We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

倫理の声明：ヒト被験者、インフォームドコンセントと倫理の承認から細胞を採取する前に取得する必要があります。 1.ステインを準備注：実験日の汚れを準備します。 <ol><li> 15mlの遠心チューブに2.5ミリリットルのリン酸緩衝生理食塩水（PBS）に5μlのエチジウム（または4μM）および5μlのカルセインAM（または4μM）を追加します。ミックス。使用するまで室温で光とストアから遮蔽するためにアルミホイルで包みます。 </li></ol> 2.細胞を回収<ol><li>そのパッケージから細胞培養インサートを取り外し、各まぶたをサンプリングするためにラベルを付けます。永久的なマーカーを用いた膜プラスチックホルダー側の細胞収集のマークの向き。 ICを受ける地域の適切な健康状態を確認するために、適度な倍率（約20倍）で、スリットランプ検査を実施します。 </li><li> EAの下結膜嚢に局所麻酔薬（0.5％プロパラカイン塩酸塩）の1滴を分配chの目と目を保つために、参加者に指示するには、1分間閉じました。 </li><li>エバート上部眼瞼とまつ毛により所定の位置に保持します。蓋ワイパー領域との接触を避けます。 オプション：第二研究者の援助は、この手順を実行する必要があります。 </li><li>最小限の圧力で蓋ワイパー領域の中央部に垂直に膜を適用します。 </li><li> 5秒 - 3のための場所で膜を保持します。接触領域に半透明になった膜を観察します。この時点で、優しく膜を除去し、元の位置に戻し、「フリップ」に参加者のふたを可能にします。 </li><li>速やかに乾燥するのを防止するために、サンプルをPBSの一滴を適用します。膜は半透明に変わり、これが乾燥を示すように、いつでも不透明回すべきではありません。二次調査員は、サンプルの処理（セクション3）ですぐに進んでいます。 </li><li>結膜の整合性を確保するために、最終的な眼の検査を実施します。眼潤滑剤を分配し、参加者に指示麻酔効果が切れるまで、彼らの目をこすり避けるために（約15分）。 </li></ol> 3.サンプル処理<ol><li>マイクロはさみを使用して、垂直な細胞採取の中央から、半分（下流の分析のそれぞれに1つの半分）で膜をカット。プラスチック製のホルダーからそれを分離するための一つの膜半分の外縁に沿って切断。膜は、それ自体の上に折り畳まないことを保証を含む膜上に収集された細胞との相互作用を避けます。 </li><li>完全ホルダーから取り外す前に、ピンセットを用いて膜を固定します。 95％（v / v）エタノールを500μlを含む標識2 ml遠心チューブに膜を置き、（セクション4.2）を処理する前に、2時間の最大20分の最低固定するために残します。 </li><li>すぐに（セクション3.2で説明したように）膜の第二の半分を分離し、速やかにセクション4.1に進んでください。 </li></ol> 4.サンプル染色<ol><li> ImmunocytochemiCAL染色<ol><li>慎重に収集側を下に向けて35ミリメートルのガラスボトム培養皿に膜を置きます。膜が平坦であることを確認します。 </li><li>膜へのセクション1で組み立てられた免疫細胞化学的染色組成物のピペットを20μl。サンプルカールアップは、慎重にその縁部を押し下げることにより膜を平坦化する使い捨てのピペットチップを使用している場合。細胞収集領域との接触を避けます。 </li><li>ゆっくりとガラスカバースリップでメンブレンをカバーしています。サンプルの無駄な動きを避けてください。蓋付き皿を覆い、エッジの周りラボ・フィルムで密封します。ディッシュ（上部と下部）と試料の可視性の透明性を妨げないでください。ラボのマーカーでラベルを付けます。 </li><li>すぐにイメージング（セクション5.1）に進み、その後、膜の他の半分の細胞学的染色を継続するためにセクション4.2に戻ります。 </li></ol></li><li> 細胞学的染色 <ol><li>徐々にゆっくりとチューブのuに蒸留水500μLを加えることによって膜を水和セクション3.4に固定するためのsed。中身を取り出した後、混合するピペットチップに吸引する内容を数回。 </li><li>蒸留水500μLを加えます。削除した後、数秒間放置します。アルシアンブルー染色の500μLを加え、3分間のまま。汚れを削除し、3回連続500μlの蒸留水リンスや液体が透明にリンスするまで行います。 </li><li>ヘマトキシリン​​＃1染色の500μLを加え、3分間のまま。汚れを削除して、液体が透明リンスまで水リンスを蒸留または3回連続500μLを行います。透明な液体を吸引することにより、（セクション4.2.1の逆）を脱水。 </li><li>パパニコロウOG-6染色の500μLを加え、3分間のまま。汚れを削除し、1500μlの95％（v / v）エタノールリンスを行います。 </li><li>パパニコロウEA-65染色の500μLを加え、3分間のまま。汚れを削除し、（v / v）エタノールリンスの連続した​​3500μlの95％を実行します。 </li><li>完全に3回連続500μlの100％エタノールリンスを使用して脱水します。 TWEを使用しましたezers、溶液から試料を取り出します。感動細胞収集領域を避けます。 </li><li>スライドガラス上に膜を置きます。そのセルのコレクションラインを確保することは、並列またはイメージング中に容易に位置合わせのためのマージンをスライドする垂直のいずれかです。 </li><li> 100％エタノールのドロップを適用し、ガラススリップでカバーしています。すぐにイメージング（5.2節）に進みます。 </li></ol></li></ol> 5.サンプル画像<ol><li> イメージング蛍光免疫組織化学染色 <ol><li>画像取得ソフトウェアを実行しているコンピュータに接続されたデジタルカラーカメラを装備した11または蛍光顕微鏡12、共焦点レーザー走査顕微鏡（CLSM）を使用します。培養皿内に含まれるサンプルに、倒立顕微鏡を必要とすることができます。 </li><li>エチジウムホモダイマー-1（DNAの存在下で、617分の528 nmのEX /エム最大値）の吸収スペクトルおよび発光スペクトルとカルセインAM（517分の494 nmのEに係る顕微鏡11,12を設定しますX /エム最大値）。 </li><li> （ 例えば、2.5倍対物レンズ）顕微鏡で最も低い倍率を選択し、デジタルイメージングシステムを活性化させます。コンピュータのモニタ上に表示された試料を観察します。 </li><li>目的の細胞機能のサンプルを検査します。 </li><li>希望顕微鏡の倍率を選択し、イメージングソフトウェアを使用して画像を取得します。 </li><li>ネイティブ顕微鏡ファイル形式または非圧縮ファイル形式（ 例えば 、*の.raw、* .TIFF）のいずれかでファイルを保存します。 </li></ol></li><li> イメージング細胞学的染色 <ol><li>画像取得ソフトウェアを実行しているコンピュータに接続されたデジタルカラーカメラを装備していないフィルタを備えた標準的な実験明視野顕微鏡を、使用してください。 </li><li>顕微鏡ステージ上に配置し（ステップ4.2.8から）ロックスライドガラス。必要に応じて、全体のイメージング手順を通して、100％エタノールで試料を再水和します。 </li><li> （ 例えば 、2.5倍対物レンズ）顕微鏡で最も低い倍率を選択して、目を活性化させますEデジタルイメージングシステム。コンピュータのモニタ上に表示された試料を観察します。 </li><li>確保細胞収集は、顕​​微鏡ステージ制御のXまたはY軸のいずれかと整列されます。カバースリップを除去し、ピペットチップやピンセットを用いて試料を回転させることにより、必要に応じて調整してください。 </li><li>イメージングソフトウェアを使用して、最低の顕微鏡倍率で試料全体の画像（複数可）を取り込みます。 </li><li>適度な顕微鏡倍率（ 例えば、10 - 20X対物レンズ）に切り替えて、顕微鏡の光源とソフトウェア撮影パラメータ（露出、コントラスト、ホワイトバランスなど ）を調整し、その自動化されたソフトウェアメータリングを無効にします。将来の使用のためにこれらの設定を保存します。 </li><li>スキャンの開始点と終了点（細胞採取の例えば 、左上と右下の角）を決定します。 </li><li>イメージングソフトウェアを使用して、始点と終点内全体細胞収集領域の画像（複数可）を取り込みます。 betw 20％の側サイドと上から下への重複を確認してくださいすべての隣接する画像EEN。 </li><li>ネイティブ顕微鏡ファイル形式または非圧縮ファイル形式（ 例えば 、*の.raw、* .TIFF）を使用してファイルを保存します。 </li><li> （ 例えば、アドビPhotoshop Elementsの）選択肢の自動ステッチソフトウェアを使用して、最終的なパノラマ画像を生成します。最終的な画像に自動化されたステッチの精度を確認するために、ステップ5.2.5で撮影された参照画像）を使用します。 </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

印象の細胞学は、典型的には、混合セルロースエステル膜を用いて、眼球結膜上で実行されます。サンプルは、バルク材料のシートのサイズに切断し、滅菌、適用及び鉗子を使用して、結膜の表面から除去されます。同一の膜材料を用い、ピストン制御ICデバイスは、最近眼球結膜の表面形状に適合するように設計されており、アプリケーション13との間の一貫した圧...

眼球と対向する2ミリメートル狭い結膜地域-人間の上まぶたはわずか1で、毎日1約1万点滅を実行します。まばたきの間のワイピング動作による、眼瞼縁のこの部分は、「蓋ワイパー「領域2と呼ばれています。摩擦が原因で地球上ワイピング蓋に、常習的な点滅の間にこの領域で増加することが想定されます。これは、眼の快適で重要な役割を果たしている可能性があります。特定の経験眼の不快感のコンタクトレンズ装用者、これは3を着用レンズを停止するための主要な理由の一つです。コンタクトレンズが眼に配置されると、レンズ材料と眼の表面との間の摩擦係数を4に変更することが示されています。乾燥状態の症状は、この変更された摩擦2,5,6-に関連することができることを示唆する証拠があります。 この関連付けはまた、臨床的に観察現象に反映される可能性があり、特に蓋のwi上皮症（LWE）あたり、また上蓋マージン染色（ULMS）6と呼ばれます。 LWEは、眼の刺激の初期兆候とドライアイ疾患のための潜在的な予測因子です。これは、観察された上下の眼瞼縁の領域の生体染色によって測定されます。このグレーディングシステム2とその臨床的妥当性（自覚症状を持つ、すなわち、相関が）議論の下にまだあるが、眼の不快感は、最も重要なのは、患者にとって不便似臨床医と科学者のための難問のままと。 現在まで、少しは蓋ワイパー領域7の（サブ）携帯解剖学と生理学において臨床的に関連のバリエーションについては知られており、1つのレポートだけでは細胞学的方法8を利用します。印象細胞診（IC）は、膜9,10の適用によって眼球や瞼板結膜の上皮表面から細胞を収集するために40年以上にわたって使用されています。除去の際、接着細胞は、cを受けますytological染色および顕微鏡イメージング。蓋ワイパー領域における解剖学的および細胞性の違いのために、この技術は、最適化を必要とします。

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

プラスチックホルダーの上に張られた細胞培養膜は、蓋ワイパー結膜からの上皮細胞のハンドヘルド収集のための便利なツールです。この方法は、典型的には、IC膜を作製し、処理するために使用される追加の滅菌器具の必要性を排除する。 図1は、細胞培養インサートを使用して、蓋ワイパー領域のICを示します。我々は、新規な方法で蓋ワイパー領域?...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

蓋ワイパーエリアの印象細胞診

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

impression-cytology-of-the-lid-wiper-area

Research

JoVE Journal

Medicine

10.3K ビュー.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

ビデオ: Impression Cytology of the Lid Wiper Area

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

のTaqManワンステップRT-PCR法によるキッズスツールからノロウイルスの検出とGenogrouping

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

医学

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

膵臓腺癌組織のパラフィン包埋したスライド上のB7-H1（PD-L1）の免疫組織化学的染色

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

隔離された両心室ワーキングウサギ心臓のNADH蛍光イメージング

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

前立腺癌におけるバイオマーカーの発見への応用：個々のHERV座発現のマイクロアレイベースの識別

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

次世代シーケンシングのための収集と唾液DNAの抽出

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

梗塞エリアの中央動脈閉塞技術と可視化を使用したマウスにおける虚血性脳卒中および虚血 - 再灌流の誘導

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

[<sup&gt; 18</sup&gt; F] 3F4AP：脱髄疾患イメージングのための新規PETトレーサ

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

卵巣癌早期発見のための有望なツールとしてのファロピウス管の管細胞診

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

生体材料に対する細胞毒性と細胞応答へのアクセス

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...