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. 유전자에서 쿼리의 개시, 약물 또는 질병 약관 <ol><li> http://drug-repurposing.nationwidechildrens.org : 다음 링크에서 &quot;좋은 약 RE&quot;의 홈페이지에 액세스 할 수 있습니다. 의약품 (제네릭 의약품 명), 질병 (새 질환 표시) 또는 유전자 (공식 HGNC 유전자 기호) : 다음과 같은 세 가지 범주 중 하나에서 검색 창에 검색어를 입력하여 시작합니다. </li><li> 전용 &quot;약물 이름&quot;, &quot;새로운 질병의 표시&quot;, &quot;유전자 기호&quot;또는 &quot;전체&quot;카테고리를 포함하도록 검색 창 기능을 필터링합니다. 검색 창 쿼리 항목에 대한 자동 채우기 기능을 포함하고 있습니다. </li><li> 키워드에 넣고 &quot;검색&quot;버튼을 클릭합니다. 정렬 다음 열 중 하나에 의해 결과의 테이블 : &quot;약&quot;, &quot;등록 표시&quot;, &quot;P-값&quot;, &quot;P-값이 조정&quot;, &quot;확율 비율&quot;, &quot;연구&quot;, &quot;새로운 표시&quot;, &quot;의약품 은행 표시 &quot;,&quot;Medline을 초록의 # &quot;,&quot;CLI의 #nical 시험 등록 &quot;,&quot;잠재적 인 &quot;,&quot;SNP &quot;,&quot;유전자 &quot;또는&quot;작용 &quot;. </li><li> 약물 정보 기능을 활성화하기 위해 고급 검색 옵션으로 이동합니다. 특정 약물에 대한 &#39;정보&#39;항목에서 아이콘을 클릭합니다. 나열하는 페이지를 준수합니다 모든 협회, 질병 이름, 약 이름에 대한 P 값, 유전자 정보 (NCBI 유전자 링크) 의약품 정보 (DrugBank 링크)를 포함하여 해당 정보를 제공합니다. </li></ol> 고급 옵션 2. 탐험 <ol><li> 페이지의 오른쪽에있는 &quot;고급&quot;버튼을 클릭하고, 또한 결과를 수정하는 몇 가지 옵션이 제공됩니다. 약물, 협회, 질병, 잠재력, 유전자 및 조치 : 고급 검색 옵션은 다음에 대한 수정을 포함한다. </li><li> 수출은 페이지의 오른쪽에있는 &#39;내보내기&#39;버튼을 클릭하여 테이블을 발생합니다. 고급 검색 창을 아래로 접어하기 위해서 &quot;단순&quot;버튼을 클릭합니다. &lt;/ 리&gt; <li> 고급 옵션 &quot;약물&quot;탭에서, 특정 약물 표시 또는 결과를 필터링하는 추가 약물의 이름을 지정합니다. </li><li> 은 &quot;연결&quot;탭에서, 유의 수준의 P-값, FDR과 조정 된 P-가치, 효과 크기 (확율 비율) 및 / 또는하여 필터 결과 유형 (GWAS, PheWAS 또는 둘 다)을 공부한다. </li><li> 은 &quot;질병&quot;탭에서 예측 된 새로운 사용하기위한 특정 질병에 대한 설명을 지정합니다. </li><li> 은 &quot;전위&quot;탭에서 다음 조건 중 어느 한 항에있어서, 필터 결과는 (i) 약물 표시가 DrugBank 데이터베이스에 포함되어 있는지 여부, 약물의 동시 발생 및 질병과 Medline을 초록의 (II)의 수 ( 약물의 동시 발생과 질병 (IV) 잠재적 용도 변경과 ClinicalTrials.gov 데이터베이스 항목의 ⅲ) 수. 주 : (II)를 강하게지지 관계가 이미 DrugBank 데이터베이스에 존재 : 알려진 (I) 다음 잠재적 용도 변경 옵션은 검색의 신규성을 설명: 두 임상 시험 레지스트리와 Medline을 초록의 일부 지원, (ⅲ) 가능성 : 임상 시험 레지스트리 또는 Medline을 초록과 중 일부 지원 (ⅳ) 소설 : 임상 시험 레지스트리도 Medline을 초록의 증거. </li><li> 은 &quot;유전자&quot;탭 아래에서 SNP ID를 입력하거나 유전자 기호는 특정 약물 표적 유전자에 의한 결과를 필터링 할 수 있습니다. </li><li> &quot;작업&quot;탭에서 작용제, 길항제, 기타, 알 수없는 또는 모든 (: DrugBank 데이터베이스 소스)를 포함하는 약물 표적 (들)에 대한 약물 작용의 유형을 지정합니다. </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.

높은 처리량 약물 및 납 화합물 스크리닝을 포함하여 기존의 신약 개발 접근 방식과 관련된 비용이 많이 들고 비효율적 인 프로세스는, 환자 1, 2에 대한 치료에 대한 연구의 발견을 번역에 지연에 기여하고있다. 10 억 달러로 15 ~ 20 년 평균은 침대 옆 3 벤치에서 새로운 약을 가지고해야합니다. 또한, 약물의 52 %는 1 상 임상 시험에서 개발하는 동안 실패하고, 2 단계를 입력 화합물의 25 %는 전체 3 상 임상 시험 (4)에 진행합니다. 약물의 용도 변경 또는 약물 재배치의 목적은 실패 마약을 갱신 및 / 또는 더 빨리 환자들에게 새로운 치료법을 제공하고 높은 성공률과 함께하기 위해 승인 된 의약품에 대한 새로운 적응증을 찾는 것입니다. 의약품의 용도 변경은 3-12년 5 환자에 사용되는 약물을 사용할 수 있도록 타임 라인을 감소시킬 수있다. 약물 용도 변경을위한 중요한 의료 응용 프로그램은 다음과 같습니다 가난한 prognos과 질병하고 낮은 생존율, 약제 내성 질병, 충분한 자금 질환 연구 분야와 가난하고 소외된 환자 집단. 전산 약물의 용도 변경은 설계 및 약물 후보 6 새로운 적응증에 대한 가설을 생성 할 수있는 자동화 된 워크 플로우를 검증하는 과정으로 정의된다. 기존 컴퓨팅 약제 용도 변경 방법은 타겟 기반 기술 기반 서명 기반, 네트워크 기반의 분류 및 타겟팅 메커니즘 - 기반 및 유전자 질환 또는 약물 관점에서 배향 될 수있다. 또한, 계산 방법은 또한 용도 변경 약물 후보 7 개념 증명 검증 실험 및 소규모 임상 연구를 가속화 할 수 있습니다. 우리는 이전에 &quot;RE : 좋은 약&quot;에보고 한 약물 유전자 - 질병의 관계 (8)의 전이 이론을 바탕으로 의약품 용도 변경 가설 생성을위한 자유롭게 사용할 대화 형 웹 기반 도구입니다. 전체 g이 방법의 OAL 체계적으로 임상, 산업 및 규제 지역 사회 등 다양한 지역 사회에서 사용자의 용도 변경 약물을 사용하는 약물, 유전자 및 임상 데이터의 다양한 유형을 통합하는 것이다. 이 시스템에 대한 기초적인 방법은 이전 게놈 넓은 협회 연구 (GWAS) 연구 9,10 용도 변경 약물에 발 형체 전체 연관 연구 (PheWAS) 데이터의 사용에 대한보고되었다. 이러한 유형의 데이터의 새로운 조합은 다른 목표 기반의 방법 6,11에서 우리의 웹툴을 구분합니다. 재 : 고급 마약 시스템은 현재 916 마약, 567 유전자와 1,770 질병을 다루는 60,911 약물 용도 변경 가설이 포함되어 있습니다. 웹툴 대화 형 약물 용도 변경 가설을 검색하고 다양한 기준을 사용하여 우선 순위를 연구자를위한 사용자 친화적 인 인터페이스를 제공합니다. 예를 들어, 사용자는 생물 의학 문헌에서 지원 및 임상 시험 databa와 약물 용도 변경 가설을 필터링 할 수 있습니다그 자체로 상당한 P-값, 협회 확률 비율 또는 특정 표시로. 이 시스템에 대한 유일한 요건은 인터넷이다.

<table border="0" cellpadding="0" cellspacing="0" width="797">
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			<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.

이 예에서, 유전자 &quot;IL2RB&quot;는 유전자 기반 쿼리로 입력하고, 자동으로 자동 완성 기능 (도 1)에 의해 그렇게 인식 하였다. 도 2에 도시 된 바와 같이 IL2RB 유전자의 열두 약제 용도 변경 가설이 반환된다. 이 경우 특정 약물의 용도 변경 가설, &quot;클리&quot;에 대한 상세 정보 페이지는 &quot;정보&quot;열 (그림 3)에서 제공된다. 약물 탭은 &quot;클리&quot;약물에 대응하는 모든 결과가되었다에도 4에 도시 된 바와 같이 그 결과를 여과 하였다. 그림 5는 &quot;이식&quot;질병 용어 (: DrugBank 데이터베이스 소스)에 대한 알려진 표시 만 그 약을 보여줍니다. 협회 탭 홍보 확률의 비율로 정의 통계적 유의성 (P-값) 유전 효과 크기 (교차비)에 의해 SNP-표현형 관계를 필터링 할 수있는 사용자를 할 수 있습니다염기 다형성 대립 유전자가없는 개인 질병의 존재의 가능성을 통해 특정 유전자형 (SNP 대립 유전자)와 개인의 질병의 esence. 도 6은 0.000001 0.05로 P 값의 범위 내에서 연결 탭 필터링 된 결과를 나타낸다. 그림 7은 우리가 연구에서 발견 된 새로운 적응증에 따라 &quot;천식&quot;에 대한 특정 약물 용도 변경 가설을 보여줍니다. 잠재적 탭에서 그림 8은 결과 약물과 질병 용어의 동시 발생을 포함하는 5 Medline을 초록의 최소 숫자로 필터링합니다. 이 예에서, 유전자 탭 모든 약물 결과를 원래의 질의 용어 (도 9)에 대응하는 &quot;IL2RB&quot;유전자를 대상으로한다. 마지막으로, &quot;작업&quot;탭에서 여과 그림 10은 결과는 IL2RB 유전자에 작용 물질의 역할을 모두 약물을 반환합니다. <img alt="그림 1"SRC = &quot;/ 파일 / ftp_upload / 54948 / 54948fig1.jpg&quot;/&gt; 그림 1 : RE : 좋은 마약 대화 형 대시 보드 홈페이지. 사용자는 약 이름, 새로운 질병의 표시 또는 유전자 기호를 입력하여 조회를 시작할 수 있습니다. 링크는 또한 약물의 용도 변경 가설을 생성하는 방법을 설명하는 GWAS와 PheWAS 참조 서류 제공됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 그림 2 : 쿼리 항목에 대한 자동 채우기 기능. 예로서, 유전자 검색어 &quot;IL2RB&quot;가 자동 유전자 용어로 인식되었다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <p유지-together.within 페이지 = &quot;1&quot;&gt; : FO 클래스 = &quot;jove_content&quot; <img alt="그림 3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 그림 3 : 유전자 기반 쿼리 (예를 들어, IL2RB)에서 생산되는 의약품 용도 변경 결과를 테이블. IL2RB 유전자에 대한 십이 약물 용도 변경 가설이 생성된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 그림 4 : 결과 페이지에서 개별 의약품에 대한 정보 페이지. &#39;정보&#39;항목에서 아이콘을 클릭하면 약물 클리에 대한 상세한 정보를 보여줍니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 5 : 약물 탭에서 고급 검색 옵션은 특정 약물에 의해 필터링합니다. 이러한 예에서, 세 가지 결과는 약물 클리 위해 도시된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 그림 6 : 약 탭에서 고급 검색 옵션은 DrugBank 데이터베이스에서 특정 질병의 지시에 의해 필터링합니다. 질병 용어는 &quot;이식&quot;에 대한 알려진 표시 모든 약물이 표시됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 7 : 연결 탭에서 고급 검색 옵션은 유의 수준을 기준으로 필터링합니다. 이 경우, 팔의 연관 결과 유의 수준 0.05-0.000001의 P 값의 범위 내에 설치되어있다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 그림 8 : 질병 탭에서 고급 검색 옵션은 우리가이 연구에서 추출 된 특정 질병의 지시에 의해 필터링합니다. 이 예에서, 네 개의 결과 새로운 용도 질환 표시 천식에 대해 도시된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">큰 versi을 보려면 여기를 클릭하십시오</a>이 그림의에. <img alt="그림 9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 그림 9 : 잠재적 탭에서 고급 검색 옵션은 Medline을 초록 약물과 질병 용어의 동시 발생에 의해 필터링합니다. 이 예에서, 네 개의 결과 약물 질병 용어의 동시 발생을 함유하는 5 Medline을 초록의 최소 수에 의해 지원되는 것을 나타낸다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 그림 10 : 유전자 탭에서 고급 검색 옵션은 모든 결과는 원래의 질의에 사용되는 IL2RB 유전자에 해당하는 특정 유전자 기호에 의해 필터링합니다. 이 예에서, 모든 약물 결과유전자 탭에서 원래 검색어에 해당하는 &quot;IL2RB&quot;유전자를 대상으로하고 있습니다. 작업 탭에서 고급 검색 옵션 효능 약물에 의해서만 필터링합니다. 이 예에서, 여섯 결과는 IL2RB 유전자에 작용 물질의 역할을 모든 의약품에 대한 반환됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

은 &quot;RE를 : 좋은 약&quot;을 사용하여 의약품 용도 변경 가설 발전 시스템

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

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JoVE Journal

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9.2K 조회수.  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. 

비디오: 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.

외과 종양 표본의 Explants를 사용하여 소설 안티 - 암 의약품 개발을위한 방법


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

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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.

소설 허벌 요법을 평가하는 패혈증의 동물 모델의 사용

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.

생성 및 마우스의 동맥 병변의 3 차원 정량 광학 프로젝션 단층 촬영을 사용하여

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.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

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 / MS를 사용하여 건조 혈액 명소에 면역 억제제 타 크롤리 무스의 정량화

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.

<em&gt; 체외</em&gt; 골수에서 에스트로겐에 민감한 유방암의 휴면 모델 : 분자 메커니즘 연구 및 가설 생성을위한 도구

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 프로파일 의약품 응답 테스트를위한 3D 인간의를 Biogel 문화 시스템을 사용 Microtumors 및 환자 유래 아교 모세포종 세포의 생성

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.

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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.

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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.

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

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