This work was partially supported by the National Institutes of Health (NIH) Clinical and Translational Science Awards (CTSA) Grant (UL1TR001070) to the Ohio State University's Center for Clinical and Translational Science (CCTS) and the National Library Of Medicine under Award Number T15LM011270.

1.遺伝子からのクエリの開始、薬物または疾患利用規約<ol><li> http://drug-repurposing.nationwidechildrens.org：以下のリンクから：「ファイン薬RE」のホームページにアクセスします。薬（ジェネリック医薬品名）、疾患（新しい病気表示）または遺伝子（公式HGNC遺伝子記号）：以下の3カテゴリーのいずれかからの検索バーに検索タームを入力することによって開始します。 </li><li>唯一の「薬剤名」、「新しい病気の表示」、「遺伝子記号」または「すべて」のカテゴリを含めるように検索バー機能をフィルタリングします。検索バーは、クエリのエントリの自動入力機能を備えています。 </li><li>キーワードを入れて、「検索」ボタンをクリックしてください。次の列のいずれかによって、結果のソート表：「医薬品」、「登録指示」、「P-バリュー」、「調整p値」、「オッズ比」、「研究」、「新表示 &quot;、&quot;医薬品銀行指示」、「Medlineのアブストラクトの＃」、「CLIの＃nicalトライアルレジストリ」、「潜在的な」、「SNP」、「遺伝子」または「アクション」。 </li><li>医薬品情報機能を有効にするために、高度な検索オプションに移動します。特定の薬剤のための「情報」列のアイコンをクリックします。関連のp値、疾患名、薬剤名、遺伝子の詳細（NCBI遺伝子リンク）医薬品の詳細を含むすべての対応する情報（DrugBankリンク）を表示するページを確認します。 </li></ol>詳細オプションの2探査<ol><li>ページの右側にある「高度な」ボタンをクリックし、さらに結果を絞り込むには、いくつかのオプションが提供されています。薬剤、協会、病気、潜在的な、遺伝子およびアクション：高度な検索オプションには、以下の修正が含まれています。 </li><li>エクスポートは、ページの右側にある「エクスポート」ボタンをクリックして、テーブルをもたらします。高度な検索ウィンドウを倒しするために、「シンプル」ボタンをクリックします。&lt;/李&gt; <li>高度なオプション「薬物」タブの下で、特定の薬剤の適応症または結果をフィルタリングするための追加の薬剤名を指定します。 </li><li> 「会合」タブの下で、有意水準p値、FDRで調整p値、効果の大きさ（オッズ比）、および/またはによってフィルタ結果がタイプ（GWAS、PheWASまたは両方）を学びます。 </li><li> 「疾患」タブの下で、予測された新たな使用のための特定の疾患の説明を指定します。 </li><li> 「潜在的」タブで、次の基準のいずれかに記載のフィルタ結果は、（i）薬物指示がDrugBankデータベースに含まれているかどうか、薬剤の共起や病気、とMedlineの抄録の（ii）の数（ ⅲ）薬剤の同時発生と疾患と、（iv）再目的化の可能性を持つClinicalTrials.govデータベースエントリの数を。 注：再目的化可能性のオプションは、発見の新規性について説明します。（ⅰ）既知：関係が既にDrugBankデータベースに存在するが、（ii）は強くサポート：臨床試験レジストリとメド抄録の両方でいくつかのサポート、（iii）の可能性が高い：臨床試験のレジストリでも、Medlineの抄録で証拠：臨床試験のレジストリまたはMedlineの抄録および（iv）小説のいずれかで何らかのサポート。 </li><li> 「遺伝子」タブの下で、特定の薬物標的遺伝子によって結果をフィルタリングするSNP識別子または遺伝子記号を入力します。 </li><li> 「アクション」タブの下で、アゴニスト、アンタゴニスト、他の、未知の、またはすべて（：DrugBankデータベースソース）を含む、薬物標的（複数可）に対する薬物作用のタイプを指定します。 </li></ol>

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The authors declare that they have no competing financial interests.

The protocol described here for the RE:fine Drugs interactive dashboard can be modified in different ways according to the user's preferences. This method uniquely integrates GWAS and PheWAS data as a novel paradigm underlying drug repurposing hypothesis generation. Specifically, this system provides access to both 52,966 PheWAS associations and 7,945 GWAS associations with advanced options to filter the results by the study type, effect size and/or significance level. Another advantage of this method over existing computational drug repurposing tools is that queries may be made from drug, gene or disease perspectives. 
There are several limitations to this method. Currently, the PheWAS data is limited to primarily adult patient population from five institutions contained in the Electronic Medical Records and Genomics (eMERGE) network with a mean age of 69.5 years 12. Additionally, the "repurposing potential" feature uses co-occurrence of search terms in Medline abstracts as one of its criteria. It is well known that text mining methods using co-occurrence have limitations with respect to syntactical structure and literature bias. Thus, we recommend this feature be used as a starting point to explore the potential novelty and/or evidence supporting specific drug repurposing hypotheses and recommend additional investigation into the biomedical literature and clinical trial databases. 
Future directions for this work not described here would be to extend this database to additional sources of GWAS and PheWAS data as they become available. Similar efforts to systematically translate results from large-scale GWAS studies into drug repurposing hypotheses have been previously published 9,13-14. It may be useful to compare these different workflows to predict drug candidates from GWAS data in future studies. Additionally, several other methods exist to computationally generate drug repurposing hypotheses from different data sources, including: genomics, transcriptomics, chemical structures, drug side effect profiles, as previously summarized 6,11. Future methodological advancements could also include automating drug combination predictions and providing information on drug toxicity to guide follow up studies for drug candidates. 
Furthermore, the hypotheses generated from RE:fine Drugs may be further validated using electronic health records, before initiating clinical trials 15. Finally, future studies will be needed to compare this system to other target-based drug repurposing methods.

高スループットの薬物およびリード化合物のスクリーニングを含む伝統的な創薬アプローチに関連したコストがかかり、非効率的なプロセスは、患者1,2のための治療法の研究の発見を翻訳するの遅れに貢献しています。 10億ドルと15年から20年の平均は、ベッドサイド3にベンチから新薬をもたらすために必要とされます。さらに、薬物の52％が第1相臨床試験で開発中に失敗し、フェーズ2に入る化合物の25％のみが完全な第3相臨床試験4に進みます。薬物転用または薬物の再配置の目標は、失敗した麻薬を更新および/またはより高速な患者に新しい治療法を提供し、高い成功率とするために、承認薬のための新規適応症を見つけることです。薬物再利用は3-12年5人の患者に使用するための薬を利用できるようにタイムラインを減少させることができます。薬物再利用のための重要な医療用途は次のとおりです。貧しいprognos伴う疾患をであり、低生存率、薬剤耐性疾患、積立不足病気の研究分野や貧困と不十分な患者集団。 計算薬物転用は、薬剤候補6のための新たな適応症のための仮説を生成することができる自動化されたワークフローを設計および検証するプロセスとして定義されています。既存の計算の薬物再利用方法は、標的ベース、知識ベース、シグネチャベース、ネットワークベースの分類、およびターゲットと機構系、遺伝子、疾患または薬物の視点から配向させることができるされています。また、計算のアプローチは、さらに、概念実証検証実験と再利用薬剤候補7のための小規模な臨床研究を加速することができます。我々は以前、「RE：罰金薬」で報告している、薬物-遺伝子-疾患関係8の推移理論に基づく薬物再利用仮説生成のために自由に利用できる、インタラクティブなWebベースのツール。全体的なグラムこの方法のOALは、体系的、臨床的、業界および規制社会など多様なコミュニティからのユーザーのための薬剤の再利用を可能にするために、薬物の多様な種類、遺伝学的および臨床データを統合することです。このシステムの基本的な方法は、以前にゲノムワイド関連解析（GWAS）と研究9,10を再利用する薬剤でphenomeワイド関連研究（PheWAS）データの使用が報告されています。これらのタイプのデータの新規な組み合わせは、他のターゲットに基づく方法6,11から当社のWebツールを区別します。 RE：罰金薬システムは、現在916薬、567の遺伝子と1770疾患をカバー60911の薬物転用仮説が含まれています。 Webツールは、研究者が対話的に、薬物転用仮説を検索し、多様な基準を使用してそれらに優先順位を付けるためのユーザーフレンドリーなインターフェイスを提供します。たとえば、ユーザーが生物医学文献および臨床試験がdatabaでのサポートを有する薬物転用仮説をフィルタリングすることができますSE、有意なp値、アソシエーションオッズ比または特定の指示によって。このシステムのための唯一の要件は、インターネットへのアクセスです。

<table border="0" cellpadding="0" cellspacing="0" width="797">
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		<tr height="63">
			<td height="63" style="height:63px;width:389px;">Access the homepage for &#8220;RE:fine Drugs&#8221; at the following link: http://drug-repurposing.nationwidechildrens.org.&#160;</td>
			<td style="width:81px;">n/a</td>
			<td style="width:136px;">n/a</td>
			<td style="width:191px;">The only requirement for this system is Internet access</td>
		</tr>
	</tbody>
</table>

drug repurposing hypothesis generation using the "re:fine drugs" system

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

この例では、遺伝子「IL2RB」は、遺伝子ベースのクエリとして入力し、自動的にオートフィル機能（ 図1）によってそのように認識されました。 図2に示すように、IL2RB遺伝子について12薬物転用仮説は、返されます。この場合、特定の薬物転用仮説、「ダクリズマブ」の詳細情報ページは、「情報」欄（ 図3）から提供されています。薬物タブが「ダクリズマブ」薬剤に対応するすべての結果によるものであったの図4に示すように結果は、濾過しました。 図5は 、「移植」は、疾患の用語（：DrugBankデータベースソース）のための既知の適応症でのみ薬を示しています。関連]タブには、ユーザーが統計的有意性（P値）とPRのオッズの比として定義される遺伝的影響の大きさ（オッズ比）によって、SNP-表現型の関係をフィルタリングすることができますSNP対立遺伝子のない個人における疾患の存在のオッズ以上の特定の遺伝子型（SNP対立遺伝子）を有する個体における疾患のesence。 図6は、0.000001の0.05にP-値の範囲内の関連タブ下で濾過した結果を示しています。 図7は、我々が研究で見つかった新しい指標に基づいて、「喘息」のための特異的な薬物再利用の仮説を示しています。薬と病気の用語の共起を含む5 Medlineの抄録の最小数でフィルタリングするための潜在的なタブの下で、図8に示す結果。この例では、遺伝子タブの下にあるすべての薬の結果は、元の問合せ語句（ 図9）に対応する、「IL2RB」遺伝子を対象としています。 IL2RB遺伝子上のアゴニストとして作用するすべての薬剤を返すために、「アクション」タブの下で濾過最後に、 図 10の結果を示します。 <img alt="図1"SRC = &quot;/ファイル/ ftp_upload / 54948 / 54948fig1.jpg&quot; /&gt; 図1：RE： 罰金薬インタラクティブなダッシュボードのホームページ。ユーザーは、薬剤名、新しい病気の兆候や遺伝子記号を入力することで、クエリを開始することができます。リンクはまた、薬物再利用の仮説を生成するための方法を記述GWASとPheWAS基準紙のために提供されます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 図2： クエリのエントリの自動入力機能。例として、遺伝子の照会用語「IL2RB」が自動的遺伝子用語として認識されました。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <pキープtogether.withinページ= &quot;1&quot;&gt;：FOクラス= &quot;jove_content&quot; <img alt="図3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 図3： 遺伝子ベースのクエリ（ 例えば、IL2RB）から製造された医薬品転用結果テーブル。 IL2RB遺伝子について十二薬剤転用仮説が生成されます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 図4：結果ページから個々の薬剤のための情報ページ。 「情報」列からアイコンをクリックすると、薬物ダクリズマブの詳細情報を示しています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図5： 薬物タブの下で高度な検索オプションは、特定の薬物によってフィルタリングします。この例では、3つの結果は、薬物ダクリズマブについて示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 図6：DrugBank データベースから特定の疾患指標によってフィルタリングする薬剤タブの下で高度な検索オプション。病気の用語「移植」するための既知の適応症を持つすべての薬が示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図7：アソシエーション]タブの下で高度な検索オプションは、有意水準によってフィルタリングします。この場合には、8個の結果は、その関連有意水準は0.05 0.000001のp値の範囲内に設けられています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 図8： 病気]タブの下で高度な検索オプションは、我々はこの研究で抽出された特定の疾患の徴候によってフィルタリングします。この例では、4つの結果は、新しい使用疾患適応症として喘息のために示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">より大きなversiを表示するには、こちらをクリックしてください。</a>この図の上。 <img alt="図9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 図9：Medline の抄録での薬物および疾患用語の共起によってフィルタリングするための潜在的なタブの下で高度な検索オプション。この例では、4つの結果は、薬物や病気の用語の共起を含む5 Medlineの抄録の最小数によってサポートされていることが示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 図10： 遺伝子タブの下で高度な検索オプションは、すべての結果が元のクエリに使用IL2RB遺伝子に対応する特定の遺伝子記号によってフィルタリングします。この例では、すべての薬物結果遺伝子タブの下で、元の問合せ語句に対応する、「IL2RB」遺伝子を対象としています。唯一のアゴニスト薬によってフィルタリングするアクション]タブの[詳細検索オプション。この例では、6つの結果がIL2RB遺伝子上のアゴニストとして作用するすべての薬剤に対して戻されます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

薬物転用仮説生成の使用」RE：罰金薬「システム

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

drug-repurposing-hypothesis-generation-using-the-refine-drugs-system

Nanyang Technological University

Research

JoVE Journal

Medicine

9.2K ビュー.  The Ohio State University. Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options. 

ビデオ: Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

手術標本の腫瘍外植片を用いて新規抗がん薬の開発のための方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

医学

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

新しいハーブ治療法を評価するために敗血症の動物モデルの使用

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

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

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

次世代シーケンシングのための収集と唾液DNAの抽出

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

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

LC-MS / MSを用いて乾燥血液スポット上で免疫抑制タクロリムスの定量化

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

アン<em&gt;インビトロ</em&gt;骨髄中のエストロゲン感受性乳癌の休眠モデル：分子機構の研究と仮説生成のためのツール

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

多フッ素化された胆汁酸を用いて<em&gt;インビボ</em胆汁酸輸送を測定するために&gt;磁気共鳴イメージング

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

眼底イメージングを使用して認知症を研究するには

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

マウスの脳内へ連続的な薬剤注入システムと末梢グルコース不耐症に視床下部のインスリン シグナルを勉強

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...