Understanding the funding characteristics of research impact: A proof-of-concept study linking REF 2014 impact case studies with Researchfish grant agreements.

BACKGROUND: All parts of the research community have an interest in understanding research impact whether that is around the pathways to impact, processes around impact, methods for measurement, describing impact and so on. This proof of concept study explored the relationship between research funding and research impact using the case studies submitted to the UK Research Excellence Framework (REF) exercise in 2014 as a proxy for impact. METHODS: The paper describes an approach to link the REF impact case studies with the underpinning research grants present in the Researchfish dataset, primarily using the publications captured in both datasets. Where possible the methodology utilised unique identifiers such as Digital Object Identifiers and PubMed ID's, and where this was not possible the funding information within each publication was used. RESULTS: Through this automated approach 21% of the non-redacted case studies could be linked to a specific research grant. Additional qualitative analysis was then done for unlinked REF impact case studies, which involved reading the document to identify additional information to make the linkage. This approach was taken on 100 REF impact case studies selected at random and resulted in only seven having no identifiable research grants funding associated. The linked research grants were analysed to identify characteristics that are more frequently associated with these grants, than non-linked ones. CONCLUSIONS: This analysis did point to some interesting observations such as the grant funding linked to REF impact case studies are more likely to be longer, higher financial value, have more publications and be more collaborative (amongst other characteristics). These findings should be used with caution at present and not be over interpreted, this is due to the sample size for this proof of concept study and some potential limitations on the data which were not addressed at this stage.

Tales of 1,008 small molecules: phenomic profiling through live-cell imaging in a panel of reporter cell lines.

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥ 0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study's conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.

Endogenous gene tagging with fluorescent proteins.

Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the target's activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs.

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

Tagging of genomic STAT3 and STAT1 with fluorescent proteins and insertion of a luciferase reporter in the cyclin D1 gene provides a modified A549 cell line to screen for selective STAT3 inhibitors.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN)--mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer targets.

Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Oral delivery of insulin with the eligen technology: mechanistic studies.

The development of oral insulin using the eligen technology represents a significant advance in insulin administration which is expected to improve the quality of life of diabetic patients. As clinical studies progress, a great deal of interest has focused on the process by which this technology enables insulin absorption from the intestinal lumen into the bloodstream. The eligen technology employs low molecular weight compounds (termed drug delivery agents or carriers) which interact weakly and non-covalently with insulin, increasing its lipophilicity and thereby its ability to cross the gastrointestinal epithelium. In this study we investigated the mechanism of insulin absorption across caco-2 cell monolayers with one of these drug delivery agents, N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC). Our results show that SNAC increases insulin permeability approximately ten fold across cell monolayers and does so without affecting mannitol permeability or disrupting cell membranes. Confocal microscopy and immunocytochemistry revealed that insulin is transported transcellularly without detectable alteration of the tight junctions between adjacent cells. SNAC also appears to play some role in protecting insulin from proteolytic degradation, potentially allowing for more intact insulin to be available at the site of absorption.

Pathway of oral absorption of heparin with sodium N-[8-(2-hydroxybenzoyl)amino] caprylate.

PURPOSE: The oral bioavailability of heparin is negligible. Recent studies, however, have shown that sodium N-[8-(2-hydroxybenzoyl) amino]caprylate (SNAC) and other N-acylated amino acids enable oral heparin absorption. To investigate the mechanism by which heparin crosses the intestinal epithelium in the presence of SNAC, we have used fluorescence microscopy to follow the transport of heparin across Caco-2 cell monolayers. METHODS: The experiments were carried out on Caco-2 monolayers and Caco-2 cells grown to confluence on culture dishes, using different concentrations of SNAC. The localization of fluorescently labeled heparin was determined using epi-fluorescence and confocal microscopy. DNA dyes were used to determine the effect of SNAC on the plasma membrane integrity. F-actin was labeled with fluorescent phalloidin to investigate the stability of perijunctional actin rings in the presence of SNAC. RESULTS: Heparin was detected in the cytoplasm only after incubation of the cells with heparin and SNAC. No DNA staining was observed in cells incubated with a DNA dye in the presence of SNAC concentrations at which heparin transport occurred. In addition, no signs of actin redistribution or perijunctional ring disbandment were observed during the transport of heparin. CONCLUSIONS: The results indicate that SNAC enables heparin transport across Caco-2 monolayers via the transcellular pathway. Heparin transport in the presence of SNAC is selective and does not involve permeabilization of the plasma membrane or tight junction disruption.