ETM37と癌研究英国の実験的がん医療センターでサポートされている：チーフサイエンティストオフィス、承認番号によって運営されている上記の作家FCO、DF西澤、DJHとGDSの仕事は言及した。 ALの仕事はエディンバラロバート·トラスト、ケア、がんや医療研究評議会の治療のためのメルヴィル·トラストの外科医のロイヤルカレッジによって賄われています。 IOはマリー·キュリー·アクションと英国医学研究評議会によってcofundedエジンバラスコットランド政府フェローシップの王立協会によってサポートされています。著者らは、この論文に記載された内容の一部に彼らの有益な議論のためにSCOTRRCC共同申請者と協力者に感謝したいと思います。

1。形態学的および分子腫瘍の不均一性の識別<ol><li>腫瘍は、-80℃の冷凍庫から取り出し、ドライアイス上に保持。 </li><li>約1cm 3の部分に腫瘍を分割します。各腫瘍互いに相対セクションとユニークな名前のラベルの元の位置をマップします。ストアサンプル-80個クライオバイアル℃·使用するための準備が整うまで。 </li><li>コー​​トのサンプル10月と-22℃のクライオスタットでカット</li><li>サンプルは、ヘマトキシリン​​およびエオシン対比染色法を用いて染色した。 </li><li>彼らはccRCC自然、生存腫瘍及び格付のための（凍結切片上Fuhrmanグレード1から4を区別するために挑戦低悪性度、高悪性度または混合高/低グレード）があることを確認するように凍結切片の顕微鏡分析図1＆B（H＆E染色高い低いと混合グレード）。 </li><li>タンパク質抽出のために各々の腫瘍内の各形態学的に異なる領域から最大で4つのサンプルを選択します。 </li></ol> 2。腫瘍サンプルからのタンパク質抽出<ol><li> 10月からの腫瘍サンプルをカットします。 </li><li>溶解バッファー990μLで2 mlチューブにティッシュの代わりに50から75ミリグラム[50mMトリス - 塩酸（pH7.5）、5mMのEGTA液（pH8.5）、150mMのNaCl]アプロチニン（シグマA6279）（10ミリグラムを補充/ ml）を、ホスファターゼ阻害剤カクテル2（シグマP5716）、ホスファターゼ阻害剤カクテル3（シグマP0044）およびプロテアーゼ阻害剤カクテル（Roche、11836153001）。 </li><li>各チューブに単5ミリメートルの鋼球を追加し、二回各5分間経過後、TissueLyserを用いた均質化のレベルを確認する5分間50Hzでホモジナイズする。 </li><li>後ろに鋼球を残して、ピペットを用いて新しい2 mlチューブに均質化したサンプルを転送します。 </li><li> 4℃で30分間13000×gで遠心分離する前に、各サンプルにTriton X-100を10μlを追加</li><li>新しいマイクロチューブに上清を移してください。 </li><li> BCAアッセイ（ 図2）を用いてタンパク質濃度を決定します。 </li><li>1 mg / mlとなるようにタンパク質濃度を正常化する。 </li></ol> 3。抗体の検証<ol><li>ウェスタンブロットのために、タンパク質サンプルを（適切な細胞株や組織から抽出）を準備し、10％SDS-PAGEゲル上で実行されます。 </li><li> 4℃で一晩、ニトロセルロース膜上にサンプルを転送℃に</li><li>室温で1時間LI-CORのオデッセイブロッキングバッファー（PBSに50:50に希釈）で膜をブロックします。 </li><li>メーカーでLI-CORのオデッセイブロッキングバッファーで一次抗体（PBS中に50:50に希釈）希釈する希釈は1,000で、典型的には1をお勧めします。 </li><li> 4℃で一晩、一次抗体で膜をインキュベート</li><li> 0.1パーセントのPBS-Tween20にて（; 1ミリリットルツイーン20 / 1L PBS PBS-T）を作る。 </li><li> 5分（×3）室温でPBS-Tで洗浄する。 </li><li> 1:10,000希釈（1.5μl/15ml）でSDS（PBS中で50:50に希釈）を0.01％ブロッキングバッファーオデッセイに蛍光標識二次抗体を希釈します。 </li><li>二次で膜をインキュベート穏やかに振盪しながら45分間室温で抗体 - それが最終的にスキャンされているような時間まで光から膜を保護することが重要です。 </li><li>暗闇の中で膜を保つ5分（×3）、室温でPBS-Tで洗浄する。 </li><li>再び暗闇の中で膜を維持し、残留Tween20を削除するには、5分（×3）室温でPBS中で洗浄する。 </li><li>暗闇の中で濾紙上に平坦な膜を嘘と空気乾燥することができます - メンブレンを乾燥させると、信号を強化し、バックグラウンドを低減するが、ストリッピング、再プロービングするため、それが役に立たなくなる可能性があります。 </li><li> LiCorオデッセイスキャナー上に膜をスキャンします。それをスキャンしたようになるまで、暗闇の中で膜を保つ。 </li><li>唯一の正しい分子量の単一支配的なバンドを生成する抗体を選択する。 図3は RPPAで使用するために許容され、受け入れられない抗体の例を示します。 </li></ol> 4。 RPPAぷりnting <ol><li>タンパク質可溶化液をマイクログリッドIIロボットスポッターを用いてニトロセルロースでコーティングされたガラススライド（Fastslides-Whatman社製）上にスポットした。使用されるスライドは、試料をスポットし、その上に2つのパッドを含んでいた。各パッドは同一のサンプル、この場合は100サンプルで発見された。他の利用可能なフォーマットは、1、8、16パッドのスライドが含まれています。パッドの数字が大きいほど小さく、それぞれにスポットできるサンプル数。 </li><li> 5 2倍希釈系列を、各サンプルから作られた、それぞれがその後（サンプルあたり15箇所の合計になります）三重で発見された。 </li></ol> 5。 RPPAタンパク質検出<ol><li>過剰LiCorブロッキングバッファー（PBSで希釈50:50）でぬれたスライド。ロッキングプラットフォーム上で室温で1時間インキュベートします。 </li><li>所望の濃度のLiCorブロッキングバッファー（PBSに50:50に希釈）で一次抗体800μlのを準備し、氷上で保存する。 </li><li>は、（i）単一のフレーム·チップ·クリップでスライドをマウントタイトなシールがスライドとインキュベーションチャンバーとの間に形成されるように、または（ii） &#39;FastFrame&#39; 4ベイのスライドホルダー。 </li><li>井戸から残った液を取り除き、それぞれのウェルに600μlの一次抗体を加える。 </li><li>一晩4プラットフォームを揺らしに密封されたウェットボックス、インキュベートにスライドとチャンバーを置き℃に</li><li> 0.1パーセントのPBS-Tween20にて（;100μlのツイーン100分の20 mlのPBS PBS-T）を作る。 </li><li>寒い部屋からスライドを取り外し、慎重に各ウェルからの一次抗体を除去します。 </li><li> PBS-Tを600μlを加え、5分間（X3）をRTでプラットフォームを揺らしでスライドを洗浄してください。 </li><li>オデッセイブロッキングバッファー（PBSに50:50に希釈）0.01％第一審の1:2,000希釈（1μL/ 2ミリリットル）のSDSで希釈することにより、蛍光標識二次抗体を準備します。 </li><li>井戸からバッファを削除し、各ウェルに600μlの蛍光標識二次抗体を加える。穏やかに振とうしながら45分間室温で二次抗体をインキュベートする - それをそれが最終的にスキャンされているようになるまで、光から膜を保護することが重要です。 </li><li>室温で600μlのPBS-Tでよく洗浄し、短時間（X3）からの二次抗体を除去する。キャリアからスライドを外し、適当な容器に移し、暗闇の中で膜を維持し、15分間超過PBS-Tで洗浄してください。 </li><li>再び暗闇の中で膜を維持し、残留Tween-20を除去するために、PBS-T、15分間室温でPBSでさらに洗浄膜を取り除きます。 </li><li> 10分間、50℃のオーブンでFastslideを乾燥してから、LI-CORオデッセイのスキャナでスキャンしてください。それがスキャンされるまで、暗闇の中でスライドを保つ。 </li><li>二次抗体/使用する抗体に応じて680 nmおよび/または800 nmでスライドをスキャンします。 2色の検出のために常に交差反応性を最小限にするために高度に架橋吸着した二次抗体を使用しています。一次抗体および二次抗体を慎重に選択するには、2つの色の検出のために必要である。第一に重要での選択である2次抗体に異なるホスト種（ 例えば、ウサギ、マウス）。これは容易に区別できる発光スペクトルと色素で標識された抗ウサギ抗マウス二次抗体による差別ができます。 </li><li>画像ファイルは、。TIFFファイルとして保存されます。 図4（スキャンしたファイルの画像）。 </li></ol> 6。データ解析<ol><li> MicroVigeneソフトウェア（VigeneTech、カーライル、マサチューセッツ州、米国）を起動します。 </li><li> RPPAスライドのスキャンを含む。TIFFの画像ファイルを開きます。 </li><li> RPPAスライドの画像をオーバーレイするグリッドを持って事前に定義されたテンプレートファイルを選択します。 </li><li>クリックしてグリッドを起動するために、 関心領域 （ROI）ボタンの領域を定義します 。 </li><li> RPPAスポット上にグ ​​リッドを配置します。 図6a（画像上にグ ​​リッドの画像）。 </li><li>すべてのROIを強調表示するには、[ すべて選択]ボタンをクリックします。 </li><li> すべて検索]をクリックします。 MicroVigeneはAUTOMますatically ROIを見つけ、スポットを見つける、バックグラウンドを差し引き、ほこりを除去し、スポットを定量化する。 </li><li> RPPAスライド上のすべてのサンプルの結果を表示するために表示希釈曲線]ボタンをクリックします。 </li><li> 希釈のデータを保存]をクリックします 。 </li><li>として各サンプルはエラーのリスクを削減し、カーブフィッティングの質を向上させ、分析する15点が存在する三重に各5希釈ポイントにプリントされています。 MicroVigeneは、抗原抗体結合反応速度のS字曲線を生成するために全てのスポットを組み込んだ4パラメータロジスティックログモデル&quot;Supercurve&quot;アルゴリズム（ 図6b）を生産しています。前提は同じ抗体-抗原結合反応速度は、一般的な応答曲線に合わせてアレイ上のすべてのスポットを取ることによって、このようにしても、別のサンプルでは、各試料スポットで行われていると、カーブフィッティング10,11の自信を高めることができるということです Y = A +（（BA）/（1 + E（C * D-LN（X））） ここで、xはdilutioですnの係数とYは信号強度である。 サンプルは比較的我々の分析でsupercurveへそれらをマッピングした後のxの値の中間点でyの値に対応したY0値を用いて解析することができます。 </li><li> 図7のように、Microsoft ExcelとプロットY0でデータをエクスポートします。 </li><li>腫瘍内タンパク質変動をANOVAの枠組みの中で未処理と処理治療を受けた患者のために別々に計算された。分析されたすべてのタンパク質からのデータを組み合わせる分散分布は、Mann-Whitney検定（MWT）に比較した。個々のタンパク質の腫瘍内分散はLillefoursとFlignerテストを用いて評価し、それぞれ正常と等分散性の仮定が開催されたF検定により比較した、偽の発見率（FDR）の補正が12を適用した。未処理および処理された患者のサンプル間の差動タンパク質発現は、スチューデントのt検定を用いて、各タンパク質について試験したところ正常と等分散性の仮定sがそうでなければ、MWTを行ったところ、満たされた、ルーズベルトは、結合されたt-検定とMWT値12の上に塗布した。タンパク質のタンパク質発現と分散ピアソン相関の有意性は、[R参照]とFDR応用12の標準的なアプローチを用いて推定された。 </li></ol>

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特別な利害関係は宣言されません。

ここで紹介するRPPA法はタンパク質解析の広く使​​われているが、比較的低いスループットウエスタンブロット法に高スループットの代替を表しています。この方法は、サンプルの数は半定量的に解析し、細胞株や組織サンプルの幅広い選択を越えて重要なタンパク質の直接比較を可能にし同時に比較することができます。異なる抗体種と多重化はさらに複数の抗体を同時に使用することが...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>アプロチニン</td><td>シグマ</td><td> A6279 </td></tr><tr><td>ホスファターゼ阻害剤カクテル2 </td><td>シグマ</td><td> P5726 </td></tr><tr><td>ホスファターゼ阻害剤カクテル3 </td><td>シグマ</td><td> P0044 </td></tr><tr><td>プロテアーゼ阻害剤カクテル</td><td>ロッシュ</td><td> 11836153001 </td></tr><tr><td>トリトンX-100 </td><td>トリトン-X </td><td> T8787 </td></tr><tr><td>ブロッキングバッファーLI-CORのオデッセイ</td><td> LI-COR </td><td> 927から40000 </td></tr><tr><td> TissueLyserを</td><td>キアゲン</td><td> 85600 </td></tr><tr><td>マイクログリッドIIロボットスポッター</td><td> Biorobotics </td><td></td></tr><tr><td> FastFrame &#39;4ベイのスライドホルダー</td><td>ワットマン</td><td> 10486001 </td></tr><tr><td> FASTスライド - 2 - パッド</td><td>ワットマン</td><td> 10485317 </td></tr><tr><td> IRDye 680LTヤギ抗マウスIgG </td><td> Licor </td><td> 926から68020 </td></tr><tr><td> IRDye 800CWヤギ抗ウサギIgG </td><td> Licor </td><td> 926から32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

現在、転移性明細胞腎細胞癌に対する根治的治療法、病気の最も一般的な変形はありません。この治療抵抗性の重要な要因は、病気の1の分子複雑であると考えられている。例えば、チロシンキナーゼ阻害剤（TKI）スニチニブとして標的療法が利用されてきたが、患者のわずか40％は1年以内に2回再発、これらの患者の圧倒的多数で、対応させていただきます。腎細胞癌患者における内因性および獲得耐性のような質問のように、関連性の高い3です。 効果的な、パーソナライズされた治療法の開発を最終目標として、TKIsに対する抵抗性を研究するために、標的療法の具体的な期間の後、順次組織は、慢性骨髄性白血病4で成功した証明したアプローチが必要になります。しかし腎細胞癌におけるそのような戦略のアプリケーションは、bの高レベルによって複雑になる腎細胞癌5,6だけでなく、他の固形腫瘍7の特徴であるOTH間および腫瘍内不均一性。トランスクリプトームとの遺伝的差異に起因Intertumoral異質性は十分にあっても同様のプレゼンテーション、ステージと腫瘍の悪性度の患者では確立されています。それに加えて、さらに大きな分子多様性を表現する可能性がある、RCCに大きな形態学（腫瘍）異質性が、そこにあることは明らかである。結合された形態素解析とFuhrmanグレーディングによってRCC腫瘍の詳細なマッピングや分類は、プロテオーム解析のための代表的な領域を選択することができます。 RCC 8のタンパク質ベースの分析では、原因病理研究室でその広範な可用性に魅力的ですが、そのアプリケーションは、特異的抗体9の限られた有用性のために問題になることがあります。逆相タンパク質アレイのドットブロット性質（RPPA）、抗体の特異性がなければなりませんbに起因電子事前検証、使用する抗体のような厳密な品質管理としては極めて重要である。この制限にもかかわらず、ドットブロット形式は、単一のニトロセルローススライド上にサンプルの数の印刷が可能になり、アッセイの小型化が許可されています。印刷されたスライドは、その後、多重化を可能にする、標的特異的一次抗体と蛍光標識二次抗体を用いてウエスタン分析と同様の方法で分析することができます。スライド上のすべてのサンプル間の差動タンパク質の発現は、その後、より費用対効果の高い、高スループットの方法で蛍光の相対的なレベルを比較することによって、同時に解析することができます。

スキャンRPPAスライドの例が示されて680と800nm ​​の両方のチャネルを図4（i）で見ることができます。波長は、 図4（ii）で画像を分離することにより、解析対象とするRPPAスライドや図4（iii）を決定し、個々のタンパク質の発現に各パッドを有効にします。 図4に見られるように、（iii）のサンプル間の個々のタンパク質の発現?...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPAは、蛍光標識抗体を用いて、同時に尋問するニトロセルローススライド上に印刷されたサンプルの何百ものタンパク質の発現を可能にします。このテクニックは、明細胞腎癌内薬物治療の異質の効果を研究するために適用されています。

個々の腎細胞がんの中のタンパク質発現変動を探るために逆相タンパク質アレイ（RPPA）の使用

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

the-use-reverse-phase-protein-arrays-rppa-to-explore-protein

Research

JoVE Journal

Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K ビュー.  University of Edinburgh. RPPAは、蛍光標識抗体を用いて、同時に尋問するニトロセルローススライド上に印刷されたサンプルの何百ものタンパク質の発現を可能にします。このテクニックは、明細胞腎癌内薬物治療の異質の効果を研究するために適用されています。

ビデオ: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

隔離された腎動脈閉塞のためのハンギングウェイトシステムの利用

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

医学

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

定量的免疫蛍光法を用いて腫瘍におけるタンパク質発現の不均一性のマッピング

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

がんの生産と活性酸素種の検出（ROS）

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

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, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

癌細胞の浸潤や衛星腫瘍形成を探検するための三次元培養アッセイ

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

ヒト炎症反応の個々の変動を評価するための最小紅斑用量(MED)試験の精密実施

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

腎動脈を介した腎臓への直接薬物送達

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...