ハーバード大学医学部のバイオポリマー施設からバイオアナライザーの技術支援を受けました。この研究は、全米多発性硬化症学会の研究助成金RG-1707-28516(H.L.W.およびS.L.)によってサポートされました。

ここに記載されている研究動物を含むすべての方法は、ハーバード大学医学部ブリガムアンドウィメンズ病院の施設内動物管理および使用委員会(IACUC)によって承認されています。
ここで説明するヒト研究対象を含むすべての方法は、パートナーズヒト研究委員会が定めたガイドラインに準拠しています。
1.糞便サンプル収集
<ol><li>オートクレーブまたはスクリューキャップ付きの滅菌およびヌクレアーゼフリーの2mLマイクロ遠心チューブを実験中の各動物対象用に調製する。<ol><li>ヒト被験者に対しては、各被験者に適切でヌクレアーゼフリーの滅菌便検体採取装置を提供する。</li>
	</ol></li>
	<li>無菌環境で各動物被験者から25〜100 mg(マウス糞便サンプルの場合は約1〜4個の糞便ペレット)を収集します。. 注:最高純度のRNAを得るには、2つ以上の糞便ペレット(~50 mg以上)が好ましい。<ol><li>必要なすべての個人用保護具(PPE)と材料(例:実験用手袋、消毒スプレー、滅菌ペーパータオル)を利用して、動物研究対象が置かれている作業エリアを滅菌し、糞便サンプルの汚染を回避します。<ol><li>ヒト被験者の場合、各研究対象/コレクターに、できるだけ無菌の環境で100〜200mgの便サンプルを収集するように指示します。標準的な無菌操作を使用し、便検体の汚染を避けてください。</li>
		</ol></li>
	</ol></li>
	<li>汚染を避けるために、他の表面に触れることなく、各動物被験者から糞便サンプルをスクリューキャップ付きの2mLマイクロ遠心チューブに直接収集します。<ol><li>ヒト被験者の場合、汚染を避けるために、提供された適用可能な収集装置(例えば、滅菌便検体収集キット)に直接排便するように各被験者に指示する。 注意: トイレの表面、水、尿、またはその他の非滅菌の表面/物体による汚染を避けるように被験者に指示します。</li>
	</ol></li>
	<li>2 mLマイクロ遠心チューブで採取した糞便サンプルをすぐに-80°Cで凍結するか、ドライアイスの入ったバケツに入れてRNAの量と質を高めてから、以下の手順で説明するように糞便を再懸濁します。<ol><li>ヒト糞便検体の場合は、200 mgの新鮮な検体をスクリューキャップ付きの2 mLマイクロ遠心チューブに分注し、以下に説明するように糞便を再懸濁する前に-80°Cで凍結します。<ol><li>スクリューキャップ付きの2 mLマイクロ遠心チューブにない便検体を保管する場合は、以下に説明するように糞便を再懸濁する前に、凍結検体をそれぞれ100〜200 mgの重量を量り、スクリューキャップ付きの個別の2 mLマイクロ遠心チューブに移します。 注:ヒトの糞便標本の場合、次の手順で問題が発生する可能性があるため、RNA分離のために200mgを超える便標本を服用しないでください。2 mLのマイクロ遠心チューブに過負荷のサンプルがあると、水相、有機相、および界面相が明確に形成および分離されない場合があります。手順はここで一時停止できます。</li>
		</ol></li>
	</ol></li>
</ol>2.洗浄液の調製
<ol><li>miRNAアイソレーションキット( 材料の表を参照)に含まれている洗浄液1に21 mLの米国化学会(ACS)グレードの100%エタノールを加えて、ボトルに示されているように最終容量30 mLに到達します。すべてがボトルに溶けるまで渦を巻きます。</li>
	<li>ボトルに示されているように、提供された洗浄液2/3に40 mLのACSグレード100%エタノールを加えて、最終容量の50 mLに到達します。5秒間、または最終混合物がよくブレンドされるまで渦巻きます。</li>
</ol>3.機器と材料の準備
<ol><li>作業エリアと機器、たとえば、実験室のベンチ、化学ヒュームフード内の作業エリア、マイクロ遠心チューブラックに、リボヌクレアーゼ(RNase)除染溶液(市販のRNase除染ソリューションなど)をスプレーします。汚染を避けるために、必要に応じていつでもどこでもRNase除染溶液を表面に塗布してください。</li>
	<li>清潔な白衣を着用し、フェイシャルマスクを着用し、適切な実験用手袋を着用して、糞便サンプル中のRNAを人間の皮膚に存在するヌクレアーゼから保護します。手袋にRNase除染液をスプレーし、汚染を避けるために手袋を頻繁に交換してください。</li>
	<li>糞便の再懸濁前の解凍を防ぐために、-80°Cで保存された糞便サンプル用のドライアイスのバケツと、貯蔵寿命を延ばすための材料用の氷のバケツを準備します。 注意: プロトコルで使用される培地を含む材料がヌクレアーゼの汚染なしに無菌であることを確認してください。</li>
</ol>4.糞便の再懸濁
<ol><li>25〜100 mgの糞便サンプルを600 μLの滅菌1xダルベッコリン酸緩衝生理食塩水(DPBS)に再懸濁します。 注意:糞便サンプルは、サンプル中の細胞が融解すると氷の結晶が細胞コンパートメントの内側と外側の両方で破裂するため、RNaseと細胞RNAの放出を最小限に抑えるために、部分的な解凍もせずに-80°Cから解凍したらすぐに処理する必要があります。<ol><li>室温(RT)の糞便サンプルを含むスクリューキャップ付きの2 mLマイクロ遠心チューブに600 μLの1x DPBSを追加します。</li>
		<li>600 μLの1x DPBSに沈めた糞便サンプルの混合物を、RTで30分間キャップした2 mLマイクロ遠心チューブでインキュベートします。</li>
		<li>1 mL ピペットチップでマッシュアップして混合物を再懸濁し、2 mL マイクロ遠心チューブをキャップした状態でよくボルテックスします。RNAの量と質を最適化して向上させるには、S4000(または4000 rpm)および45秒で1サイクルの設定でホモジナイザーで混合物を再懸濁します。</li>
	</ol></li>
</ol>5.有機抽出
注意: 酸性フェノールを使用して、ステップ6までの次の手順には、危険な化学物質のヒュームフードを使用してください:クロロホルムとACSグレードの100%エタノールは、毒性と可燃性のため。危険物を取り扱うときは、必要に応じてPPEを交換し、適切な標準の予防措置に従ってください。
<ol><li>600 μLの酸-フェノール:クロロホルムでRNAを抽出します(必要な酸-フェノール:クロロホルムの量は、ステップ4.1で添加した1x DPBSの初期量に相当します)。<ol><li>ステップ4.1の懸濁液に600 μLの酸-フェノール:クロロホルムを加えます。 注:上相が水性緩衝液と混合されるため、酸-フェノール:クロロホルムをボトルの下相から引き出します。これら2つの相の間の間相が乱されている場合は、汚染を避けるために間相が再確立したときにのみ、酸 - フェノール:クロロホルムを待って引き抜いてください。</li>
	</ol></li>
	<li>混合物を60秒間ボルテックスして完全に混合します。あるいは、収量中のRNA量を最適化および増加させるために、S4000および45秒で1サイクルの設定でホモジナイザーを使用して混合する。</li>
	<li>RTで10,000 x g で15分間遠心分離し、微量遠心分離機で水相と有機相を分離します。遠心分離後、間期はコンパクトになるはずです。そうでない場合は、遠心分離を繰り返します。 注:遠心分離を数回繰り返した後、添加された酸-フェノール:クロロホルムの量に対する初期体積の比率が不均一である可能性があるため、間期が希望するほどコンパクトでなかった場合は、汚染を避けるためにより注意して水相を回収してください。</li>
	<li>水相を回収し、ヒンジキャップ付きの新しい2 mLマイクロ遠心チューブに移します(miRNA分離キットには付属していません)。<ol><li>下相を乱さずに水性(または上)相を慎重に取り除き、ヒンジキャップ付きの新しい2mLマイクロ遠心チューブに移します。転送された容量に注意してください(例:~500 μL)。 注:間相がコンパクトで、上相が明確に分離されている場合、水相の上部にいくつかの小さな残留粒子が浮遊している可能性があります。ピペットでこれらの残留物を避け、目に見えて明確に分離された水相のみを回収して、少量の水相しか得られない場合でも、高品質のRNA収量を確保します。</li>
	</ol></li>
</ol>6. 最終的なRNA単離
<ol><li>2 mLマイクロ遠心チューブの水相に1.25容量のRT ACSグレード100%エタノールを追加します(たとえば、ステップ5.4から500 μLの水相を回収した場合は、625 μLの100%エタノールを追加します)。渦3秒。</li>
	<li>水相/エタノール混合物を、miRNA分離キットに付属のフィルターカートリッジに通します。<ol><li>サンプルごとに、フィルターカートリッジをキット付属の収集チューブの1つに入れます。<ol><li>ピペットで、600 μLの水相/エタノール混合物をフィルターカートリッジに入れます。 注:ピペッティングする前に、混合物を短時間ボルテックスしてエタノールを水相と完全に混合します。一度にロードできる水相/エタノール混合物は700 μL以下です。</li>
		</ol></li>
		<li>10,000 x g で90秒間遠心分離し、混合物をろ過します。高速で回転すると、フィルターが損傷する可能性があります。</li>
		<li>ろ液を廃棄し、連続したアプリケーションですべての混合物が同じフィルター膜でろ過されるまで、手順6.2.1から6.2.2を繰り返します。以下の洗浄手順で同じ収集チューブを保管して再利用します。</li>
		<li>700 μLのmiRNA洗浄液1でフィルターを洗浄します。 注意: miRNA洗浄液1には、皮膚の炎症や深刻な目の損傷を引き起こす可能性のあるグアニジンチオシアン酸塩が含まれています。手袋、フェイスシールド、防護白衣など、必要なPPEを着用してください。必要に応じて頻繁に手袋を交換してください。<ol><li>ACSグレードの100%エタノールで調製した作業溶液であるmiRNA洗浄溶液1を700 μLのフィルターカートリッジに塗布します。</li>
			<li>60秒間遠心分離して、miRNA洗浄溶液1をフィルターカートリッジを通してろ過します。</li>
			<li>収集チューブからろ液を廃棄し、同じフィルターカートリッジを同じ収集チューブに入れます。</li>
		</ol></li>
		<li>フィルターを洗浄液2/3で、700 μL、500 μL、250 μLの容量でそれぞれ1回ずつ連続して洗浄します。<ol><li>ACSグレードの100%エタノールで調製した作業溶液である洗浄液2/3の700μLをフィルターカートリッジに塗布します。<ol><li>10,000 x g で1分間遠心分離します。</li>
				<li>収集チューブからろ液を廃棄し、同じフィルターカートリッジを同じ収集チューブに入れます。</li>
			</ol></li>
			<li>500 μLの洗浄液2/3をフィルターカートリッジに塗布します。<ol><li>10,000 x g で1分間遠心分離します。</li>
				<li>収集チューブからろ液を廃棄し、同じフィルターカートリッジを同じ収集チューブに入れます。</li>
			</ol></li>
			<li>250 μLの洗浄液2/3をフィルターカートリッジに塗布します。<ol><li>10,000 x g で1分間遠心分離します。</li>
				<li>ろ液を回収チューブから捨てます。</li>
			</ol></li>
			<li>フィルターカートリッジを新しい収集チューブに移し、アセンブリを5分間回転させて、フィルターから残留液を取り除きます。</li>
		</ol></li>
	</ol></li>
</ol>7. 50 μLのヌクレアーゼフリー水でRNAを溶出
<ol><li>フィルターカートリッジを新しい収集チューブに移します。50 μLのヌクレアーゼフリー水をフィルターの中央にピペットで入れ、回収チューブにキャップをします。<ol><li>RTで10分間インキュベートします。</li>
		<li>8,000 x g で5分間スピンして、RNAを新しい収集チューブに回収します。</li>
		<li>蛍光光度計を使用して、回収された糞便RNAの濃度と純度を決定します。回収した糞便RNAは-80°Cで保存できます。</li>
	</ol></li>
</ol>

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著者らは、このプロトコルペーパーに記載されている研究方法に関連する関連または重要な金銭的利益を宣言しません。

分離中のRNase汚染を防ぐために、RNaseフリー技術を使用することが重要です7。遠心分離とコンパクトな間相の形成後、水相を回収するときは、水相、下相、および水相の上部に浮遊する粒子汚染物質を回避することが重要です。さらに、500 μLと250 μLの洗浄液2/3による2つの洗浄ステップが追加され、フィルター膜内の汚染物質を除去して品質を最適化します。さらに、200 mg?...

細胞外RNAは、多くの生物学的プロセスを媒介する重要な因子として認識されつつあります1。糞便中の細胞外RNAは、2008年に結腸癌および活動性潰瘍性大腸炎のマーカーとして最初に報告され2、最近、腸管腔および糞便の正常な成分として明らかにされ、宿主と微生物のコミュニケーションを媒介します3,4,5。このRNA分離プロトコルの目的は、動物およびヒトの被験者から収集された糞便サンプルから高品質の細胞外RNAを抽出することです。このプロトコルは、市販のmiRNAアイソレーションキットから採用されました。取得したRNAは、RNAシーケンシング、RT-PCR、マイクロアレイなどのダウンストリーム分析に利用されます。このプロトコルには、動物や人間の糞便に含まれるRNAの量と質を最大化するための重要で有用なヒントがいくつか含まれています。このRNA(マイクロRNAを含む)単離方法を開発および最適化する理由は、糞便中の微生物RNAを減らし、調査研究の変数を制限し、さまざまな交絡因子や汚染源を考慮せずに腸内のRNA組成を分析するためです。注目すべきことに、このRNA単離は、生細胞および生きた微生物からのRNAの放出を最小限に抑えます(細胞RNA)。腸細胞によって放出された、または食物摂取によって獲得された細胞外RNAに焦点を当てています。主に、この方法は微生物トランスクリプトームが調査される研究には適していません。

<table>
	<tbody>
		<tr>
			<td>Acid-Phenol: Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fischer Scientific</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mirVana miRNA Isolation Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM1561</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free microcentrifuge tubes (1.5 mL, 2 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (Not DEPC-treated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor and Nuclease-Free Pipette tips (with filter)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>QIAGEN</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal (micro) rna isolation

RNAは、動物やヒトの腸管腔や糞便に存在することが明らかになりつつあります。以下に説明するプロトコルは、動物およびヒト被験者の糞便サンプルからマイクロRNAを含む全RNAを単離する。目的は、RNAシーケンシング、RT-PCR、マイクロアレイなどのダウンストリーム分析のために、高純度および量のトータルRNAを単離することです。miRNA単離におけるこの最適化されたプロトコルの利点は、説明されている追加の洗浄ステップで高度に精製されたRNA製品を単離する能力、サンプルの再懸濁における改良された方法で得られるRNAの量の増加、および除染の重要なヒントです。1つの制限は、これらのサンプルサイズが間期の明確な形成を困難にするため、200 mgを超えるより大きなサンプルを処理および精製できないことです。その結果、サンプルサイズが大きいと、プロトコルに記載されているように、抽出される水相が、最終的に単離されたRNAの品質に影響を与える有機物で汚染される可能性があります。ただし、ほとんどのダウンストリーム分析には、最大200 mgのサンプルからのRNA分離で十分です。

代表的なRNAをそれぞれ50 mgのマウス糞便サンプル(2つのマウス糞便ペレット)および100 mgのヒト糞便標本から単離し、50 μLのヌクレアーゼフリー水で溶出しました。濃度の分光光度計分析は、49μgおよび16μgのRNAの総量がそれぞれ単離されたことを示唆している(表1)。RNA純度は、A260/A280比が~2.0、A260/A230比が~1.8であることで示されるように高かった(表1)。報告<sup class="xref...

Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

Fecal micro RNA Isolation

このプロトコルは、動物およびヒト被験者の糞便サンプルから高品質のトータルRNAを分離します。市販のmiRNA分離キットは、最適化された量と質で純粋なRNAを単離するために、大幅に適応して使用されます。RNA分離株は、シーケンシング、マイクロアレイ、RT-PCRなどのほとんどのダウンストリームRNAアッセイに適しています。

糞便(マイクロ)RNA単離

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

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Immunology and Infection

Unit 1

Fecal (micro) RNA Isolation

Diagnostic and Radiological Studies of the GI System

SCIENTISTS IN ACTION

4.3K ビュー.  Brigham and Women's Hospital and Harvard Medical School. このプロトコルは、動物およびヒト被験者の糞便サンプルから高品質のトータルRNAを分離します。市販のmiRNA分離キットは、最適化された量と質で純粋なRNAを単離するために、大幅に適応して使用されます。RNA分離株は、シーケンシング、マイクロアレイ、RT-PCRなどのほとんどのダウンストリームRNAアッセイに適しています。

ビデオ: Fecal micro RNA Isolation

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

ダニのRNA干渉

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

免疫・感染学

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

ヌクレオカプシドアニーリング媒介電気泳動（NAME）アッセイは、HIV-1ヌクレオカプシド阻害剤の迅速な同定を可能にする

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量的マイクロ中和アッセイの最適化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

ヒトの胎児微生物叢に対する介入レジメンの影響を推定するインビトロバッチ培養モデル

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

ヒト胎児標本におけるカンピロバクター・イェジュニの輸送培地における検出と生存の限界を決定する培養方法

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

糸球体腎炎における細胞外DNAの半定量化のための教師付き機械学習

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

インターロイキン10欠損マウスモデルにおける糞便微生物叢移植の治療的評価

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...