The Australian experience of World Wide Views on global warming: The first global deliberation process

World Wide Views on Global Warming was the first ever global-scale citizen deliberation process, held on 25-26 September 2009 and involving approximately 4,000 citizens in 38 countries. WWViews sought to provide citizens with a voice in the 2009 UN Climate Summit in Copenhagen (COP15) by engaging them in a deliberative process about global political positions on climate change. The process produced clear, comparable results across all participating countries that were given to COP15 negotiators. The Danish Government agencies, the Danish Board of Technology and the Danish Cultural Institute, initiated the global process. Organisers in each participating country ran events using the same standardised process. The University of Technology Sydney, the organisers of the Australian WWViews event, paid special attention to several elements of the process to maximise participation and impact within the local context. This paper outlines the standardised global process used for this deliberative event and describes and reflects upon the tailored approaches developed for Australia. It examines in detail the objectives, processes and outcomes of recruiting and supporting participants and recruiting, training and coordinating facilitators, communications and dissemination of results and specific features of the Australian event. It includes the organisers' reflections on success factors, challenges and surprises, as well as feedback from facilitators and participants. This paper concludes with a number of critical questions arising from the Australian experience of World Wide Views on Global Warming that are pertinent for practitioners designing other deliberative forums and particularly anyone concerned about future prospects for global deliberative democracy. Copyright © 2011 The Berkeley Electronic Press. All rights reserved

POS0361 DNA METHYLATION SIGNATURES CHARACTERIZE PSORIASIS AND PSORIATIC ARTHRITIS IN MONOZYGOTIC TWINS DISCORDANT FOR THE DISEASE

Background:Psoriatic disease is a chronic inflammatory disorder spanning from skin disease (psoriasis) to psoriatic arthritis (PsA). The genetic background is insufficient to explain disease onset as illustrated by not very informative Genome Wide Association Studies and monozygotic (MZ) twin studies recently performed. It is strongly assumed that epigenetics may contribute to disease susceptibility modulating gene expression. DNA methylation has been found involved in several autoimmune inflammatory rheumatic diseases. Here we have analysed the DNA methylation profile of a selected cohort of MZ twins discordant for psoriasis/PsA.Objectives:To identify the methylome associated with psoriasis and PsA in the peripheral blood of MZ twins discordant for these conditions.Methods:Peripheral blood from 7 couples of MZ twins discordant for psoriatic disease was collected and DNA extracted for a genome-wide evaluation of the DNA methylation profile, with the Infinium MethylationEPIC BeadChip. Minfi and the packages of the Bioconductor were used to analyse the data obtained. Quality control and exclusion criteria were applied to the raw data having a final number of 762.451 probes, which accounts for 88% of the total.Results:The approach first identified 2564 differentially methylated positions (DMPs; *p<0.005) with 19 genes potentially affected (with at least two DMPs within 1 kb of distance), including SMAD3 and SMARCA4/BRG1 involved in the Interferon and TGFβ pathways. Gene Ontology (GO) analysis of DMP-associated genes showed a significative enrichment (*p<0.005) in transcription factor binding, transcription corepressor and transcription coactivator activity, SMAD binding and histone -lysine-N-methyltransferase activity. To further validate the results, 5'-methylcytosine immunoprecipitation (MedIP) followed by Real Time PCR was performed to assess the methylation level of SMAD3 and SMARCA4/BRG1 promoters in the same cohort of MZ twins. We found significantly DNA methylation enrichment in SMARCA4/BRG1 promoter in psoriatic disease twins (p<0.05). SMAD3 and SMARCA4/BRG1 mRNA expression was also assessed to evaluate any inverse correlation with promoter methylation level, on the MZ cohort used for the EPIC array (n=4) and on a cohort of PsA/Ps patients (n=8) and appropriate healthy controls (n=3). Reduced mRNA expression (p<0.05) was demonstrated for SMARCA4/BRG1 (n=4). Conversely, no changes were found for SMAD3.Conclusion:We report the first DNA methylation approach in MZ twins discordant for psoriatic disease. We believe that the observed changes in SMAD3 and SMARCA/BRG1 genes may suggest an epigenetic imbalance of chromatin remodelling factors involved in inflammation pathways with a potential role in PsA/psoriasis immunopathogenesis.Disclosure of Interests:None declare

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions

A consensus research agenda for optimising nasal drug delivery

Nasal drug delivery has specific challenges which are distinct from oral inhalation, alongside which it is often considered. The next generation of nasal products will be required to deliver new classes of molecule, e.g. vaccines, biologics and drugs with action in the brain or sinuses, to local and systemic therapeutic targets. Innovations and new tools/knowledge are required to design products to deliver these therapeutic agents to the right target at the right time in the right patients. We report the outcomes of an expert meeting convened to consider gaps in knowledge and unmet research needs in terms of (i) formulation and devices, (ii) meaningful product characterization and modeling, (iii) opportunities to modify absorption and clearance. Important research questions were identified in the areas of device and formulation innovation, critical quality attributes for different nasal products, development of nasal casts for drug deposition studies, improved experimental models, the use of simulations and nasal delivery in special populations. We offer these questions as a stimulus to research and suggest that they might be addressed most effectively by collaborative research endeavors

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy

ANNINE-6 and ANNINE-6plus are voltage-sensitive dyes that when combined with two-photon microscopy are ideal for recording of neuronal voltages in vivo, in both bulk loaded tissue and the dendrites of single neurons. Here, we describe in detail but for a broad audience the voltage sensing mechanism of fast voltage-sensitive dyes, with a focus on ANNINE dyes, and how voltage imaging can be optimized with one-photon and two-photon excitation. Under optimized imaging conditions the key strengths of ANNINE dyes are their high sensitivity (0.5%/mV), neglectable bleaching and phototoxicity, a linear response to membrane potential, and a temporal resolution which is faster than the optical imaging devices currently used in neurobiology (order of nanoseconds). ANNINE dyes in combination with two-photon microscopy allow depth-resolved voltage imaging in bulk loaded tissue to study average membrane voltage oscillations and sensory responses. Alternatively, if ANNINE-6plus is applied internally, supra and sub threshold voltage changes can be recorded from dendrites of single neurons in awake animals. Interestingly, in our experience ANNINE-6plus labeling is impressively stable in vivo, such that voltage imaging from single Purkinje neuron dendrites can be performed for 2 weeks after a single electroporation of the neuron. Finally, to maximize their potential for neuroscience studies, voltage imaging with ANNINE dyes and two-photon microscopy can be combined with electrophysiological recording, calcium imaging, and/or pharmacology, even in awake animals

Mitochondrial Morphogenesis, Dendrite Development, and Synapse Formation in Cerebellum Require both Bcl-w and the Glutamate Receptor δ2

Bcl-w belongs to the prosurvival group of the Bcl-2 family, while the glutamate receptor δ2 (Grid2) is an excitatory receptor that is specifically expressed in Purkinje cells, and required for Purkinje cell synapse formation. A recently published result as well as our own findings have shown that Bcl-w can physically interact with an autophagy protein, Beclin1, which in turn has been shown previously to form a protein complex with the intracellular domain of Grid2 and an adaptor protein, nPIST. This suggests that Bcl-w and Grid2 might interact genetically to regulate mitochondria, autophagy, and neuronal function. In this study, we investigated this genetic interaction of Bcl-w and Grid2 through analysis of single and double mutant mice of these two proteins using a combination of histological and behavior tests. It was found that Bcl-w does not control the cell number in mouse brain, but promotes what is likely to be the mitochondrial fission in Purkinje cell dendrites, and is required for synapse formation and motor learning in cerebellum, and that Grid2 has similar phenotypes. Mice carrying the double mutations of these two genes had synergistic effects including extremely long mitochondria in Purkinje cell dendrites, and strongly aberrant Purkinje cell dendrites, spines, and synapses, and severely ataxic behavior. Bcl-w and Grid2 mutations were not found to influence the basal autophagy that is required for Purkinje cell survival, thus resulting in these phenotypes. Our results demonstrate that Bcl-w and Grid2 are two critical proteins acting in distinct pathways to regulate mitochondrial morphogenesis and control Purkinje cell dendrite development and synapse formation. We propose that the mitochondrial fission occurring during neuronal growth might be critically important for dendrite development and synapse formation, and that it can be regulated coordinately by multiple pathways including Bcl-2 and glutamate receptor family members

In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors

Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 \ub5M all-trans Retinoic Acid (ATRA), 5 \ub5M Phenyl Butyrate (PB), and 200 \ub5M diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and \u3b1-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f) current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors