Implementation strategies for improving vitamin D status and increasing vitamin D intake in the UK: current controversies and future perspectives: proceedings of the 2nd Rank Prize Funds Forum on vitamin D.

A multi-disciplinary expert group met to discuss vitamin D deficiency in the UK and strategies for improving population intakes and status. Changes to UK Government advice since the 1st Rank Forum on Vitamin D (2009) were discussed, including rationale for setting a reference nutrient intake (10 µg/d; 400 IU/d) for adults and children (4+ years). Current UK data show inadequate intakes among all age groups and high prevalence of low vitamin D status among specific groups (e.g. pregnant women and adolescent males/females). Evidence of widespread deficiency within some minority ethnic groups, resulting in nutritional rickets (particularly among Black and South Asian infants), raised particular concern. Latest data indicate that UK population vitamin D intakes and status reamain relatively unchanged since Government recommendations changed in 2016. Vitamin D food fortification was discussed as a potential strategy to increase population intakes. Data from dose-response and dietary modelling studies indicate dairy products, bread, hens' eggs and some meats as potential fortification vehicles. Vitamin D3 appears more effective than vitamin D2 for raising serum 25-hydroxyvitamin D concentration, which has implications for choice of fortificant. Other considerations for successful fortification strategies include: (i) need for 'real-world' cost information for use in modelling work; (ii) supportive food legislation; (iii) improved consumer and health professional understanding of vitamin D's importance; (iv) clinical consequences of inadequate vitamin D status and (v) consistent communication of Government advice across health/social care professions, and via the food industry. These areas urgently require further research to enable universal improvement in vitamin D intakes and status in the UK population.

Vitamins D3 and D2 have marked but different global effects on gene expression in a rat oligodendrocyte precursor cell line.

BACKGROUND: Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but it is unclear whether vitamin D supplementation improves the clinical course of MS, and there is uncertainty about the dose and form of vitamin D (D2 or D3) to be used. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with vitamin D3 at 24 h and 72 h following onset of differentiation. METHODS: Gene expression in differentiating CG4 cells in response to vitamin D2 or D3 was quantified using Agilent DNA microarrays (n = 4 replicates), and the transcriptome data were processed and analysed using the R software environment. Differential expression between the experimental conditions was determined using LIMMA, applying the Benjamini and Hochberg multiple testing correction to p-values, and significant genes were grouped into co-expression clusters by hierarchical clustering. The functional significance of gene groups was explored by pathway enrichment analysis using the clusterProfiler package. RESULTS: Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for Gene Ontology (GO) categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway. CONCLUSIONS: This study shows that vitamin D3, compared with D2, changes the expression of a larger number of genes in OLs. Identification of genes affected by D3 in OLs should help to identify mechanisms mediating its action in MS.

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor.

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and 'translatome'; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as 'potentiation'. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

Tools, techniques, organisation and culture of the CADD group at Sygnature Discovery.

Computer-aided drug design encompasses a wide variety of tools and techniques, and can be implemented with a range of organisational structures and focus in different organisations. Here we outline the computational chemistry skills within Sygnature Discovery, along with the software and hardware at our disposal, and briefly discuss the methods that are not employed and why. The goal of the group is to provide support for design and analysis in order to improve the quality of compounds synthesised and reduce the timelines of drug discovery projects, and we reveal how this is achieved at Sygnature. Impact on medicinal chemistry is vital to demonstrating the value of computational chemistry, and we discuss the approaches taken to influence the list of compounds for synthesis, and how we recognise success. Finally we touch on some of the areas being developed within the team in order to provide further value to the projects and clients.

Exploiting human and mouse transcriptomic data: Identification of circadian genes and pathways influencing health.

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

Mistimed sleep disrupts circadian regulation of the human transcriptome.

Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep-wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep-wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep-wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.

Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome.

Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, including obesity, cardiovascular disease, and cognitive impairment, but the mechanisms involved remain largely unexplored. Twenty-six participants were exposed to 1 wk of insufficient sleep (sleep-restriction condition 5.70 h, SEM = 0.03 sleep per 24 h) and 1 wk of sufficient sleep (control condition 8.50 h sleep, SEM = 0.11). Immediately following each condition, 10 whole-blood RNA samples were collected from each participant, while controlling for the effects of light, activity, and food, during a period of total sleep deprivation. Transcriptome analysis revealed that 711 genes were up- or down-regulated by insufficient sleep. Insufficient sleep also reduced the number of genes with a circadian expression profile from 1,855 to 1,481, reduced the circadian amplitude of these genes, and led to an increase in the number of genes that responded to subsequent total sleep deprivation from 122 to 856. Genes affected by insufficient sleep were associated with circadian rhythms (PER1, PER2, PER3, CRY2, CLOCK, NR1D1, NR1D2, RORA, DEC1, CSNK1E), sleep homeostasis (IL6, STAT3, KCNV2, CAMK2D), oxidative stress (PRDX2, PRDX5), and metabolism (SLC2A3, SLC2A5, GHRL, ABCA1). Biological processes affected included chromatin modification, gene-expression regulation, macromolecular metabolism, and inflammatory, immune and stress responses. Thus, insufficient sleep affects the human blood transcriptome, disrupts its circadian regulation, and intensifies the effects of acute total sleep deprivation. The identified biological processes may be involved with the negative effects of sleep loss on health, and highlight the interrelatedness of sleep homeostasis, circadian rhythmicity, and metabolism.

A terD domain-encoding gene (SCO2368) is involved in calcium homeostasis and participates in calcium regulation of a DosR-like regulon in Streptomyces coelicolor.

Although Streptomyces coelicolor is not resistant to tellurite, it possesses several TerD domain-encoding (tdd) genes of unknown function. To elucidate the function of tdd8, the transcriptomes of S. coelicolor strain M145 and of a tdd8 deletion mutant derivative (the Δtdd8 strain) were compared. Several orthologs of Mycobacterium tuberculosis genes involved in dormancy survival were upregulated in the deletion mutant at the visual onset of prodiginine production. These genes are organized in a putative redox stress response cluster comprising two large loci. A binding motif similar to the dormancy survival regulator (DosR) binding site of M. tuberculosis has been identified in the upstream sequences of most genes in these loci. A predicted role for these genes in the redox stress response is supported by the low NAD(+)/NADH ratio in the Δtdd8 strain. This S. coelicolor gene cluster was shown to be induced by hypoxia and NO stress. While the tdd8 deletion mutant (the Δtdd8 strain) was unable to maintain calcium homeostasis in a calcium-depleted medium, the addition of Ca(2+) in Δtdd8 culture medium reduced the expression of several genes of the redox stress response cluster. The results shown in this work are consistent with Tdd8 playing a significant role in calcium homeostasis and redox stress adaptation.

A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing.

Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression.

Deciphering the regulon of Streptomyces coelicolor AbrC3, a positive response regulator of antibiotic production.

The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by SCO4598, SCO4597, and SCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in Streptomyces coelicolor and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene, abrC3 (SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of abrC3 in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the ΔabrC3 mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the actII-ORF4 promoter region; this was independently verified by in vitro DNA-binding assays. This binding is dependent on the sequence 5'-GAASGSGRMS-3'. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the redZ or redD promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of SCO0736, bdtA (SCO3328), absR1 (SCO6992), and SCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ΔabrC3 mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.

Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets.

Streptomycetes sense and respond to the stress of phosphate starvation via the two-component PhoR-PhoP signal transduction system. To identify the in vivo targets of PhoP we have undertaken a chromatin-immunoprecipitation-on-microarray analysis of wild-type and phoP mutant cultures and, in parallel, have quantified their transcriptomes. Most (ca. 80%) of the previously in vitro characterized PhoP targets were identified in this study among several hundred other putative novel PhoP targets. In addition to activating genes for phosphate scavenging systems PhoP was shown to target two gene clusters for cell wall/extracellular polymer biosynthesis. Furthermore PhoP was found to repress an unprecedented range of pathways upon entering phosphate limitation including nitrogen assimilation, oxidative phosphorylation, nucleotide biosynthesis and glycogen catabolism. Moreover, PhoP was shown to target many key genes involved in antibiotic production and morphological differentiation, including afsS, atrA, bldA, bldC, bldD, bldK, bldM, cdaR, cdgA, cdgB and scbR-scbA. Intriguingly, in the PhoP-dependent cpk polyketide gene cluster, PhoP accumulates substantially at three specific sites within the giant polyketide synthase-encoding genes. This study suggests that, following phosphate limitation, Streptomyces coelicolor PhoP functions as a 'master' regulator, suppressing central metabolism, secondary metabolism and developmental pathways until sufficient phosphate is salvaged to support further growth and, ultimately, morphological development.

The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in streptomyces coelicolor.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that generally relate to the control of primary metabolism, whereby its member glucose kinase acts as the central control protein in carbon control in Streptomyces. Here, we show that deletion of SCO6008 (rok7B7) strongly affects carbon catabolite repression (CCR), growth, and antibiotic production in Streptomyces coelicolor. Deletion of SCO7543 also affected antibiotic production, while no major changes were observed after deletion of the rok family genes SCO0794, SCO1060, SCO2846, SCO6566, or SCO6600. Global expression profiling of the rok7B7 mutant by proteomics and microarray analysis revealed strong upregulation of the xylose transporter operon xylFGH, which lies immediately downstream of rok7B7, consistent with the improved growth and delayed development of the mutant on xylose. The enhanced CCR, which was especially obvious on rich or xylose-containing media, correlated with elevated expression of glucose kinase and of the glucose transporter GlcP. In liquid-grown cultures, expression of the biosynthetic enzymes for production of prodigionines, siderophores, and calcium-dependent antibiotic (CDA) was enhanced in the mutant, and overproduction of prodigionines was corroborated by matrix-assisted laser desorption ionization-time-of-flight analysis. These data present Rok7B7 as a pleiotropic regulator of growth, CCR, and antibiotic production in Streptomyces.

Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation.

BACKGROUND: The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. RESULTS: We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. CONCLUSION: Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

An example of designed multiple ligands spanning protein classes: dual MCH-1R antagonists/DPPIV inhibitors.

A ligand-based approach to identify potential starting points for a dual MCH-1R antagonist/DPPIV inhibitor medicinal chemistry program was undertaken. Potential ligand pairs were identified by analysis of MCH-1R and DPPIV in vitro data. A highly targeted synthetic effort lead to the discovery of pyridone 11, a dual MCH-1R antagonist/DPPIV inhibitor with selectivity over DPP8 and DPP9.

Design and synthesis of potent carboxylic acid DGAT1 inhibitors with high cell permeability.

A series of potent carboxylic acid DGAT1 inhibitors with high permeability were developed from a virtual screening hit. Strategies were employed to reduce Pgp substrate recognition and increase passive permeability, resulting in the discovery of a series showing good inhibition of cellular triglyceride synthesis. The mutagenic potential of prospective metabolites was evaluated in the Ames assay, and one aniline was shown to be devoid of mutagenicity.

Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity.

Calcium-dependent antibiotics (CDA) are cyclic lipopeptides assembled by nonribosomal peptide synthetase (NRPS) enzymes. Active site modification of the 3-methyl glutamate activating adenylation (A) domain of the CDA NRPS enables the incorporation of synthetic 3-methyl glutamine into CDA. This provides the first example of how A-domains can be engineered to introduce synthetic "non-natural" amino acids into nonribosomal peptides.

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome.

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D2 and D3 share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D2 and D3. Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D2 or D3 daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D3 or D2 on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D2 or D3-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D2 or vitamin D3, with only vitamin D3 having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D2 and vitamin D3 is warranted.

Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

UK Nutrition Research Partnership 'Hot Topic' workshop: Vitamin D-A multi-disciplinary approach to (1) elucidate its role in human health and (2) develop strategies to improve vitamin D status in the UK population.

Vitamin D is truly unique-not a 'vital' amine in the true sense of the word, but rather a prohormone, which is produced in the skin during exposure to sunlight (UVB radiation at 290-315 nm) and which can also be obtained from food and from supplements. A high prevalence of low vitamin D status has been reported across the world in a wide range of population groups, and this includes communities living in low latitude areas despite the abundance of sunlight. It is accepted that vitamin D status is reflected by the level of the circulating metabolite 25-hydroxyvitamin D (25[OH]D), which is produced by hepatic hydroxylation of vitamin D, derived either from the skin from UV exposure or the gut from oral intake. Vitamin D has been associated with a wide range of health outcomes, but controversies remain as to their exact nature and extent and whether associations are in the causal pathway. In order to enable wider discussions on this nutrient, a 'Hot Topic' Vitamin D Workshop achieved funding from the UK Nutrition Research Partnership Medical Research Council call. The objectives of the workshop were (1) to elucidate the role of vitamin D in human health and (2) develop strategies to improve vitamin D status in the UK population. This paper provides a detailed resume of the discussions of the workshop; of the presentations and concomitant Q&As; and of identified areas for future research.