我々 は実験にミス新楽しい呉を感謝したいと思います。本研究は、チャン庚医学研究プログラム (CMRPG3C0371、CMRPG3C0372、CMRPG3C0373) および IRB 100-4470A3、104-2462A3、台湾からの助成金によって支えられました。

臨床研究室改善改正 (CLIA) 1988 年の規制、制度検討委員会の長庚記念病院による承認の要件に従って行われた実験室で行ったテストと(ライセンス番号 100 - 4470A3 ・ 104 2462A3) の大学。すべての患者からインフォームド コンセントを得た。
1. 血液検体採取
<ol><li>市販の EDTA 処理管に全血を収集します。穏やかに混合し、処理まで 4 ° c の血液サンプルを格納します。</li>
</ol>2. 末梢血から DNA 抽出法
ゲノム DNA の抽出の DNA 抽出キットを使用 (材料の表を参照してください)。
<ol><li>ソリューション 1 の 10 mL を 4.0 mL の血液サンプルに追加します。10 分間氷のサンプルをインキュベートします。</li>
	<li>4 ° C で 5 分間 2,000 x g でサンプルを遠心分離します。削除し、上澄みを慎重に廃棄します。ソリューション 1 の 3 mL にペレットを再懸濁します。</li>
	<li>再び 4 ° C で 5 分間 2,000 x g でサンプルを遠心し、上清を捨てます。ソリューション 2 の 3 mL にペレットを再懸濁します。</li>
	<li>最終濃度 100 μ G/ml を取得してよく混ぜるプロナーゼ原液 (225 mg/mL) の 10 μ L を追加します。37 ° C で一晩インキュベートします。</li>
	<li>10 分追加 0.8 mL ソリューション 3 のサンプルに氷の上管を冷やすし、反転の 3 - 5 回。5 分間氷の上にサンプルを配置します。</li>
	<li>4 ° C で 15 分間 3,000 x g でサンプルを遠心分離します。慎重に 15 mL 滅菌円錐形遠心チューブに上清を転送します。</li>
	<li>最終濃度 20 μ G/ml を得るため RNase 原液 (10 mg/mL) の 6 μ L を追加します。37 ° c 15 分間インキュベートします。</li>
	<li>DNA を沈殿させるために数回を軽く反転して徹底的にイソプロピル アルコールとミックスの 2.5 mL を追加 (白、綿状の素材の鎖となる)。大口径ピペットを使用して、新しい 1.5 mL 遠心チューブに DNA 鉛塩法を転送します。</li>
	<li>4 ° C で 3 分間、12,000 × g で遠心分離慎重に上清を捨てます。70% エタノール 500 μ L を追加することによって洗浄の DNA の餌。</li>
	<li>1 分間 12,000 × g で遠心分離し、上澄みを廃棄します。空気常温乾燥 DNA の餌。DdH2O の 100 μ L を追加し、DNA を再懸濁しますに 15 分の 65 ° C で孵化させなさい。</li>
</ol>3. ゲノム DNA の定量化
分光光度計を使用して (材料の表を参照) ゲノム DNA の質と量を検出します。
<ol><li>分光光度計のマシンに接続されているコンピューターにログインします。ソフトウェアを開きます。</li>
	<li>分光光度計マシンを初期化します。<ol>
<li>分光光度計ソフトウェアを「核酸」をクリックします。</li>
		<li>使い捨ての紙ワイプと精製水で台座をクリーンアップします。</li>
	</ol>
</li>
	<li>空白の分光光度計。<ol>
<li>台座に精製水 2 μ L をロードします。</li>
		<li>分光光度計のアッパー アームを下げ、それを校正するための「空白」をクリックします。</li>
		<li>それが終わったら、アッパー アームを持ち上げて拭くと台座を乾燥します。</li>
	</ol>
</li>
	<li>このサンプルを次のように測定します。<ol>
<li>サンプルの種類に「DNA 50」をクリックします。</li>
		<li>2 μ L の台座に DNA のサンプルをロードします。</li>
		<li>アッパー アームを下げ、A260/A280 比とサンプルの濃度を測定する「測定」をクリックします。</li>
		<li>ワイプと精製水とそれぞれの実行の間に台座をクリーンアップします。</li>
	</ol>
</li>
	<li>データを収集する「印刷画面」をクリックします。</li>
</ol>4. 遺伝の突然変異解析
PCR4ターゲット DNA を増幅します。25 μ L 反応で PCR を行う DNA ポリメラーゼを用いた混合キット (材料の表を参照してください)。TTR 遺伝子のイントロンのプライマーを表 1に示します。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>遺伝子</td>
			<td>プライマー シーケンス</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5'-TCAGATTGGCAGGGATAAGC-3' R: 5'-GCAAAGCTGGAAGGAGTCAC-3'</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5'-TCTTGTTTCGCTCCAGATTTC-3' R: 5'-TCTACCAAGTGAGGGGCAAA-3'</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5'-GTGTTAGTTGGTGGGGGTGT-3' R: 5'-TGAGTAAAACTGTGCATTTCCTG-3'</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5'-GACTTCCGGTGGTCAGTCAT-3' R: 5'-GCGTTCTGCCCAGATACTTT-3'</td>
		</tr>
</tbody></table>テーブル 1。TTR 遺伝子イントロン プライマー 。(NCBI 参照シーケンス: NC_000018.10)
<ol><li>0.2 mL PCR チューブに表 2に試薬を追加します。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>コンポーネント</td>
			<td>(Μ l)</td>
			<td>最終濃度</td>
		</tr>
<tr>
<td>10 x バッファー</td>
			<td>2.5</td>
			<td>1 x</td>
		</tr>
<tr>
<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1.5 mM</td>
		</tr>
<tr>
<td>360 GC エンハンサー</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360 DNA ポリメラーゼ</td>
			<td>0.2</td>
			<td>2 U/反応</td>
		</tr>
<tr>
<td>dNTP ミックス (25 mM)</td>
			<td>2</td>
			<td>2 mM</td>
		</tr>
<tr>
<td>プライマー F (10 μ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>プライマー R (10 μ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>PCR グレード水</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
<tr>
<td>総容積</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>表 2。PCR 反応条件。
<ol start="2"><li>穏やかに混合、ベンチトップ遠心管の底にすべてのコンポーネントを収集するために簡単に遠心力場します。たちにチューブを置きます。</li>
	<li>30 サイクルの PCR を実行します。各サイクルは 30 の変性のステップで構成されています 94 ° c、プライマーアニー リングの 30 s 58 ° C、および 45 のポリメラーゼ拡張子 s 72 ° c s (表 3参照)。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>ステップ</td>
			<td>温度 (° C)</td>
			<td>時間</td>
			<td>サイクル</td>
		</tr>
<tr>
<td>初期変性</td>
			<td>95</td>
			<td>5 分</td>
			<td>1</td>
		</tr>
<tr>
<td>DNA の変性のステップ</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 サイクル</td>
		</tr>
<tr>
<td>プライマーのアニーリング ステップ</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 サイクル</td>
		</tr>
<tr>
<td>ポリメラーゼ拡張ステップ</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 サイクル</td>
		</tr>
<tr>
<td>ポスト延長のステップ</td>
			<td>72</td>
			<td>10 分</td>
			<td>1</td>
		</tr>
<tr>
<td>PCR のサイクリング</td>
			<td>4</td>
			<td>無期限</td>
			<td></td>
		</tr>
</tbody></table>表 3。PCR の製品を増幅するための条件をサイクリングします。
<ol start="4"><li>ゲル電気泳動法に可視化して PCR の製品を検証します。<ol>
<li>1.2% の agarose のゲルを準備します。<ol>
<li>アガロース粉末 1.2 g を混ぜて 0.5 100 mL 電子レンジのフラスコの中の x TBE。電子レンジ 2 分、アガロースを完全に溶解するまで。55 ° C に agarose の混合物を冷却します。</li>
			<li>Agarose の混合物にエチジウム ブロマイド溶液 (10 mg/mL) の 2 μ L を加え、軽く混ぜます。注ぎ agarose ゲル トレイ 5-7 mm 程度にし、場所に、comb(s) を追加します。Comb(s) を慎重に取り外します (30 分以上) を固めることができます。</li>
		</ol>
</li>
		<li>サンプルをロードし、agarose のゲルを実行します。<ol>
<li>電気泳動セルにゲルとトレイを配置します。電気泳動セルに 0.5 を入力 x TBE ゲルが覆われるまで。</li>
			<li>慎重に積載 2 μ L 100 bp の DNA の梯子分子量マーカーとゲルの 1 つの車線に (材料の表を参照してください)。負荷に追加車線の染料/サンプル混合物 (1 μ L 6 × 読み込み染料でに PCR プロダクト ミックスの 5 μ L)。</li>
			<li>電源をオンにします。100 V で 25 分のためのゲルを実行します。</li>
			<li>電気泳動セルからゲルを削除します。イメージング システムでの紫外光下で DNA のフラグメントを視覚化します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. DNA の配列
<ol><li>市販のキットを使用して PCR の製品の浄化し、商業的または自動シーケンサーのシーケンス (材料の表を参照してください)。</li>
	<li>塩基配列を塩基配列データベース10ヌクレオチド クエリを比較する NCBI BLAST サーバーに送信します。結果が表示されたら、配列アラインメントを実行し、予想される突然変異を識別します。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective,+observational+study+of+patients+with+uncommon+distal+symmetric+painful+small-fiber+neuropathy.">A prospective, observational study of patients with uncommon distal symmetric painful small-fiber neuropathy.</a> PLoS ONE. 12 (9), 0183948 (2017).</li><li>Salvi, F., Pastorelli, F., Plasmati, R., Bartolomei, I., Dall'Osso, D., Rapezzi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+phenotypic+correlation+in+an+Italian+population+of+hereditary+amyloidosis+TTR-related+(HA-TTR):+Clinical+and+neurophysiological+aids+to+diagnosis+and+some+reflections+on+misdiagnosis.">Genotypic and phenotypic correlation in an Italian population of hereditary amyloidosis TTR-related (HA-TTR): Clinical and neurophysiological aids to diagnosis and some reflections on misdiagnosis.</a> Amyloid. 19, 58-60 (2012).</li><li>Suhr, O. B., Lundgren, E., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mutation,+two+distinct+disease+variants:+Unravelling+the+impact+of+transthyretin+amyloid+fibril+composition.">One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition.</a> Journal of Internal Medicine. 281 (4), 337-347 (2017).</li><li>Vil&#224;-Rico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+post-translational+modifications+in+human+serum+transthyretin+associated+with+familial+amyloidotic+polyneuropathy+by+targeted+LC-MS+and+intact+protein+MS.">Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.</a> Journal of Proteomics. 127, 234-246 (2015).</li></ol>

著者が明らかに何もありません。

プロトコル内で 2 つの重要な手順があります。まず、白血球細胞の十分な数を持つために hemodiluted 試験片は回避11をする必要があります。第二に、適切な PCR のプライマーの使用は信頼性の高い結果12を取得する基本的です。プライマー ブラスト web ツールを使用してプライマー4,13; を設計するには4 つの TTR エク...

トランスサイレチン (TTR) アミロイドーシス (ATTR) 遺伝性の全身性アミロイドーシスの1、最も一般的な形式し、トランスサイレチン (TTR) 遺伝子2常染色体優性遺伝の突然変異によって引き起こされることができます。TTR 変異 4 量体タンパク質の構造を不安定にし、アミロイド線維2に再アセンブル単量体への解離に 。以上 100 アミロイド TTR の突然変異は報告されている世界的な1をされています。ポリメラーゼの連鎖反応 (PCR) 方法と遺伝学的解析は TTR ポイント突然変異の存在を確認、南しみ3と比較して放射能によって分類されたプローブの処理を回避するなどの利点があります。PCR は、現代の科学4における多数のフィールドに適用されている高速、簡単、安価で、信頼性の高い手法です。
その表現型の多様性を与えられて、この進行と致命的な病気の早期診断は困難です。注意と新興治療5と同様、ATTR の初期症状の広範な認識に伴い、TTR 遺伝子検査を含む適切な診断学的意義、予後を改善するために非常に基本的なしたがって。さらに、異なる突然変異形質の異なる浸透度、発症年齢、進行疾患の重症度、生存期間中央値、肝移植や TTR スタビライザー2,6の有効性のパターンに関連付けられているし、遺伝カウンセリング7,8,9に大きな影響を与える神経と心の関与の可変度。その上、高精度の遺伝子検査は ATTR の 2 つのタイプを区別する唯一のツール: 遺伝性 (突然変異) と野生型 (非突然変異体の形態、老人性全身性アミロイドーシス、SSA)7。2に治療法が異なるため、属性の種類を確認することが不可欠です。したがって、TTR 遺伝子検査の段階的なプロトコルを記述する必要性がますますあります。
突然変異を検出する分子のアプローチは、例として Ala97Ser、台湾では、最も一般的な風土病突然変異を使用して説明されます。DNA 抽出手順の変更は使用される 3 つのソリューションの量を減らすし、DNA の十分な量が得られます。このプロトコルでは 5' 上流、3' 下流、プロモーター、イントロンを含む地域中のすべての 4 つの TTR エクソンが行ったし、非翻訳領域 (UTR) クローニングされたないです。

<table border="0" cellpadding="0" cellspacing="0" style="width:951px;" width="951">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">EDTA-treated tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:347px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA Extraction Kit</td>
			<td style="width:199px;">Stratagen</td>
			<td style="width:347px;">200600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">NanoDrop ND2000 spectrophotometer&#160;</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:347px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Delicate Task Wipers</td>
			<td style="width:199px;">Kimberly-Clark</td>
			<td style="width:347px;">Kimtech Science Kimwipes</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">AmpliTaq Gold 360 DNA Polymerase kit</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">4398823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">TTR gene intronic primers&#160;</td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">thermocycler</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">electrophoresis cell&#160;</td>
			<td style="width:199px;">ADVANCE</td>
			<td style="width:347px;">Mupid-2plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA ladder</td>
			<td style="width:199px;">Protech</td>
			<td style="width:347px;">PT-M1-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">dye</td>
			<td style="width:199px;">BioLabs</td>
			<td style="width:347px;">B7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">AlphaImager EC</td>
			<td style="width:199px;">Alpha Innotech</td>
			<td style="width:347px;">AlphaImager EC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">automatic sequencer&#160;</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">3730xl DNA Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

遺伝学的検査は遺伝性トランスサイレチンの最も信頼性の高いテスト関連のアミロイドーシスとトランスサイレチン アミロイドーシス (ATTR) のほとんどのケースで実行する必要があります。ATTR が異種表現型; でまれな、致命的な病気したがって、診断が遅れることもあります。関心と新たな治療法と同様、ATTR の初期症状の広範な認識を増やすこと、適切な診断の研究、トランスサイレチン (TTR) 遺伝子を含むテスト、種類を確認して ATTR の亜種が基本的な予後の改善に。ポリメラーゼの連鎖反応 (PCR) 方法と遺伝学的解析がより迅速に TTR ポイント突然変異の存在を確認、南しみなどの従来の方法よりも安全です。ここで、ATTR Ala97Ser 突然変異、台湾で最も一般的な風土病突然変異の遺伝学的確認を示します。プロトコルには 4 つの主要なステップが装備されている: 収集全血試料、DNA の抽出、PCR とすべての 4 つ TTR エクソンの遺伝学的解析および DNA の配列。

アガロース電気泳動の 2 つの患者と (図 1) TTR 遺伝子の exon 4 の 454 bp の PCR の製品を含む、予想される規模の個々 の健康的な 1 つ明らかにバンド。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> 図 1.PCR を描いた電気泳動増幅 TTR 遺伝子。通常: 健康?...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

ここでは、例として Ala97Ser、台湾では、最も一般的な風土病突然変異を使用して遺伝性トランスサイレチンのアミロイド症の診断のための点突然変異の存在を確認するためのプロトコルを提示します。

遺伝性トランスサイレチン Ala97Ser の遺伝子解析関連アミロイド症

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

Research

JoVE Journal

Medicine

7.6K ビュー.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. ここでは、例として Ala97Ser、台湾では、最も一般的な風土病突然変異を使用して遺伝性トランスサイレチンのアミロイド症の診断のための点突然変異の存在を確認するためのプロトコルを提示します。

ビデオ: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

着床前遺伝子診断のためのFISH

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

医学

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

アルツハイマー病患者における事象関連電位を用いたエピソード記憶の特性を調べる

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

ニューロイメージングデータを用いた疾患関連空間共分散パターンの同定

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

マウスにおける変形性関節症の痛みのMonoiodoacetateモデル

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

検出とマクロファージによるマウス腸管炎症の定量化のための蛍光を介したトモグラフィー

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

単球のホーミングと末梢動脈疾患のマウスモデルにおける腫瘍関連血管新生の生体顕微鏡

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

PubMed、Scopus、および Web のサイエンス データベースを使用した書誌分析の比較

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

乳癌関連リンパ浮腫予後診断における拡張現実ツールの統合

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

ラット膝の周期的負荷誘発関節内軟骨病変モデルを生成するための非侵襲的方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

遺伝性トランスサイレチン Ala97Ser の遺伝子解析関連アミロイド症

要約

さらに動画を探す

この動画の章

着床前遺伝子診断のためのFISH

アルツハイマー病患者における事象関連電位を用いたエピソード記憶の特性を調べる

ニューロイメージングデータを用いた疾患関連空間共分散パターンの同定

マウスにおける変形性関節症の痛みのMonoiodoacetateモデル

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

検出とマクロファージによるマウス腸管炎症の定量化のための蛍光を介したトモグラフィー

単球のホーミングと末梢動脈疾患のマウスモデルにおける腫瘍関連血管新生の生体顕微鏡

PubMed、Scopus、および Web のサイエンスデータベースを使用した書誌分析の比較

乳癌関連リンパ浮腫予後診断における拡張現実ツールの統合

ラット膝の周期的負荷誘発関節内軟骨病変モデルを生成するための非侵襲的方法

遺伝性トランスサイレチン Ala97Ser の遺伝子解析関連アミロイド症

要約

さらに動画を探す

この動画の章

着床前遺伝子診断のためのFISH

アルツハイマー病患者における事象関連電位を用いたエピソード記憶の特性を調べる

ニューロイメージングデータを用いた疾患関連空間共分散パターンの同定

マウスにおける変形性関節症の痛みのMonoiodoacetateモデル

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

検出とマクロファージによるマウス腸管炎症の定量化のための蛍光を介したトモグラフィー

単球のホーミングと末梢動脈疾患のマウスモデルにおける腫瘍関連血管新生の生体顕微鏡

PubMed、Scopus、および Web のサイエンス データベースを使用した書誌分析の比較

乳癌関連リンパ浮腫予後診断における拡張現実ツールの統合

ラット膝の周期的負荷誘発関節内軟骨病変モデルを生成するための非侵襲的方法

PubMed、Scopus、および Web のサイエンスデータベースを使用した書誌分析の比較