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.基因查询发起，药物或疾病条款<ol><li>通过以下链接:http://drug-repurposing.nationwidechildrens.org:访问为"精药品RE"的主页。药（仿制药名），疾病（新病指示）或基因（官方HGNC基因符号）:由从任何以下三类进入搜索栏查询词开头。 </li><li>过滤搜索栏的功能，只包括"药品名称"，"新疾病适应症"，"基因符号"或"全部"类别。搜索栏包括查询条目自动填充功能。 </li><li>放在一个关键字，然后点击"搜索"按钮。排序通过以下任一列的结果表:"药品"，"注册指示"，"P值"，"P-值来调整"，"胜算比"，"研究"，"新标识"，"药品银行指示"，"MEDLINE文摘＃"，"CLI命令＃nical试验注册中心"，"势"，"SNP"，"基因"或"行动"。 </li><li>导航到高级搜索选项，以使药品信息功能。在"信息"栏中点击图标为一个特定的药物。观察一个网页，列出所有相应的信息，包括对价值关联，疾病名称，药品名称，基因信息（NCBI基因链接）药品的详细信息（DrugBank链接）。 </li></ol> 2.高级选项的探索<ol><li>点击位于页面的右侧的"高级"按钮，并提供多种选择，以进一步缩小的结果。高级搜索选项包括修改如下:药物，协会，疾病，潜在的基因和行动。 </li><li>出口通过点击页面右侧的"导出"按钮，结果表。点击以向下折叠高级搜索窗口中的"简单"按钮。&lt;/ LI&gt; <li>在高级选项"毒品"选项卡中，指定特定的药物适应症或额外的药名来筛选结果。 </li><li>在"关联"选项卡中，筛选的显着性水平P值，调整后的P值与FDR，影响大小（比值比），和/或研究型（GWAS，PheWAS或两者）。 </li><li>在"治未病"选项卡中，指定新的预测使用某种疾病的描述。 </li><li>下的"潜在"标签中，根据以下任一条件过滤结果:（i）药物指示是否被包含在DrugBank数据库，（ⅱ）数量MEDLINE摘要与药物的共现和疾病中，（ ⅲ）数量与药物的共现和疾病以及（iv）重新利用潜在ClinicalTrials.gov数据库条目。 注:重新为潜在选项描述了发现的新颖性:（一）已知:关系已存在于数据库DrugBank，（二）大力支持:一些支持在临床试验注册和MEDLINE摘要;（三）可能是:一些支持在任何临床试验注册表或MEDLINE摘要及（iv）小说:在临床试验注册也不在MEDLINE摘要没有证据。 </li><li>在"基因"标签，输入SNP标识符或基因符号，以滤除特定的药物靶基因的结果。 </li><li>在"操作"选项卡，指定打击毒品目标（S），其中包括激动剂，拮抗剂等，未知或全部（来源:DrugBank数据库）药物作用的类型。 </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.

与传统药物发现方法，包括高通量药物和先导化合物筛选相关的昂贵和低效的过程，是造成延误翻译研究发现转化为治疗病人1,2。 1十亿美元和15 - 20年的平均时需携带从板凳上一个新的药物床边3。此外，药品52％的第一阶段临床试验在开发过程中失败了，只有25％即进入第二阶段的化合物进行充分三期临床研究4。再利用药物或药物重新定位的目标是续约失败的药物和/或找到批准的药品适应症新颖，以提供新的治疗方法给患者更快的速度和更高的成功率。药品再利用可能会降低时间表，使现有的药物在患者中使用，以3-12岁5。药品再利用的重要医疗应用包括:与差PROGNOS疾病是低存活率，抗药性疾病，资金不足的疾病的研究领域和贫困和缺医少药的病人群体。 计算药物再利用被定义为设计和验证自动化的工作流程，可以生成用于为候选药物6新适应症假设的过程。现有的计算药物再利用的方法已被归类目标为基础，以知识为基础的，基于签名的，基于网络和基于目标的机制，并可以从基因，疾病或药物的角度来导向。此外，计算方法可能会进一步加速证明了概念验证性实验和小规模临床研究为改变用途的候选药物7。我们曾报道的"RE:精药品"，一个免费的，互动式的网上药品再利用产生假设基于药物基因与疾病的关系8的传递理论工具。整体摹这种方法的OAL是系统集成不同类型的药物，遗传和临床资料，以使药物再利用从不同的社区，包括临床，工业和监管社区用户。该系统的基本方法先前已经报道了利用全基因组关联分析（GWAS），并在药物phenome范围内的关联研究（PheWAS）数据再利用的研究9,10。这些类型的数据的新组合，我们区别于其他基于目标的方法6,11 webtool。 该RE:精药物制度目前包含60911药物再利用假设覆盖916药品，567个基因和1,770疾病。该webtool为研究人员提供交互式搜索药物重复利用的假设和使用不同的标准优先考虑他们一个友好的用户界面。例如，用户可以过滤药物再利用假设用在生物医学文献支持和临床试验databaSE，显著p值，关联比值比或具体指示。此系统的唯一要求是上网。

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

在这个例子中，基因"IL2RB"被输入为基于基因的查询，以及由自动填充功能（ 图1）中的自动识别。对于IL2RB基因十二药物重新利用假设被返回， 如图2。对于特定的药物再利用假说"，达珠单抗"在这种情况下，详细信息页，从"信息"栏（ 图3）提供。结果被过滤的药物标签是通过对应于"赛尼哌"药物的所有结果， 如图4。 图5显示了只有这些药物具有已知的指示，为"移植"长期疾病（来源:DrugBank数据库）。关联选项卡允许用户过滤通过统计学意义（P值）和遗传效应大小（比值比），SNP-表型的关系，限定为公关的几率的比率在与特定基因型（SNP等位基因）以上的个体疾病的存在的可能性，而不与SNP等位基因的个体的疾病的esence。 图6示出的P值范围内的关联标签下过滤的0.000001至0.05的结果。 图7显示了具体的基础上，我们在研究中发现的新适应症"哮喘"药品再利用假设。 图8表示的电位标签下的结果通过含有的药物和疾病方面共现5 MEDLINE摘要的最小数目进行过滤。在这个例子中，基因标签下所有药物的结果针对所述"IL2RB"基因，对应于原始查询字词（ 图9）。最后，"行动"选项卡下过滤， 如图 10所示的成绩来回报充当对IL2RB基因兴奋剂所有药物。 <img alt="图1"SRC ="/文件/ ftp_upload / 54948 / 54948fig1.jpg"/&gt; 图1:RE:精药品的交互式仪表板主页。用户可以通过输入药品名称，新的疾病指示或基因符号开头的查询。还提供了用于说明用于产生药物再利用假设方法的GWAS和PheWAS参考文件的链接。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 图 2: 查询条目自动填充功能。作为一个例子，所述基因查询词"IL2RB"被自动识别为一个基因术语。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 图3: 从基于基因的查询（ 例如，IL2RB）产生药物再利用的结果的表。对于IL2RB基因药物十二假设再利用产生。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 图4:从结果页个别药物信息页面。点击从"信息"列中的图标显示为药物达珠单抗的详细信息。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5: 药品标签下的高级搜索选项通过特定的药物进行筛选。在此示例中，三个结果示为药物达珠单抗。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 图6: 药品标签下的高级搜索选项从DrugBank数据库中的特定疾病的指示进行过滤。与用于疾病的术语"移植"公知的指示所有药物被示出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图7:关联选项卡下的高级搜索选项，通过显着性水平进行筛选。在这种情况下，设置八个结果其关联显着性水平的0.000001至0.05的P值范围内。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 图8: 疾病标签下高级搜索选项，通过我们在本研究中提取一个特定的疾病的指示进行筛选。在这个例子中，四结果示哮喘作为新使用的疾病的指示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">请点击此处查看大VERSI</a>这个数字的。 <img alt="图9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 图9: 潜在选项卡下的高级搜索选项，通过在MEDLINE摘要药物和疾病方面共现过滤。在这个例子中，四结果示由含有的药物和疾病方面共现5 MEDLINE摘要的最小数目的支持。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 图10: 基因标签下高级搜索选项由特定基因符号，其中所有结果对应于用于原始查询IL2RB基因进行筛选。在这个例子中，所有的药物的结果该基因标签下被定位为"IL2RB"基因，对应于原始查询字词。操作选项卡下的高级搜索选项只受体激动剂的药物筛选。在这个例子中，六结果返回为充当对IL2RB基因激动剂所有药物。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</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.

药品再利用产生假设使用"RE:精药品"系统

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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Medicine

9.2K 次观看.  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. 

视频: 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.

新型抗癌药物使用手术标本肿瘤植的发展方法


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

科研

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.

脓毒症动物模型，用于评估新型草药疗法

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.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

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.

收集和唾液DNA提取用于新一代测序

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.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

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.

一个<em&gt;在体外</em&gt;对雌激素敏感的乳腺癌休眠模式在骨髓中:分子机理研究与假设生成的一个工具

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.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

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.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

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.

利用视网膜成像研究痴呆

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.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

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