著者らは、スキャンソフトウェアの改善との継続的なサポートのためのマスター参照モデルとAlicona会社を製造するための歯科技工士ニコラLanfranconiに感謝します。

1。試料の準備/ベース<ol><li>標本の周りフラットベースを適用します。スキャンテーブルの上に試料を置きます。水平面（ 図1）に咬合面を配向。 </li></ol> 2。ソフトウェア解析<ol><li>プログラムを起動した後、実験室測定モジュール（ 図2） </li><li>スキャンテーブルの中心に試料を置きます。 </li><li>正しい倍率レンズを選択してください。大きなオブジェクトの場合は、完全なアーチスキャンのように、5倍の対物レンズを使用しています。 </li><li>試験片の表面はライブビューウィンドウ（ 図3）上に表示されるまで、3Dマウスを使って、スキャン光学系を移動します。 </li><li>最適な走査パラメータを達成するための露出とコントラストを設定するためにセンサ制御を使用してください。金属表面の場合は、400および800秒と0.3と0.8の間のコントラストの間に露出します（ 図4）を使用します。 </li><li>ボタンをチェックすることにより、画像品質を確認「ライブプレビューで表示画質」。 </li><li>の「測定制御」で正しいパラメータを設定します。測定タイプが「3Dデータセット」である。に、ImageFieldタイプ」は、一般に、ImageField」（ 図5）である。 </li><li> 「新しいのImageField」をクリックします。 </li><li>スキャンボリュームを定義します。ソフトウェアは、メジャーボリュームの境界座標を必要とします。 <ol><li>最高と最低のスキャンレベルに試料を移動し、両方の点で、「追加位置」をクリックしてください。 「Z範囲」の値は、スキャンボリューム（ 図6）の実際の高さを示している。 </li><li>走査空間のXY boundingsに試料を移動します。 X軸およびY軸の長さを定義するために、「追加位置」をクリックしてください。スキャンボリュームの寸法は、「情報」タブで示され、X軸及びY軸が1cmで試験片の寸法を超えるべきである。 </li></ol></li><li> 「ポイント」の番号を確認してください。ソフトウェアは、1つ &quot;のImageField「スキャンにおける走査億表面の点が可能である。実数この点の制限が超えています。ポイントサイズの「横ダウンサンプリング」によって計測点の数を「デシメーション」。 <ol><li> 「詳細設定」（ 図7）をクリックします。 </li><li> 「ポイント」数は100、M（ 図7）以下に減少するまで、右に「ラテラルダウンサンプリング」のスライダを移動します。ダウンサンプリングは、走査対象のより大きな画素サイズで、その結果、表面の点の横方向の解像度を低下させる。最適なスキャン結果を確実にするために、垂直方向の解像度が増加する。 </li></ol></li><li> 「測定開始」をクリックします。これは「プレビューモード」を起動します。ソフトウェアは、選択されたXおよびY軸寸法のプレスキャンを実行する。 </li><li>プリスキャンが完了した後、関心領域を選択する。これは、ファイルサイズと走査時間（ 図8）を低減するのに役立つ。 </li><li>フラットベース（ 図9）を含む、試料の周りの試料と2小節のフィールドを除いて、プレスキャンのすべての部分を選択します。 </li><li>スキャンを開始するには「スタート」をクリックします。 </li><li>マウスを使って「ImageViewerの」でスキャンを制御し、マウスの左ボタン（ 図10）を押します。 </li><li>ウィンドウを閉じ、「オンリービュー疑似色」をクリックしてください。 </li><li> 「設定」をクリックして、「擬似カラーリング」と「再現性」を選択します。 </li><li> 「最大」に設定0.2に付加価値と「適用範囲」をクリックしてください。 </li><li>再現性を制御するには、同じ傾斜と材料と地域のために等しくなければなりません。具体的には、試料の周りのベースは、均質な再現性（ 図11）を表示する必要があります。 </li><li> 「データベース」をクリックし、適切なフォルダにスキャンを保存します。 </li><li>必要に応じて、さまざまなファイル形式へのスキャンをエクスポートします。 「File/Export/3Dデータとして/ ...」をクリックしますプロトコルは、その時点で一時停止し、後に継続することができる。 </li></ol> 3。差分解析<ol><li>ベースを離れてカットする「3D-Editor &quot;を使用してください。このエリアには、違いのために使用されることはありません分析。 </li><li> 2つのスキャンを比較し、分析するために、「差測定」ソフトウェア（ 図12）を起動します。 </li><li>比較する2番目のモデルを選択します。 </li><li>モデルの最初の一致を実行するために「自動ラフアライメント」をクリックします。 </li><li> 「手動調整」をクリックして、それらが同じ向き（ 図13）になるまで回転させて移動してモデルを揃える。 </li><li> 「自動アライメント」をクリックして、最適なモデルマッチングに最適なアルゴリズムを開始するには「適用」。 </li><li> 2マッチしたモデルの差分マップを表示するには、 &quot;違い&quot;をクリックしてください。 </li><li> （ 図14）の偏差を表示するには、適切な色の範囲を選択します。分析用のスクリーンショットのような視覚偏差パターンを保存します。 </li></ol>差の統計値を表示する「統計」をクリックします。 1ミクロンクラスのサイズを選択して、統計的な比較のためにテキストファイルにヒストグラムデータを保存する（ 図15）。

<ol>
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Mater. 19 (1), 19-24 (2003).</li><li>Mehl, A., Ender, A., M&#246;rmann, W., Attin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+testing+of+a+new+intraoral+3D+camera.">Accuracy testing of a new intraoral 3D camera.</a> International Journal of Computerized Dentistry. 12 (1), 11-28 (2009).</li><li>Fickl, S., 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=Dimensional+changes+of+the+ridge+contour+after+socket+preservation+and+buccal+overbuilding:+an+animal+study.">Dimensional changes of the ridge contour after socket preservation and buccal overbuilding: an animal study.</a> J. Clin. Periodontol. 36 (5), 442-448 (2009).</li><li>Vlaar, S. T., vander Zel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+dental+digitizers.">Accuracy of dental digitizers.</a> Int. Dent. J. 56 (5), 301-309 (2006).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+complete-arch+dental+impressions:+a+new+method+of+measuring+trueness+and+precision.">Accuracy of complete-arch dental impressions: a new method of measuring trueness and precision.</a> 109 (2), 121-128 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+scanning+strategies+on+the+accuracy+of+digital+intraoral+scanning+systems.">Influence of scanning strategies on the accuracy of digital intraoral scanning systems.</a> Int. J. Comput. Dent. 16 (1), 11-21 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+arch+scans:+conventional+versus+digital+impressions--an+in-vitro+study.">Full arch scans: conventional versus digital impressions--an in-vitro study.</a> Int. J. Comput. Dent. 14 (1), 11-21 (2011).</li><li>Meer, W. J., Andriessen, F. S., Wismeijer, D., Ren, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+intra-oral+dental+scanners+in+the+digital+workflow+of+implantology.">Application of intra-oral dental scanners in the digital workflow of implantology.</a> PLoS One. 7 (8), (2012).</li></ol>

著者は、彼らが競合する経済的利益やその他の利害関係がないことを宣言します。

精度は、歯科医学の基本的な需要がある。基準スキャナは、高真度と精度で小規模および大規模なオブジェクトをスキャンすることができます。最適な走査方法であっても個々の歯の表面の詳細な形態学は、解像度が高く再現性でスキャンすることができる。スキャナの異なった倍率レベルで、マクロとミクロの形態学的構造を取得することができる。これは、モデルの様々な材料を走査す?...

精度は、歯科医学の多くの分野での主要な関心である。歯の硬組織を交換すると、適切な機能を確保し、さらに残りの歯の構造1,2を破壊防ぐために、正確なフィッティングプロテーゼを必要とします。固定部分義歯と総補綴物は、準備された歯やインプラント3のような構造を支援する正確なフィッティングのために特に重要である。非常に正確な再現が必要な理由は、特に、歯科印象と歯科技工所のワークフローの分野である。しかし、歯科治療の他の分野は、治療の成功を確認し、新たな治療戦略などソフトとハードの組織増強、腐食や磨耗監視、歯周治療、および矯正治療4,5を評価するために、真実かつ正確な韻律成果から利益を得る。これらの分野の多くでは、現在の検証手順は、キャリパーや顕微鏡6,7との直線距離の測定値である。これらのメタODSは、少数の測定点と検査部位の三次元（3D）の変化の限定された情報に限定される。新しい測定方法は、試験対象物8,9の表面全体の光または放射線撮影が挙げられる。ここで、全表面又は体積を測定し、コンピュータ画面上の3Dオブジェクトとして表示される。リニア測定が可能であるだけでなく、様々なスキャン時間からモデルの重ね合わせ。この重ね合わせにより、各スキ​​ャン点における表面変化の評価が可能である。これは、特定の領域を監視したり、すべての3座標軸の変形を表示する可能性を可能にします。また、体積変化が10を測定することができる。これらの新しい方法に限定する点は、試験対象物を捕捉するために使用されるスキャナの精度である。基準スキャナの精度内の変更のいずれも、試験対象または走査誤差の変化に分割することができない。スキャン精度は、多くの場合、MANUによって与えられた値であり、小さ ​​な、校正されたオブジェクト11をスキャンすることに由来しで経済。歯列弓のような大きなオブジェクトをスキャンする場合、この最小限のスキャンエラーが異なります。精度は、真度と精度で構成されています。真実は、その本当のジオメトリからスキャンされたオブジェクトの偏差です。精度を繰り返しスキャン（ISO 5725から1）との偏差である。本研究では、焦点変動走査技術に基づく新しい光学的基準スキャナは、最高の精度で完全なアーチモデルに単一の歯から検体をスキャンするために導入した。この基準スキャナは、従来とデジタル技術12月14日からと歯科咬合と歯科材料の摩耗に関する実際のプロジェクトのための歯科用印象の精度を比較した、いくつかの研究のためのベースとして使用された。本研究の目的は、基準スキャナおよび歯科研究の分野においてこのデバイスを使用するいくつかの可能性の精度の基本的な情報を提供することであった。

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr>
			<td>Reference model</td>
			<td></td>
			<td></td>
			<td>individual non-precious metal model, derived from a patient impression</td>
		</tr>
		<tr>
			<td>Araldit repair</td>
			<td>Huntsmen Advanced Material, Basel, Switzerland</td>
			<td></td>
			<td>used for making the base of the reference model</td>
		</tr>
		<tr>
			<td>CamBase</td>
			<td>Dentona, Dortmund, Germany</td>
			<td></td>
			<td>Type IV dental ston for pouring conventional impressions</td>
		</tr>
		<tr>
			<td>Identium</td>
			<td>Kettenbach, Eschenburg, Germany</td>
			<td></td>
			<td>Vinylsiloxanether impression material for conventional impression</td>
		</tr>
		<tr>
			<td>inEOS model holder</td>
			<td>Sirona Dental Systems, Bensheim, Germany</td>
			<td></td>
			<td>used for fixing stone models at the reference scanner</td>
		</tr>
		<tr>
			<td>Accutrans</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>used for making the base of thestone models</td>
		</tr>
		<tr>
			<td>President putty</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>mix with accutrans for betterstability of the base</td>
		</tr>
		<tr>
			<td>Alicona Infinite Focus</td>
			<td>Alicona Imaging, Graz, Austria</td>
			<td></td>
			<td>Reference scanner&#160;</td>
		</tr>
	</tbody>
</table>

accuracy in dental medicine, a new way to measure trueness and precision

基準スキャナは、手順の多くを確認するために、歯科医療において使用されている。主な関心は、彼らが歯科修復のための拠点として機能したような印象の方法を確認することです。多くの参考スキャナの電流制限は、完全な歯列弓のような大きな物体を走査し、精度の不足、または詳細歯の表面を評価するための限定された可能性である。焦点変動走査技術に基づいて、新たな基準スキャナは、最も一般的なローカルおよび正確性に関して評価した。特定の走査プロトコルは、歯科印象から元の歯の表面を走査するために試験した。また、別のモデル材料を確認した。結果は、完全なアーチスキャンの場合の精度のための真度および1.6±0.6μmの5.3±1.1ミクロンの平均偏差と基準スキャナの高いスキャニング精度を示した。 T現在の歯科用印象方法ははるかに高い偏差（12.5±2.5ミクロン：20.4±2.2μmで、精度の正確さ）を示したハン基準スキャナの内部スキャンの精度。単一の歯の表面のような小さなオブジェクトは、びらん性および研磨歯の表面損失を評価するためのシステムを可能にする、より高精度で走査することができる。基準スキャナは、歯科研究分野の多くのための違いを測定するために使用することができる。高い局所および全身精度と組み合わさ種の異なった倍率レベルは、完全なアーチに変化するまで、単一の歯または修復の変化を評価するために使用することができる。

図16aは、石膏試料のスキャンを示しています。最適なスキャンパラメータを確認するには、材料のための再現性を制御します。標本のセクションでは、さまざまなコントラストと明るさの設定でスキャンされ、再現性は、最適なスキャン設定を見つけるために、各スキャン後にチェックされている。金属表面は、高いコントラストでコントラストも低い1.0石膏や樹脂材料で最高?...

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Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

精度は、歯科医療の主要な需要である。正確性を検証するために、リファレンス·スキャナが必要とされている。この記事では、高い真度と精度を有する歯科用の形態の多種多様なを取得するように調整スキャン方法を使用して新しい基準スキャナを提示します。

歯科医学、真度と精度を測定するための新しい方法の精度

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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13.3K ビュー.  University of Zürich. 精度は、歯科医療の主要な需要である。正確性を検証するために、リファレンス·スキャナが必要とされている。この記事では、高い真度と精度を有する歯科用の形態の多種多様なを取得するように調整スキャン方法を使用して新しい基準スキャナを提示します。 

ビデオ: Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

人間の舌から茸（味）乳頭を収集する手法

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

全血におけるエノキサパリン抗凝固効果の迅速なポイントオブケアアッセイ

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

急速にマウス腸のホールマウントを準備するための簡単​​な装置

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

ゼブラフィッシュ行動症候群のための大胆、攻撃的、および浅水アッセイ

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

マイクロバイにおける健常児の口腔バイオ フィルム サンプリング

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

最大及び最大下運動生理・生物学的ヒトにおける急性運動レスポンスを測定するテストを実施

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...