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. Initiation of Queries from Gene, Drug or Disease Terms
<ol>
	<li>Access the homepage for &#34;RE:fine Drugs&#34; at the following link: http://drug-repurposing.nationwidechildrens.org. Begin by entering a query term in the search bar from any of the following three categories: drug (generic drug name), disease (new disease indication) or gene (official HGNC gene symbol).</li>
	<li>Filter the search bar function to include only &#34;Drug Name&#34;, &#34;New Disease Indication&#34;, &#34;Gene Symbol&#34; or &#34;All&#34; categories. The search bar includes an auto-fill function for query entries.</li>
	<li>Put in a keyword, and click on the &#34;Search&#34; button. Sort the table of results by any of the following columns: &#34;Drug&#34;, &#34;Registered Indication&#34;, &#34;P-Value&#34;, &#34;P-Value Adjusted&#34;, &#34;Odds Ratio&#34;, &#34;Study&#34;, &#34;New Indication&#34;, &#34;Drug Bank Indication&#34;, &#34;# of Medline Abstracts&#34;, &#34;# of Clinical Trial Registry&#34;, &#34;Potential&#34;, &#34;SNP&#34;, &#34;Gene&#34; or &#34;action&#34;.</li>
	<li>Navigate to the advanced search option in order to enable the drug information feature. Click on the icon in the &#34;Info&#34; column for a particular drug. Observe a page that lists the all the corresponding information including p-Value for the association, Disease name, Drug name, Gene details (NCBI Gene link) and Drug details (DrugBank link).</li>
</ol>
2. Exploration of Advanced Options
<ol>
	<li>Click on the &#34;advanced&#34; button located on the right side of the page, and several options to further refine the results are provided. The advanced search options include modifications to the following: drug, association, disease, potential, gene and action.</li>
	<li>Export results tables by clicking on the &#34;Export&#34; button on the right side of the page. Click on the &#34;Simple&#34; button in order to fold down the advanced search window.</li>
	<li>Under the advanced option &#34;drug&#34; tab, specify a particular drug indication or an additional drug name to filter results.</li>
	<li>Under the &#34;association&#34; tab, filter results by the significance level P-Value, Adjusted P-Value with FDR, effect size (Odds Ratio), and/or Study type (GWAS, PheWAS or Both).</li>
	<li>Under the &#34;disease&#34; tab, specify a certain disease description for the predicted new use.</li>
	<li>Under the &#34;potential&#34; tab, filter results according to any of the following criteria: (i) whether the drug indication is contained in the DrugBank database, (ii) number of Medline abstracts with co-occurrence of the drug and the disease, (iii) number of ClinicalTrials.gov database entries with co-occurrence of the drug and the disease and (iv) Repurposing Potential. 
	NOTE: The Repurposing Potential option describes the novelty of the discovery: (i) Known: relationship already exists in the DrugBank database, (ii) Strongly supported: some support in both clinical trial registry and the Medline abstracts, (iii) Likely: some support in either clinical trial registry or the Medline abstracts and (iv) Novel: no evidence in clinical trial registry nor in Medline abstracts.</li>
	<li>Under the &#34;gene&#34; tab, enter a SNP identifier or Gene Symbol to filter results by specific drug target genes.</li>
	<li>Under the &#34;action&#34; tab, specify the drug action type against the drug target(s), including agonist, antagonist, other, unknown or all (source: DrugBank database).</li>
</ol>

<ol>
	<li>Borisy, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+discovery+of+multicomponent+therapeutics.">Systematic discovery of multicomponent therapeutics.</a> Proc Natl Acad Sci U S A. 100 (13), 7977-7982 (2003).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+synergy+screening+identifies+microbial+metabolites+as+combination+agents+for+the+treatment+of+fungal+infections.">High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections.</a> Proc Natl Acad Sci U S A. 104 (11), 4606-4611 (2007).</li><li>Adams, C. P., Brantner, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+cost+of+new+drug+development:+is+it+really+802+million+dollars?.">Estimating the cost of new drug development: is it really 802 million dollars?.</a> Health Aff (Millwood). 25 (2), 420-428 (2006).</li><li>Bunnage, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+pharmaceutical+R&amp;D+back+on+target.">Getting pharmaceutical R&amp;D back on target.</a> Nat Chem Biol. 7 (6), 335-339 (2011).</li><li>Ashburn, T. T., Thor, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+repositioning:+identifying+and+developing+new+uses+for+existing+drugs.">Drug repositioning: identifying and developing new uses for existing drugs.</a> Nat Rev Drug Discov. 3 (8), 673-683 (2004).</li><li>Hurle, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+drug+repositioning:+from+data+to+therapeutics.">Computational drug repositioning: from data to therapeutics.</a> Clin Pharmacol Ther. 93 (4), 335-341 (2013).</li><li>Jin, G., Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+better+drug+repositioning:+prioritizing+and+integrating+existing+methods+into+efficient+pipelines.">Toward better drug repositioning: prioritizing and integrating existing methods into efficient pipelines.</a> Drug Discov Today. 19 (5), 637-644 (2014).</li><li>Moosavinasab, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;RE:fine+Drugs&amp;#34;:+An+Interactive+Dashboard+to+Access+Drug+Repurposing+Opportunities.">34;RE:fine Drugs&amp;#34;: An Interactive Dashboard to Access Drug Repurposing Opportunities.</a> Database (Oxford). , (2016).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genome-wide+association+studies+for+cancer+research+and+drug+repositioning.">Use of genome-wide association studies for cancer research and drug repositioning.</a> PLoS One. 10 (3), e0116477 (2015).</li><li>Rastegar-Mojarad, M., Ye, Z., Kolesar, J. M., Hebbring, S. J., Lin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+for+drug+repositioning+from+phenome-wide+association+studies.">Opportunities for drug repositioning from phenome-wide association studies.</a> Nat Biotechnol. 33 (4), 342-345 (2015).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+current+trends+in+computational+drug+repositioning.">A survey of current trends in computational drug repositioning.</a> Brief Bioinform. 17 (1), 2-12 (2016).</li><li>Denny, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+phenome-wide+association+study+of+electronic+medical+record+data+and+genome-wide+association+study+data.">Systematic comparison of phenome-wide association study of electronic medical record data and genome-wide association study data.</a> Nat Biotechnol. 31 (12), 1102-1110 (2013).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+drug-disease+relationships+as+a+complement+to+medical+genetics-based+drug+repositioning:+Where+a+recommendation+system+meets+genome-wide+association+studies.">Mining drug-disease relationships as a complement to medical genetics-based drug repositioning: Where a recommendation system meets genome-wide association studies.</a> Clin Pharmacol Ther. 97 (5), 451-454 (2015).</li><li>Sanseau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genome-wide+association+studies+for+drug+repositioning.">Use of genome-wide association studies for drug repositioning.</a> Nat Biotechnol. 30 (4), 317-320 (2012).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validating+drug+repurposing+signals+using+electronic+health+records:+a+case+study+of+metformin+associated+with+reduced+cancer+mortality.">Validating drug repurposing signals using electronic health records: a case study of metformin associated with reduced cancer mortality.</a> J Am Med Inform Assoc. 22 (1), 179-191 (2015).</li></ol>

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.

The costly and inefficient processes associated with traditional drug discovery approaches, including high-throughput drug and lead compound screening, are contributing to delays in translating research discoveries into therapies for patients 1,2. An average of 1 billion U.S. dollars and 15-20 years is required to bring a new drug from the bench to the bedside 3. Further, 52% of drugs fail during development in phase 1 clinical trials, and only 25% of compounds that enter phase 2 proceed into full phase 3 clinical studies 4. The goal of drug repurposing or drug repositioning is to renew failed drugs and/or find novel indications for approved drugs in order to deliver new therapies to patients faster and with a higher success rate. Drug repurposing may decrease the timeline to make drugs available for use in patients to 3-12 years 5. Important medical applications for drug repurposing include: diseases with poor prognosis and low survival rates, drug-resistant diseases, underfunded disease research areas and impoverished and underserved patient populations.
Computational drug repurposing is defined as the process of designing and validating automated workflows that can generate hypotheses for new indications for a drug candidate 6. Existing computational drug repurposing methods have been categorized target-based, knowledge-based, signature-based, network-based, and targeted-mechanism-based, and can be oriented from gene, disease or drug perspectives. Furthermore, computational approaches may further accelerate proof-of-concept validation experiments and small-scale clinical studies for repurposed drug candidates 7. We have previously reported on &#34;RE:fine Drugs&#34;, a freely available, interactive web-based tool for drug repurposing hypothesis generation based on the transitive theory of Drug-Gene-Disease relationships 8. The overall goal of this method is to systematically integrate diverse types of drug, genetic and clinical data to enable drug repurposing for users from diverse communities, including clinical, industry and regulatory communities. The foundational methods for this system have been previously reported for the use of genome-wide association study (GWAS) and phenome-wide association study (PheWAS) data in drug repurposing research 9,10. The novel combination of these types of data distinguishes our webtool from other target-based methods 6,11.
The RE:fine Drugs system currently contains 60,911 drug repurposing hypotheses covering 916 drugs, 567 genes and 1,770 diseases. The webtool provides a user-friendly interface for researchers to interactively search drug repurposing hypotheses and prioritize them using diverse criteria. For instance, users can filter drug repurposing hypotheses with support in the biomedical literature and clinical trials database, significant p-values, association odds ratios or by specific indications. The only requirement for this system is Internet access.

<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.

In this example, the gene &#34;IL2RB&#34; was entered as a gene-based query, and was automatically recognized as such by the auto-fill function (Figure 1). The twelve drug repurposing hypotheses for the IL2RB gene are returned, as shown in Figure 2. Detailed information page for a particular drug repurposing hypothesis, &#34;daclizumab&#34; in this case, is provided from the &#34;Info&#34; column (Figure 3). The results were filtered on the drug tab was by all results corresponding to the &#34;daclizumab&#34; drug, as shown in Figure 4. Figure 5 shows only those drugs with a known indication for the &#34;transplant&#34; disease term (source: DrugBank database). The association tab allows the user to filter SNP-Phenotype relationships by statistical significance (P-value) and genetic effect size (odds ratio), defined as the ratio of the odds of presence of disease in individuals with a specific genotype (SNP allele) over the odds of presence of disease in individuals without the SNP allele. Figure 6 shows the results filtered under the association tab within the P-Value range of 0.000001 to 0.05. Figure 7 shows drug repurposing hypotheses specific for &#34;asthma&#34; based on the new indications we found in the study. Figure 8 shows results under the potential tab to filter by a minimum number of 5 Medline abstracts containing a co-occurrence of drug and disease terms. In this example, all drugs results under the gene tab are targeted for the &#34;IL2RB&#34; gene, corresponding to the original query term (Figure 9). Finally, Figure 10 shows results filtered under the &#34;action&#34; tab to return all drugs that act as agonists on the IL2RB gene.
<img alt="Figure 1" src="/files/ftp_upload/54948/54948fig1.jpg" /> 
Figure 1: The RE:fine Drugs interactive dashboard homepage. Users may begin a query by entering a drug name, new disease indication or gene symbol. Links are also provided for the GWAS and PheWAS reference papers describing methodologies for generating drug repurposing hypotheses. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 
Figure 2: Auto-fill function for query entries. As an example, the gene query term &#34;IL2RB&#34; was automatically recognized as a gene term. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 
Figure 3: Drug repurposing results table produced from a gene-based query (e.g., IL2RB). Twelve drug repurposing hypotheses for the IL2RB gene are produced. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 
Figure 4: Information page for individual drugs from results page. Clicking on the icon from the &#34;Info&#34; column shows detailed information for the drug daclizumab. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54948/54948fig5.jpg" /> 
Figure 5: Advanced search option under drug tab to filter by a specific drug. In this example, three results are shown for the drug daclizumab. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 
Figure 6: Advanced search option under drug tab to filter by a specific disease indication from the DrugBank database. All drugs with a known indication for the disease term &#34;transplant&#34; are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54948/54948fig7.jpg" /> 
Figure 7: Advanced search option under association tab to filter by significance level. In this case, eight results are provided whose association significance level falls within the P-Value range of 0.000001 to 0.05. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 
Figure 8: Advanced search option under disease tab to filter by a specific disease indication we extracted in this study. In this example, four results are shown for asthma as a new use disease indication. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 
Figure 9: Advanced search option under potential tab to filter by a co-occurrence of drug and disease terms in Medline abstracts. In this example, four results are shown that are supported by a minimum number of 5 Medline abstracts containing a co-occurrence of drug and disease terms. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 
Figure 10: Advanced search option under gene tab to filter by a specific gene symbol, where all results correspond to the IL2RB gene used for the original query. In this example, all drugs results under the gene tab are targeted for the &#34;IL2RB&#34; gene, corresponding to the original query term. Advanced search option under action tab to filter by agonist drugs only. In this example, six results are returned for all drugs that act as agonists on the IL2RB gene. <a href="http://cloudflare.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</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.

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 ...

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9.2K Views.  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. 

Video: 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.


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

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.

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.

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.

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.

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.

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.

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.

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.

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 ...