of Oral Science. 59, 201-206 (2017).</li><li>Wade, W. 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+oral+microbiome+in+health+and+disease.">The oral microbiome in health and disease.</a> Pharmacological Research. 69, 137-143 (2013).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+resource+for+cell+line+authentication,+annotation+and+quality+control.">A resource for cell line authentication, annotation and quality control.</a> Nature. 520, 307 (2015).</li><li>Pelaseyed, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mucus+and+mucins+of+the+goblet+cells+and+enterocytes+provide+the+first+defense+line+of+the+gastrointestinal+tract+and+interact+with+the+immune+system.">The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.</a> Immunological Reviews. 260, 8-20 (2014).</li><li>Bengoa, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+gastrointestinal+conditions+increase+adhesion+ability+of+Lactobacillus+paracasei+strains+isolated+from+kefir+to+Caco-2+cells+and+mucin.">Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.</a> Food Research International. 103, 462-467 (2018).</li><li>Kleiveland, C. R., et al., Verhoeckx, K., et al. <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 Impact of Food Bioactives on Health: in vitro and ex vivo models. , 197-205 (2015).</li><li>Laparra, J. M., Sanz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+in+vitro+models+to+study+bacterial+adhesion+to+the+intestinal+epithelium.">Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium.</a> Letters in Applied Microbiology. 49, 695-701 (2009).</li><li>Gagnon, M., Berner, A. Z., Chervet, N., Chassard, C., Lacroix, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Caco-2,+HT-29+and+the+mucus-secreting+HT29-MTX+intestinal+cell+models+to+investigate+Salmonella+adhesion+and+invasion.">Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion.</a> Journal of Microbiological Methods. 94, 274-279 (2013).</li><li>Gro&#223;kopf, T., Soyer, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microbial+communities.">Synthetic microbial communities.</a> Current Opinion in Microbiology. 18, 72-77 (2014).</li></ol>

著者がある何も開示するには

口腔マイクロバイは、最近いくつか著者20,21によって報告された人間の健康の重要な要素です。前の調査結果は、大量の細菌を含む唾液の摂取が免疫のプライミングのためメインのサイトの 1 つである小腸の微生物の生態系に影響を与えることができますをお勧めします。ホスト上の微生物のコンポーネントの影響を解明して腸の上皮と粘液分泌細胞によって表されるホスト インターフェイスで静的な上部消化管消化モデルの組み合わせです。
In vitroモデルは、機構研究を実施し、背景知識を減らすため、意図を達成するための研究を行う研究生体内でより効率的より良いターゲットの基本的なツールです。このため、純度、生存率、および合成微生物コミュニティを確立する前に無菌共生栽培の最適な成長を確保する重要です。生存率と分析にバイアスを避けるために、細胞培養への暴露前に細菌群集組成の評価をお勧めします。破損した細菌の数が多い可能性があります接着を妨げるし、さらに細胞モデルの整合性に影響を与えます。さらに、セル行誤認、汚染、および貧しい注釈、22実験の再現性を高めるセルラインの信頼できるソースの使用が最小限になります。
粘液産生細胞などにより、小腸に似ている、粘液とムチンは消化管23の最初防衛線を提供します。さらに、粘膜は細菌の代謝産物の特徴づける二国間輸送を可能にする細菌の植民地化に適しています。また、模擬消化管条件はカコ 2 とムチンに24、我々 のモデルで消化器系のプロセスを組み込むことの関連性を支えるラクトバチルスパラカゼイ系統の接着能力を高めます。
以前に再検討された25として、免疫ホスト コンポーネントを組み込むモデルでさらなる改善を導入できます。ただし、上皮および免疫細胞の現在の共培養システム使用されていないこれらのシステムがない場合があることを示す可能性がありますホスト微生物相互作用のアプリケーション (例えば、プロバイオティクス食品の生理活性化合物の評価)再現性25。
カコ 2、HT29 MTX または共培養前の研究は、プロバイオティクスや哺乳類細胞26,27に病原性株の付着を評価しました。単一を使用して汚れや微生物のいくつかの団体があります限られた概要ホスト微生物の相互作用、それは人間の消化管の複雑さの代表ではないです。自然の微生物社会の使用より体内のシナリオの代表をすることができます、彼らは特徴付けるため勉強することは困難。対照的に、我々 の方法論は、複雑な代表的な小宇宙を使用して、複数のホスト ・微生物相互作用を評価する機会を提供しています。定義された合成微生物群集は、複雑さの軽減28システムの世代の使用します。社会の変化は、個々 の実験においてアイソレータケージ条件の結果をすることができます。したがって、このモデルの微生物のコンポーネント簡単にスケールできます上または下選択的な力としての環境の影響を分解するため。最後に、サンプリング アクセシビリティを保証さらにひと細胞と関連する微生物群集の機能的な活動の性格描写。
過去数年間における技術に基づく新しい体外モデルが開発されています。Organoids が表される一次組織の主要な機能のいくつかを組み込む 3 D 細胞培養です。腸 organoids 成体幹細胞と組織を勉強するために近い生理的なシステムですが、このようなモデルには、いくつかの制限もあります。腸 organoids 制限を使用して免疫細胞を欠けていると同時に感染症や薬物、炎症反応に似ているのです。また、organoids、生存率、サイズ、および表現型スクリーニングおよび標準化を複雑なレンダリングを妨げる形状に関する異種。不死化細胞株の in vitro健全なティッシュのモデリングのための制限にもかかわらず、よ特徴付けられた、安定したセルラインを使用は、再現性、再現性、低コストなどの利点を提供しています。これらの利点は、いくつかの条件の同時上映が、データの橋渡しの使用は物議を醸す。一次電池より、生体内の生理; の代表があります。ただし、個体間のばらつきが大きい、完全に特徴付けされていません、異種、限られている時間のスパン。これらの要因など複数の条件のための欠点は、または 1 つの試金で複製。
高い柔軟性と基本的なセル装備研究所で応用より代表まで再現性と再現性のあるモデルの開発に着目した細胞を組み合わせて複雑な微生物群集の挑戦を表しているよう利用可能なモデルになりよく特徴付けられました。腸インターフェイス特徴づける異物への影響、例えば、上部消化管におけるプロバイオティクスの挙動を評価するためにこれらのマルチコンパートメント モデルを使用して多様な細菌群集の機能評価を行った.したがって、プロバイオティクス、薬、または関連して定義された体外生態系内の食品化合物の多種多様なさらに試金のための拠点としてこのモデルを提案します。

人間は、細菌細胞の1と同じ数であると同居します。したがって、それは人間のマイクロバイの包括的な理解を得ることが重要重要なは。こうして様々 な細菌やそれらの別の場所でバイオ フィルムを含むいくつかの小さい生息地に分かれていることで口腔内はユニークな環境です。オープンなエコシステムをされて、口の中にいくつかの種は、一時的な訪問者にあります。ただし、特定の微生物を誕生とフォーム編成バイオ2後はすぐに植民地化します。これらは上記の歯肉溝、歯肉縁下の隙間、舌、粘膜表面と歯科補綴、充填3歯の表面で発見されます。細菌はフロックと歯管の内腔の浮遊性細胞壊死の歯髄組織と混合または流体相の中断として存在できます。
アクティブな連続的な宿主細胞と常駐微生物叢4間クロストークがあります。内および種の細菌間で通信し、他の細菌がこれらの主な植民にアタッチしながら、自然の植民の小さい割合だけが組織に付着することが。例えば、微生物間のセル セルの結合、口腔バイオ フィルム、二次植民に統合し、相互作用する微生物電池4の複雑なネットワークの構築にとって重要です。約唾サンプルの細菌の集合体の 70% は正体不明のバクテロイデス門、 Veillonella sp. Prevotellaの sp連鎖球菌sp.についての sp によって形成されます。F. nucleatumは、ポケット内のバイオ フィルムと歯周炎5に関与しているp、t. へとTannerella レンギョウ、後半の植民と集計中間植民です。さらに、連鎖球菌可鍛鋳鉄 s. 血統とs. gordonii歯3を植民地化ことを好む中粘膜と歯科の生息地を占めています。したがって、s. 血統にある下の切歯と犬歯、上部前歯6に放線菌 naeslundiiが見つかったとき。
さらに、先住民マイクロバイは2人の健康を維持する上で役割を果たしています。免疫教育、病原体の膨張を防止する常駐微生物叢が参加しています。この植民地化の抵抗は、サーフェスにアタッチするのに適応しより効率的な成長のための利用可能な栄養素の代謝でのネイティブの細菌をより良い可能性があるために発生します。プロバイオティクス菌株は消化管の通過を生き残るため、アクティブのまま、消化管の上部の場所から飲み込んだ土着菌の永続性が完全に記載されていません。したがって、シミュレートされた胃腸管内輸送条件に人工コミュニティ、口腔生態系の代表を受けます。細菌の細胞の生存率は、腸上皮に似たマルチコンパートメント モデルを使用して評価されました。現在腸シミュレータは、内腔の微生物コミュニティ7の解析の観点から適切な再現性を提供しています。ただし、細菌の付着やホスト微生物間相互作用別に対処、微生物と細胞を組み合わせて、8に挑戦します。対照的に、腸内のインターフェイスで報告されるイベントの成功植民地化の潜在的な機械論的説明を提供するフレームワークを提案する.確かに、このモデル ホスト表面上の微生物の影響を評価する、共同で腸の静的モデルを使用できます。

<table>
	<tbody>
		<tr>
			<td>STRAINS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregatibacter actinomycetemcomitans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 43718</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium nucleatum </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10953</td>
			<td></td>
		</tr>
		<tr>
			<td>Porphyromonas gingivalis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33277</td>
			<td></td>
		</tr>
		<tr>
			<td>Prevotella intermedia </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25611</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mutans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25175</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sobrinus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33478</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomyces viscosus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15987</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus salivarius&#160; TOVE-R</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mitis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49456</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sanguinis </td>
			<td>BCCM/LMG Bacteria Collection</td>
			<td>LMG 14657</td>
			<td></td>
		</tr>
		<tr>
			<td>Veillonella parvula </td>
			<td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures</td>
			<td>DSM 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus gordonii </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49818</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>86010202</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29-MTX cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>12040401</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS AND CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) broth</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar 2</td>
			<td>Oxoid</td>
			<td>CM0055</td>
			<td>Blood Agar medium</td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M9429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>H9039</td>
			<td></td>
		</tr>
		<tr>
			<td>5% sterile defibrinated horse blood</td>
			<td>E&#38;O Laboratories Ltd,</td>
			<td>P030</td>
			<td></td>
		</tr>
		<tr>
			<td>InnuPREP PCRpure Kit</td>
			<td>Analytik Jena</td>
			<td>845-KS-5010250</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>Big Dye</td>
			<td>Applied Biosystems</td>
			<td>4337454</td>
			<td>Dye for sequencing</td>
		</tr>
		<tr>
			<td>ABI Prism BigDye Terminator v3.1 cycle sequencing kit</td>
			<td>Applied Biosystems</td>
			<td>4337456</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 culture flasks uncoated, cell-culture treated, vented, sterile</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 
			0.4%, liquid, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture media, with GlutaMAX and Pyruvate</td>
			<td>Life technologies</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3450-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach Type II&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactivated fetal bovine serum</td>
			<td>Greiner Bio One</td>
			<td>758093</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100 for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium, magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce LDH Cytotoxicity Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>88953</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning HTS Transwell-24 well, pore size 0.4 &#181;m</td>
			<td>Corning Costar Corp</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Serva Electrophoresis</td>
			<td>28539010</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber improved</td>
			<td>Carl Roth</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>653118</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>MoBio</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Flush system</td>
			<td>Custom made</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>InnOva 4080 Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1007</td>
			<td>Shaker for 2.10</td>
		</tr>
		<tr>
			<td>Memmert CO2 incubator</td>
			<td>Memmert GmbH &#38; Co.</td>
			<td>ICO150med</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS (Electrical Resistance System)</td>
			<td>EMD Millipore, Merck KGaA</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Milli-Q academic, ultra pure water system</td>
			<td>Millipore, Merck KGaA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker (ROCKER 3D basic)</td>
			<td>IKA</td>
			<td>4000000</td>
			<td>Shaker for 6.10</td>
		</tr>
	</tbody>
</table>

assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface

ホストと細菌叢との間の相互作用を長い間認識されており、記載されています。口の中は、常駐細菌が発生し、外因性の細菌によって植民地化を防ぐことができます、消化管の他のセクションに似ています。確かに、600 種以上の細菌が口腔内にあるし、単一の個人はいつでも異なる 100 前後を運ぶことができます。口腔内細菌は、居住者の微生物のコミュニティおよび支持の成長そして存続の内で統合になって口腔生態系における様々 なニッチを遵守する能力を有する.しかし、腸内細菌のバランスを乱すに嚥下時に腸に細菌の流れが提案されています。実際には、 pの経口投与は回腸内細菌叢の細菌の組成をシフトしました。生存とシミュレートされた胃腸管内輸送条件を受ける口腔内細菌の生存率を明らかにするのに自然な口腔生態系の簡略表示として合成コミュニティを使用しました。14 種は選択体外唾液、胃、および腸の消化プロセスを受けるおよび腸粘膜上皮をシミュレートするカコ 2 と MTX HT29 細胞を含む細胞のマルチコンパートメント モデルを提示しました。このモデルは、腸肝循環に関与する細胞に飲み込んだ細菌の影響を解明するため提供しています。合成のコミュニティを使用して、制御性と再現性のことができます。したがって、この方法論は、病原体の生存率と関連付けられている炎症変化、プロバイオティック混合物の植民地化の能力を評価するために合わせることができる、最終的には、潜在的な細菌に広範囲 presystemic の循環に影響を与えます。

このプロトコルは、生存とシミュレートされた胃腸管内輸送条件を受ける口腔内細菌の生存率の解明に適したモデルの生成に します。個々 の緊張からそのままなセルのカウントは約 108細胞 mL-1前合成のコミュニティでは、コミュニティ (確立中に実行可能なセルの 90% 以上含まれている多種間の縮図の作成図 1Aおよび1 b)。細菌の生存率ライブ/死者の定量化に基づく、各消化手順 (図 2) 後減少します。これは、生理的条件下で発生する、小腸消化液中に含まれる胆汁酸塩の胃の通過中に酸性の pH の結果をすることができます。In vitroとin vivoの両方消化消化を通過中に生存環境の変動に依存するかもしれません (例えば、 対供給絶食条件)、あるいは細菌保護プロセス (胞子形成や腸溶性コーティング)。
流れフローサイト メーター カウントは、熱殺された細菌や正確な生菌定量化18を実行する、背景にサンプルなどの適切な制御と比較する必要があります。胃や小腸の消化の過酷な条件では、どちらか成長するか分割する生きたバクテリアである可能な培養細胞に細菌を切り替えることができます。このため、プレート カウントは、フローサイトメトリーを用いて生菌によって異なります。例えば、図 3、粘膜のインターフェイスから回復した生菌は流れの cytometry プロットに青色で着色されます。ただし、成長が認められなかったときこれらの粘液サンプルされたメッキ (結果は示されていない)。
細菌が生存率を表示分数は、プレート カウント (図 4) によって明らかにされた、細胞の残骸 (S7) です。コロニー数の変数がありますが、代謝活性細菌の存在が少ない希薄サンプルで発見しました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57699/57699fig1.jpg"> 図 1: 細胞の in vitro消化消化の初めに多種間微生物群集を構成する細菌の生存率です。ライブ/死染色とフローサイトメトリーによる定量化(A)生菌数 (ログ単位) (平均 ± 標準偏差, n = 3)。(B)代表的な流れ cytometry プロット多種間微生物共培養 48 時間後の。それぞれ緑、赤、および黒を表す実行可能で、破損している、背景または非分類細菌のドットします。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57699/57699fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57699/57699fig2.jpg"> 図 2: 模擬消化管消化後の細菌の生存率をセルします。バーは、口腔、胃、および小さい腸の消化手順後生菌 (ログ単位) の数を表す (平均 ± 標準偏差, n = 3)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57699/57699fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57699/57699fig3.jpg"> 図 3: 粘膜の相間で細菌細胞の代表的なプロットします。赤いドットは、背景と青のドット実行可能なセルを表します。条件は、小道具らに基づいて設立されたフローサイトメトリーします。(2016)19。 粘膜サンプル希釈 1: 100 (v/v)、Sybr グリーン/Propidium ヨウ化で、15 分間染色し 100 μ l のサンプルを FL1 で測定した: 533/30 nm、FL2: 585/40 nm、FL3: > 670 nm ロングパスとを FL4: 675/25 nm。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57699/57699fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57699/57699fig4.jpg"> 図 4: コロニー形成単位 (CFU) で細胞の残骸から成長が BHI プレートを変更します。3 生物学的複製は細菌の付着試金のため使用され、CFU 定量化のため 3 つの技術的な複製を行った。細胞の残骸 (S7) の-6 希薄に-1 を含む 2 つのレプリケート プレートは図のとおりです。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57699/57699fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>細胞培養</td>
			<td>付着性/懸濁液</td>
			<td>養殖中</td>
			<td>一般的なサプリメント</td>
			<td>特定のサプリメント</td>
			<td>倍加時間</td>
			<td>培養条件</td>
		</tr>
<tr>
<td>カコ 2 (ECACC 86010202)</td>
			<td>付着性</td>
			<td rowspan="2">4.5 G/L グルコース ダルベッコ変更イーグル培地 (DMEM)</td>
			<td rowspan="2">ウシ胎児血清 (10 %v/v) を不活化 ペニシリン (100 U/mL) * ストレプトマイシン (0.1 mg/mL) * アムホテリシン (0.0025 mg/mL) *</td>
			<td>Glutamax (4 mM) 非必須アミノ酸 (1 %v/v) ピルビン酸 (1 mM)</td>
			<td>65-72 h</td>
			<td rowspan="2">95% 湿度と 10% CO2 CO2インキュベーター (材料の表を参照)</td>
		</tr>
<tr>
<td>HT29 MTX (ECACC 12040401)</td>
			<td>付着性</td>
			<td>HEPES (1 mM)</td>
			<td>20-24 h</td>
		</tr>
<tr>
<td colspan="7">* 抗生物質や抗真菌薬が、アッセイを開始する前に 2 日間の細胞培養媒体から削除されました。</td>
		</tr>
</tbody></table>表 1: 細胞文化の維持の細胞ラインおよびメディア組成の説明。

Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

腸宿主微生物間相互作用は、合成経口コミュニティ、体外消化消化、および小腸上皮のモデルを組み合わせる手法を使用して評価されました。病原体と複数種バイオ フィルム細胞浸潤を評価したり、プロバイオティクス製剤の生存性をテストするために合わせることができる方法を提案します。

培養腸宿主微生物インターフェイスで合成細菌コンソーシアムの実行可能性を評価します。

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

assessing-viability-synthetic-bacterial-consortium-on-vitro-gut-host

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

11.5K ビュー.  Ghent University. 腸宿主微生物間相互作用は、合成経口コミュニティ、体外消化消化、および小腸上皮のモデルを組み合わせる手法を使用して評価されました。病原体と複数種バイオ フィルム細胞浸潤を評価したり、プロバイオティクス製剤の生存性をテストするために合わせることができる方法を提案します。

ビデオ: Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

のpH依存性活性の検出<em&gt;エシェリヒア・コリ</em&gt;シャペロンHdeB<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

生化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

ビオチン化細胞表面タンパク質の精製<em&gt; Rhipicephalus microplus</em&gt;上皮腸細胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

体外初期エンドソームの F-アクチンの重合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

カエノラブディティス ・ エレガンスにおけるハイスループット化合物スクリーニングのための簡単な方法

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

植物の鉄含有量を測定するための比色定量法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

格子光シート顕微鏡による表面T細胞受容体ダイナミクスの可視化4次元的な研究

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

支持浮遊ブロックを用いた透過電子顕微鏡におけるサンプル支持膜の作製

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...