Broadening diversity through creative involvement to identify research priorities.

BACKGROUND: Patient and public involvement (PPI) can help with steering and shaping research prioritisation and execution. However, some groups of people may not be encouraged to take part and their voices may be seldom listened to in the production of research. This is important to consider because they may have poorer healthcare experiences. We tried using art as a vehicle for including individuals not necessarily invited to be part of research priority setting. METHODS: We contacted existing groups and organisations to reach people not routinely supported to be part of PPI. We targeted individuals: a) with dementia, b) with a mental and physical health condition, c) of South Asian heritage. We ran a workshop with each group at which individuals shared their experiences of healthcare. A young amateur artist also attended, who produced a piece of artwork afterwards that reflected the research priorities raised. We held a Twitter chat to discuss these pieces of art and the processes involved in their generation. RESULTS: From each workshop, we produced a list of research priorities. These included: a) improving coordination of care for people with dementia, b) information needs and anxiety/guilt around accessing care for people with physical and mental health conditions, c) supporting discussion of women's health issues in South Asian communities. These priorities were reflected in three pieces of art, which can be viewed online. Feedback from those at workshops suggested that the artwork helped them to feel that their voice had been heard and triggered their interest in how research is developed. Those involved in the Twitter chat commented that art was one means through which researchers could connect with a range of groups in a PPI context when preparing and producing a study. CONCLUSIONS: We found the medium of art to be an effective way of including a range of people in research prioritisation setting. This approach could be useful for future PPI, building on what we have learnt from the project described in this paper.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels.

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Automated cell plating and sample treatments for fixed cells in high content assays.

Robust and reliable methods for the manipulation of neural cell lines, by passaging, plating, dye labeling, imaging, fixation, and immunocytochemistry, are required to enable consistent, reproducible screens to be performed. We describe herein procedures and processes we have established to maximize the level of consistency of cell plating, fixation, and dye or antibody labeling, to ensure that assays which we are running on a routine basis remain consistent across long periods of time. These procedures involve a variety of fully or semiautomated steps, using high-quality commercially available liquid handling and dispensing technology.

Measurement and analysis of calcium signaling in heterogeneous cell cultures.

High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

A morphology- and kinetics-based cascade for human neural cell high content screening.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional high-throughput screening approaches.

High content kinetic assays of neuronal signaling implemented on BD pathway HT.

A great deal of information can be gained from kinetic fluorescence-based measurement of cellular responses; however, until recently the use of such approaches has been limited by the manual nature of the instrumentation available. Higher-throughput kinetic studies of signaling pathways are greatly facilitated by new confocal, liquid handling-enabled, high content screening (HCS) platforms. In the present work, we have implemented one such instrument, the BD(TM) Pathway HT bioimager (BD Biosciences, Rockville, MD), for studying regulation of neuronal signaling pathways. We have established a neuronal calcium oscillation model, whereby rate of oscillation, amplitude of oscillation, and level of synchronicity across the culture can be measured. We have implemented membrane potential measurement using fluorescence resonance energy transfer-based dyes, for single cell characterization on this platform, showing the benefits of a truly flexible excitation and recording system; this dye combination cannot be readily implemented on all HCS platforms because of constraints of excitation wavelengths. We have validated long-term intracellular calcium imaging experiments, using innovative dyes and BD Pathway HT's spinning disk-based confocal excitation. To maximize both throughput and reproducibility, walk-away automation integration of this bioimaging technology has been implemented, producing an affordable, compact platform for fully automated kinetic HCS.

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF.

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.

Quantitative assays of chemotaxis and chemokinesis for human neural cells.

Cell migration is vital for many physiological processes, and its modulation is likely to be of therapeutic benefit. In this study we have developed fluorescence image-based chemokinesis and chemotaxis assays on the Cellomics (Pittsburgh, PA) ArrayScan platform, which would be suitable for industrial drug discovery in a variety of fields. Studying the migratory characteristics of neural stem cells is of interest for understanding the therapeutic potential of these cells, in terms of both cellular transplantation and the activation of endogenous populations of stem cells. Growth conditions were identified whereby human neural precursors could be maintained as neurospheres and plated out into microtitre plates for high-content assays. Chemokinesis was assessed using fluorescent bead-coated 96-well microtitre plates, whilst chemotaxis was assessed using BD Biosciences (Oxford, UK) Fluoroblok 24-well plates. Assays for both chemokinesis and chemotaxis were developed that were quantified automatically using the ArrayScan and appropriate algorithms. Using the two complementary techniques, foetal bovine serum was observed to have chemokinetic effects on the cells, whilst platelet-derived growth factor isoform AB was chemotactic. The two assays described here are suitable for screening for novel modulators of cell migration, or for performing more detailed mechanistic follow-up studies. These assays enable us to perform cell motility studies with minimal laboratory handling, in an automated manner, thereby allowing quantitative studies of cell behaviour to be incorporated in a routine drug discovery screening cascade.

The search for novel TRPV1-antagonists: from carboxamides to benzimidazoles and indazolones.

Based on a series of diaryl amides the corresponding inverse amides have been found to be potent TRPV1 receptor antagonists. Benzimidazole and indazolone derivatives prepared retained good potency in vitro and indazolone 4a was identified as a novel TRPV1 receptor antagonist suitable for evaluating orally in animal models of analgesia.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signáis that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signáis involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of reléase from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.