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Vigilance, or sustained attention tasks involve detecting critical signals, embedded amid more frequent neutral signals, over an extended period of time. A decline in performance, engagement, and arousal over time, as well as high workload and stress, are common outcomes of such tasks. Exposure to broad-spectrum or short wavelength bright light has been found to positively impact alertness, speed of information processing, and mood, but has not been extensively explored in the vigilance domain. The present study explored whether a light therapy lamp could mitigate the negative vigilance outcomes found in both performance and affective state. Results indicated that the therapy light did not prevent a decline in detection of critical signals over time, nor significantly impact workload, sleepiness, or subjective stress state compared to a dim light condition. However, mood questionnaire results suggest that lighting may impact separate constructs of arousal and tiredness, warranting further research.

Objective To explore vigilance task performance, cerebral blood flow velocity (CBFV), workload, and stress in a within-subjects, two-session experiment. Background Vigilance, or sustained attention, tasks are often characterized by a decline in operator performance and CBFV with time on task, and high workload and stress. Though performance is known to improve with practice, past research has not included measures of CBFV, stress, and workload in a within-subjects multi-session design, which may also provide insight into ongoing theoretical debate. Method Participants performed a vigilance task on two separate occasions. Performance, CBFV, workload, and self-reported stress were measured. Results Within each session, results were consistent with the vigilance profile found in prior research. Across sessions, performance improved but the time on task decrement remained. Mean CBFV and workload ratings did not differ between sessions, but participants reported significantly less distress, worry, and engagement after session two compared to one. Conclusion Though practice may not disrupt the standard vigilance profile, it may serve to improve overall performance and reduce stress. However, repeated exposure may have negative implications for engagement and mind-wandering. Application It is important to better understand the relationship between experience, performance, physiological response, and self-reported stress and workload in vigilance because real-world environments often require operators to do the same task over many occasions. While performance improvement and reduced distress is an encouraging result, the decline in engagement requires further research. Results across sessions fail to provide support to the mind-wandering theory of vigilance.

In many human performance tasks, researchers assess performance by measuring both accuracy and response time. A number of theoretical and practical approaches have been proposed to obtain a single performance value that combines these measures, with varying degrees of success. In this report, we examine data from a common paradigm used in applied human factors assessment: a go/no-go vigilance task (Smith et al., 2019). We examined whether 12 different measures of performance were sensitive to the vigilance decrement induced by the design, and also examined how the different measures were correlated. Results suggest that most combined measures were slight improvements over accuracy or response time alone, with the most sensitive and representative result coming from the Linear Ballistic Accumulator model. Practical lessons for applying these measures are discussed.

CD4+ T-cells represent a heterogeneous collection of specialised sub-types and are a key cell type in the pathogenesis of many diseases due to their role in the adaptive immune system. By investigating CD4+ T-cells at the single cell level, using RNA sequencing (scRNA-seq), there is the potential to identify specific cell states driving disease or treatment response. However, the impact of sequencing depth and cell numbers, two important factors in scRNA-seq, has not been determined for a complex cell population such as CD4+ T-cells. We therefore generated a high depth, high cell number dataset to determine the effect of reduced sequencing depth and cell number on the ability to accurately identify CD4+ T-cell subtypes. Furthermore, we investigated T-cell signatures under resting and stimulated conditions to assess cluster specific effects of stimulation. We found that firstly, cell number has a much more profound effect than sequencing depth on the ability to classify cells; secondly, this effect is greater when cells are unstimulated and finally, resting and stimulated samples can be combined to leverage additional power whilst still allowing differences between samples to be observed. While based on one individual, these results could inform future scRNA-seq studies to ensure the most efficient experimental design.

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca 2+ ([Ca 2+] c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca 2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca 2+] c, whereas pharmacological inhibition of these channels reduced Ca 2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca 2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.

Despite the success of TNF-inhibitor therapy in rheumatoid arthritis treatment, up to 40% of patients fail to respond adequately. This study aimed to identify transcriptome-based biomarkers of adalimumab response in rheumatoid arthritis (RA) to aid timely switching in non-responder patients and provide a better mechanistic understanding of the pathways involved in response/non-response.The Affymetrix Human Transcriptome Array 2.0 (HTA) was used to measure the transcriptome in whole blood at pre-treatment and at 3 months in EULAR good- and non-responders to adalimumab therapy. Differential expression of transcripts was analysed at the transcript level using multiple linear regression. Differentially expressed genes were validated in independent samples using OpenArray™ RT-qPCR.In total, 813 transcripts were differentially expressed between pre-treatment and 3 months in adalimumab good-responders. No significant differential expression was observed between good- and non-responders at either time-point and no significant changes were observed in non-responders between time-points. OpenArray™ RT-qPCR was performed for 104 differentially expressed transcripts in good-responders, selected based on magnitude of effect or p value or based on prior association with RA or the immune system, validating differential expression for 17 transcripts.An early transcriptome signature of DAS28 response to adalimumab has been identified and replicated in independent datasets. Whilst treat-to-target approaches encourage early switching in non-responsive patients, registry evidence suggests that this does not always occur. The results herein could guide the development of a blood test to distinguish responders from non-responders at 3 months and support clinical decisions to switch non-responsive patients to an alternative therapy.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

Vigilance, or the ability to sustain attention for extended periods of time, has been of interest to the human factors and ergonomics community for 70 years. During this time, several theories have...

Objective A dual-task study was performed to explore the performance effects for swimming, word recall, and the combination of the two tasks performed simultaneously. Background Dual-task interference studies have been performed for a variety of tasks; however, there has not been much dual-task interference research where one of the tasks is a naturalistic physically strenuous task. Swimming is a unique physical task that requires spatial orientation on three dimensional axes, similar to that of flying, but has no risk of falling. Previous studies have been conducted in other activity combinations with word-free recall, such as running and climbing, but swimming has yet to be explored. Method A verbal memory recall task and swimming task were performed in isolated (single-task) and simultaneous conditions. A comparison of effects across these different activities was also explored. Results Swimming and the word-recall task resulted in significant dual-task interference: almost as much as when word recall was paired with another verbal task, but more than running and less than climbing. Conclusion Consistent with other dual-task studies, this study observed dual-task interference between the physical swimming task and the cognitive verbal memory task. Application Future technologies and training for personnel who engage in water rescue or commercial diving, such as underwater welding and fiber optic cable, may be improved by these findings.

We examined the impact of performing a cognitive task of varying loads and kayaking simultaneously, compared to performance on the same tasks individually.When two tasks are performed together, performance often suffers compared to performance on either task alone. Interference not only occurs in competing cognitive tasks, but has also been found with certain physically demanding activities, such as climbing.Skilled kayakers performed a kayak course on open water, performed a high and a low cognitive load tone counting task, and also performed the kayak and counting tasks simultaneously.Despite some past research finding dual task facilitation with laboratory aerobic activities, simultaneous kayaking and tone counting led to dual-task interference.Concurrent counting and kayaking led to performance impairments in both tasks, relative to single task performance.The present results are applicable to occupations involving concurrent demanding physical activity and cognitive task performance, such as the work of first responders and military operators. PRéCIS: Kayaking, like climbing, appears to hinder cognitive performance more than the low-risk physical tasks carried out in laboratory conditions.

Background/Aims Juvenile idiopathic arthritis (JIA) is a childhood-onset rheumatic disease, which is associated with increased risk of uveitis. Approximately 80% of childhood chronic anterior uveitis cases are JIA-associated (JIAU), where it is considered a serious complication with the potential to lead to permanent blindness. Studies have reported associations of increased risk of JIAU to genetic variation within human leukocyte antigen (HLA) genes, in particular amino acid positions of HLA-DRB1. Here we report the results of the largest fine-mapping study of HLA genes in JIAU to date. Methods Genotyping was performed using Illumina Infinium CoreExome and Infinium OnmiExpress arrays. Samples were excluded with a call rate 0.9 and MAF > 0.01 using logistic regression, or omnibus test for multi-allelic markers, including 3 PCs as covariates. Independent effects were identified using forward stepwise logistic regression by inclusion of previously identified variants as covariates. Results We analysed 7,425 markers across the HLA region in 450 JIAU cases and 2,024 JIA cases. We defined study-wide significance at a Bonferroni corrected threshold of 6.7x10-6. The most significant association was at amino acid position 13 of DRB1 (p-value = 3.0x10-30) where the presence of serine (OR 1.7, 95% CI 1.4-1.9) or glycine (OR 2.0, 95% CI 1.6-2.5) were associated with increased risk of uveitis. Conditioning on DRB1 position 13, found a further association to amino acid position 67 of DRB1 (p-value = 2.4x10-6, unconditioned p-value = 3.2x10-23). The presence of isoleucine (OR 1.5, 95% CI 1.2-1.9) and phenylalanine (OR 1.8 95%, CI 1.4-2.2) at position 67 were associated with increased risk. No further study-wide associations were found at DRB1. Conditioning on all HLA-DRB1 alleles identified an independent effect at amino acid position 69 in DPB1 (p-value = 5.3x10-7, unconditioned p-value = 1.1x10-10). The presence of glutamic acid was associated with increased risk (OR 1.7, 95% CI 1.4-2.0). Conclusion This is the largest genetic study of HLA regions in JIAU and further resolves the genetic risk factors in this key susceptibility region. The analysis in this study has independently validated the association signal at position 13 of HLA-DRB1 (highly correlated with position 11) that had been reported in a previous study. Conditional analysis of HLA-DRB1 revealed a novel secondary association signal at DRB1 to amino acid position 67. Conditional analysis on all DRB1 alleles confirmed association to position 69 of HLA-DPB1 where previous reports of this signal had been only modestly associated. Disclosure M. Tordoff: None. S.L. Smith: None. E. Lopez-Isac: None. A. Morris: None. S. Eyre: None. W. Thomson: None. J. Bowes: None.

Background Despite the success of TNF-inhibitor therapy in rheumatoid arthritis treatment, up to 40% of patients fail to respond adequately. This study aimed to identify transcriptome-based biomarkers of adalimumab response in rheumatoid arthritis (RA) to aid timely switching in non-responder patients and provide a better mechanistic understanding of the pathways involved in response/non-response. Methods The Affymetrix Human Transcriptome Array 2.0 (HTA) was used to measure the transcriptome in whole blood at pre-treatment and at 3 months in EULAR good- and non-responders to adalimumab therapy. Differential expression of transcripts was analysed at the transcript level using multiple linear regression. Differentially expressed genes were validated in independent samples using OpenArray™ RT-qPCR. Results In total, 813 transcripts were differentially expressed between pre-treatment and 3 months in adalimumab good-responders. No significant differential expression was observed between good- and non-responders at either time-point and no significant changes were observed in non-responders between time-points. OpenArray™ RT-qPCR was performed for 104 differentially expressed transcripts in good-responders, selected based on magnitude of effect or p value or based on prior association with RA or the immune system, validating differential expression for 17 transcripts. Conclusions An early transcriptome signature of DAS28 response to adalimumab has been identified and replicated in independent datasets. Whilst treat-to-target approaches encourage early switching in non-responsive patients, registry evidence suggests that this does not always occur. The results herein could guide the development of a blood test to distinguish responders from non-responders at 3 months and support clinical decisions to switch non-responsive patients to an alternative therapy.

ObjectivesJuvenile idiopathic arthritis (JIA) is the most prevalent form of juvenile rheumatic disease. Our understanding of the genetic risk factors for this disease is limited due to low disease prevalence and extensive clinical heterogeneity. The objective of this research is to identify novel JIA susceptibility variants and link these variants to target genes, which is essential to facilitate the translation of genetic discoveries to clinical benefit.MethodsWe performed a genome-wide association study (GWAS) in 3305 patients and 9196 healthy controls, and used a Bayesian model selection approach to systematically investigate specificity and sharing of associated loci across JIA clinical subtypes. Suggestive signals were followed-up for meta-analysis with a previous GWAS (2751 cases/15 886 controls). We tested for enrichment of association signals in a broad range of functional annotations, and integrated statistical fine-mapping and experimental data to identify target genes.ResultsOur analysis provides evidence to support joint analysis of all JIA subtypes with the identification of five novel significant loci. Fine-mapping nominated causal single nucleotide polymorphisms with posterior inclusion probabilities ≥50% in five JIA loci. Enrichment analysis identified RELA and EBF1 as key transcription factors contributing to disease risk. Our integrative approach provided compelling evidence to prioritise target genes at six loci, highlighting mechanistic insights for the disease biology andIL6STas a potential drug target.ConclusionsIn a large JIA GWAS, we identify five novel risk loci and describe potential function of JIA association signals that will be informative for future experimental works and therapeutic strategies.

Summary Background Juvenile idiopathic arthritis (JIA) is a heterogeneous disease, the signs and symptoms of which can be summarised with use of composite disease activity measures, including the clinical Juvenile Arthritis Disease Activity Score (cJADAS). However, clusters of children and young people might experience different global patterns in their signs and symptoms of disease, which might run in parallel or diverge over time. We aimed to identify such clusters in the 3 years after a diagnosis of JIA. The identification of these clusters would allow for a greater understanding of disease progression in JIA, including how physician-reported and patient-reported outcomes relate to each other over the JIA disease course. Methods In this multicentre prospective longitudinal study, we included children and young people recruited before Jan 1, 2015, to the Childhood Arthritis Prospective Study (CAPS), a UK multicentre inception cohort. Participants without a cJADAS score were excluded. To assess groups of children and young people with similar disease patterns in active joint count, physician's global assessment, and patient or parental global evaluation, we used latent profile analysis at initial presentation to paediatric rheumatology and multivariate group-based trajectory models for the following 3 years. Optimal models were selected on the basis of a combination of model fit, clinical plausibility, and model parsimony. Finding Between Jan 1, 2001, and Dec 31, 2014, 1423 children and young people with JIA were recruited to CAPS, 239 of whom were excluded, resulting in a final study population of 1184 children and young people. We identified five clusters at baseline and six trajectory groups using longitudinal follow-up data. Disease course was not well predicted from clusters at baseline; however, in both cross-sectional and longitudinal analyses, substantial proportions of children and young people had high patient or parent global scores despite low or improving joint counts and physician global scores. Participants in these groups were older, and a higher proportion of them had enthesitis-related JIA and lower socioeconomic status, compared with those in other groups. Interpretation Almost one in four children and young people with JIA in our study reported persistent, high patient or parent global scores despite having low or improving active joint counts and physician's global scores. Distinct patient subgroups defined by disease manifestation or trajectories of progression could help to better personalise health-care services and treatment plans for individuals with JIA. Funding Medical Research Council, Versus Arthritis, Great Ormond Street Hospital Children's Charity, Olivia's Vision, and National Institute for Health Research.

The Sustained Attention to Response Task (SART) is a go/no-go task where participants must respond frequently to target stimuli and withhold responses from infrequent neutral stimuli. Researchers have shown that the fast and frequent responding characteristic of SART is typically associated with difficulty withholding responses to no-go stimuli. Imposing additional cognitive demands has been shown to further impair task performance. In the present research, participants completed a modified SART task, a narrative memory task, and a dual-task condition where both were done simultaneously. No significant performance impairments were found in the dual- compared to single-task conditions. The tasks’ non-overlapping resource demands, alongside a potential arousing benefit of the memory task, may explain the lack of notable dual-task interference. Future research is needed to better understand the effects of arousal and other factors that may help to uncouple errors of commission from response time, particularly in tasks with high ecological validity.

Objective We examined the impact of increasing cognitive load on climbing performance and the impact of climbing on concurrent cognitive task performance. Background Generally when two tasks are performed simultaneously performance of one or both suffers relative to performance of each alone. Such dual task decrement is not confined to competing cognitive tasks, but has also been found when one task involves demanding physical activity. Method Skilled climbers performed a traverse climb alone and in combination with low and high cognitive load counting tasks, which were also performed alone. Results In more realistic physical settings, physical and cognitive tasks will interfere, unlike what some literature using laboratory physical tasks may indicate. Conclusion Compared to single task (climb only) performance concurrent counting and climbing resulted in impaired performance. However, climbers appeared to prioritize climbing over cognitive task performance. Application The results and this program of research have implications for occupations that involve concurrent demanding physical activity and cognitive task performance. Precis High risk, physical tasks in real world conditions appear to hinder cognitive performance more so than low-risk physical tasks carried out in laboratory conditions.

ObjectivesJuvenile idiopathic arthritis (JIA) is an autoimmune disease and a common cause of chronic disability in children. Diagnosis of JIA is based purely on clinical symptoms, which can be variable, leading to diagnosis and treatment delays. Despite JIA having substantial heritability, the construction of genomic risk scores (GRSs) to aid or expedite diagnosis has not been assessed. Here, we generate GRSs for JIA and its subtypes and evaluate their performance.MethodsWe examined three case/control cohorts (UK, US-based and Australia) with genome-wide single nucleotide polymorphism (SNP) genotypes. We trained GRSs for JIA and its subtypes using lasso-penalised linear models in cross-validation on the UK cohort, and externally tested it in the other cohorts.ResultsThe JIA GRS alone achieved cross-validated area under the receiver operating characteristic curve (AUC)=0.670 in the UK cohort and externally-validated AUCs of 0.657 and 0.671 in the US-based and Australian cohorts, respectively. In logistic regression of case/control status, the corresponding odds ratios (ORs) per standard deviation (SD) of GRS were 1.831 (1.685 to 1.991) and 2.008 (1.731 to 2.345), and were unattenuated by adjustment for sex or the top 10 genetic principal components. Extending our analysis to JIA subtypes revealed that the enthesitis-related JIA had both the longest time-to-referral and the subtype GRS with the strongest predictive capacity overall across data sets: AUCs 0.82 in UK; 0.84 in Australian; and 0.70 in US-based. The particularly common oligoarthritis JIA also had a GRS that outperformed those for JIA overall, with AUCs of 0.72, 0.74 and 0.77, respectively.ConclusionsA GRS for JIA has potential to augment clinical JIA diagnosis protocols, prioritising higher-risk individuals for follow-up and treatment. Consistent with JIA heterogeneity, subtype-specific GRSs showed particularly high performance for enthesitis-related and oligoarthritis JIA.

Background Juvenile idiopathic arthritis (JIA) is a rheumatic disease, onset before the age of sixteen. JIA is a common complex disease, with contributions from both genetic and environmental risk factors. However, given the young age of onset for JIA, it is possible that genetic risk factors influence susceptibility to a greater extent than environmental risk factors. While several susceptibility loci have been identified for JIA, they are mainly genetic variants that are common in the population. The role of rare genetic variants has not yet been fully explored in JIA. Therefore, the aims of this research were to use rare ( Methods A total of 2,162 UK JIA cases were genotyped using the Illumina HumanCoreExome array and the Understanding Society’s UK household longitudinal study provided the genotype data for 9196 controls. Single variant analysis was used to calculate the association of variants to JIA susceptibility. Gene-based aggregation analysis, specifically the SKAT-O method, were used to calculate the association of whole gene regions to JIA cases. This gene-based aggregation method increases the statistical power of associations, which is valuable when attempting to identify rare variants. Study-wide significance for this research was calculated using a Bonferroni corrected threshold (P = 3.34x10-6). Results Several strongly associated candidate gene regions were identified using SKAT-O analysis. Firstly, the region mevalonate kinase (MVK) (P = 3.95x10-10) was found to be strongly associated. MVK has been reported in mevalonate kinase deficiency, a rare juvenile disease with a phenotype akin to systemic JIA. PYCR1 (P = 1.97x10-9) was an additional region of interest. Variants in this region have been reported in geroderma osteodysplasticum; a disease that results in dysplasia of the hips, hyperextensible joints and skeletal changes. The region WISP3 (P = 8.52x10-9) was also strongly associated. Variants from this region have previously been reported in progressive pseudorheumatoid dysplasia; a disease that can be clinically misdiagnosed with JIA due to similarities in phenotypes. Furthermore, LILRA2 (P = 2.89x10-7) presented as a strong candidate region, with the gene being upregulated in synovial tissue of rheumatoid arthritis patients. Conclusion This analysis has identified promising candidate genes that influence susceptibility to JIA and highlights two key points. Firstly, gene-burden tests of rare variants have the potential to identify novel susceptibility genes for juvenile-onset disease. While MVK and WISP3 have previously been reported to be associated with JIA, these associations were modest, and the current study provides compelling evidence to support their importance. Secondly, the results highlight the importance of investigating the contribution of monogenic genes in juvenile-onset common complex disease. Disclosures M.K. Tordoff None. T.A. Briggs None. S.L. Smith None. A. Yarwood None. W. Thomson None. J. Bowes None.

Background Juvenile idiopathic arthritis (JIA) is a clinically heterogeneous group of childhood onset inflammatory joint diseases with strong evidence to support a genetic contribution to susceptibility. JIA is divided into seven clinical subgroups based on observed patterns of clinical symptoms using the International League of Associations for Rheumatology (ILAR) classification system. The genetic overlap between these groups is not completely understood and this lack of knowledge typically leads to the different ILAR groups being analysed as discrete entities potentially reducing the power of genetic association studies. The aim of this study was to conduct a large case-control association study on susceptibility to JIA to identify novel susceptibility loci and to investigate differences in these associations between the different ILAR groups for the purposes of maximising power. Methods JIA participants were genotyped on the Illumina Infinium CoreExome or OmniExpress arrays. UK population control genotype data was obtained from the Understanding Society Longitudinal Study. Quality control of data was performed conforming to conventional standards and imputation was performed using the Haplotype Reference Consortium panel on the Michigan Imputation Server. Association testing of all SNPs was performed with an additive model incorporating imputation uncertainty using SNPTEST. Testing for specificity and sharing across ILAR groups was performed using Bayesian multinomial regression in the Trinculo software package. Loci passing the suggestive significance threshold (5x10-6) were selected for meta-analysis with previously published JIA GWAS data assuming fixed effects and weighted by inverse-variance using the software package GWAMA. Results Following quality control the dataset consisted of > 7.4 million SNPs for 3305 JIA cases and 9196 controls. We found no strong evidence to support association of any SNP to a specific ILAR group with most of the SNPs showing evidence for sharing across multiple groups. Association testing in a combined dataset of all ILAR groups identified seven SNPs associated at genome-wide significance (5x10-8); six of these have previously been reported for JIA while one is a novel association. The novel association (rs497523, p-value = 7.12x10-9) maps to chromosome 16p11 and is located within intron one of the CCDC101 gene. Meta-analysis identified association to rs7647909 which is an intronic variant in the FOXP1 gene (p-value 2.0x10-9) and rs2614258 within the AHI1 gene (p-value 9.5x10-12). Conclusion We identified novel associations to SNPs in the CCDC101 and FOXP1 genes. In addition, we report a genome-wide significant association to AHI1, previously reported at suggestive significance levels. Fine mapping with the integration of genomic data will be required to identify the true causal gene at each of these loci. The results provide little evidence to support ILAR subgroup specificity for any of the associated variants and combined analysis of data across subgroups maximised power to identify novel associations. Disclosures J. Bowes None. M.C. Marion None. S. Smith None. A. Yarwood None. M. Sudman None. D. Tarasek None. C.D. Langefeld None. S.D. Thompson None. W. Thomson None.

Poster presentation Tuesday 8 October Background Juvenile idiopathic arthritis (JIA) is a clinically heterogeneous group of childhood onset inflammatory joint diseases with strong evidence to support a genetic contribution to susceptibility. JIA is divided into seven clinical subgroups based on observed patterns of clinical symptoms using the International League of Associations for Rheumatology (ILAR) classification system. The genetic overlap between these groups is not completely understood and this lack of knowledge typically leads to the different ILAR groups being analysed as discrete entities and reducing the overall power of genetic association studies. The aim of this study was to conduct a large case-control association study on susceptibility to JIA to identify novel susceptibility loci and to investigate differences in these associations between the different ILAR groups. Methods JIA participants were genotyped on the Illumina Infinium CoreExome or OmniExpress arrays at the University of Manchester. UK population control genotype data was obtained from the Understanding Society Longitudinal Study. Quality control of data was performed conforming to conventional standards based on call rate, cryptic relatedness and ancestry outliers. Imputation was performed using the Haplotype Reference Consortium panel on the Michigan Imputation Server followed by exclusion of SNPs with low imputation accuracy (r2 < 0.5) and low minor allele frequency (< 1%). Association testing of all SNPs was performed with an additive model incorporating imputation uncertainty using SNPTEST. A subset of SNPs independently associated with all JIA (p-value < 5x10-6) were tested to evaluate if they were specific to a particular ILAR group or shared across multiple ILAR groups using Bayesian multinomial regression and model selection methods implemented in the Trinculo software package. Results Following quality control, the dataset consisted of > 7.4 million SNPs for 3,305 JIA cases and 9,196 controls. Association testing in a combined dataset of all ILAR groups identified seven SNPs associated at genome-wide significance (5x10-8); six of these have previously been reported for JIA while one is a novel association. The novel association (rs497523, p-value = 7.12x10-9, OR 0.85, 95% CI 0.8:0.9) maps to chromosome 16p11 and is located within intron one of the CCDC101 gene. In a subset of 44 independently associated SNPs we found no strong evidence to support association of any SNP to a specific ILAR group with the majority of the SNPs showing evidence for sharing across multiple groups. Conclusion In a large case control association study for susceptibility to JIA, we identified a novel association to a SNP in the intron of CCDC101. This gene is involved in transcriptional regulation through histone acetyltransferase. The results support a general model of sharing of loci across multiple ILAR groups. The combined analysis of data across subgroups, informed by model sharing, will maximise power to identify novel associations. Conflicts of Interest The authors declare no conflicts of interest.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2–activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16−, CD56dimCD16+CD57−, and CD56dimCD16+CD57+. CD56dimCD16+CD57− cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon–responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

Background Juvenile idiopathic arthritis (JIA) is a clinically heterogeneous group of childhood onset inflammatory joint diseases with strong evidence to support a genetic contribution to susceptibility. JIA is divided into seven clinical subgroups based on observed patterns of clinical symptoms using the International League of Associations for Rheumatology (ILAR) classification system. The genetic overlap between these groups is not completely understood and this lack of knowledge typically leads to the different ILAR groups being analysed as discrete entities and reducing the overall power of genetic association studies. Objectives The aim of this study was to conduct a large case-control association study on susceptibility to JIA to identify novel susceptibility loci and to investigate differences of these associations between the ILAR groups. Methods JIA participants were genotyped on the Illumina Infinium CoreExome or OmniExpress arrays at The University of Manchester. UK population control genotype data was obtained from the Understanding Society Longitudinal Study. Quality control of data was performed conforming to conventional standards based on call rate, cryptic relatedness and ancestry outliers. Imputation was performed using the Haplotype Reference Consortium panel on the Michigan Imputation Server followed by exclusion of SNPs with low imputation accuracy (r2 Results Following quality control the dataset consisted of > 7.4 million SNPs for 3305 JIA cases and 9196 controls. Association testing in a combined dataset of all ILAR groups identified seven SNPs associated at genome-wide significance (5x10-8); six of these have previously been reported for JIA while one is a novel association. The novel association (rs497523, p-value = 7.12x10-9, OR 0.85, 95% CI 0.8:0.9) maps to chromosome 16p11 and is located within intron one of the CCDC101 gene. In a subset of 44 independently associated SNPs we found no strong evidence to support association of any SNP to a specific ILAR group with the majority of the SNPs showing evidence for sharing across multiple groups. Conclusion In the largest case control association study for susceptibility to JIA performed to date, we identified a novel association to a SNP in the intron of CCDC101. This gene is involved in transcriptional regulation through histone acetyltransferase. CCDC101 may be the causal gene at this locus; however introns contain enhancer elements that regulate other genes in the transcriptional domain. Therefore fine mapping with the integration of genomic data is currently being performed for this locus. The results provide little evidence to support ILAR subgroup specificity for any of the associated variants; on the contrary the results support a general model of sharing across multiple groups. The combined analysis of data across subgroups, informed by model sharing, will maximise power to identify novel associations. Disclosure of Interests None declared

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering the mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here, we present an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (µ-CT). We show how rapid processing of intact animals, at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use µ-CT imaging to study growth defects in the Drosophila brain through the characterization of abnormal spindle (asp) and WD repeat domain 62 (Wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. Our work demonstrates the power of combining µ-CT with traditional genetic, cellular and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering the mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here, we present an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (µ-CT). We show how rapid processing of intact animals, at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use µ-CT imaging to study growth defects in the Drosophila brain through the characterization of abnormal spindle (asp) and WD repeat domain 62 (Wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. Our work demonstrates the power of combining µ-CT with traditional genetic, cellular and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.

Background:Juvenile idiopathic arthritis (JIA) is a childhood onset rheumatic disease which is classified into seven different clinical subtypes based upon the ILAR classification criteria. The most common extra articular manifestation of JIA is its associated uveitis (JIAU); particularly chronic anterior uveitis (CAU). Uveitis is a serious complication with the potential to lead to visual impairment and blindness. The rheumatoid factor negative polyarthritis and oligoarthritis ILAR subtypes, often referred to as the “polygo” subgroup, are at a higher risk for developing JIAU, with up to 30% of polygos afflicted by CAU. The HLA region has long been reported as a genetic risk factor for JIA susceptibility, with evidence suggesting that different amino acids of HLA genes infer risk to different JIA subtypes.Objectives:Investigate the association of amino acids and genetic variants in the HLA region with susceptibility to JIAU and the ILAR clinical subtypes.Methods:Samples were genotyped using the Illumina Infinium CoreExome and Infinium Onmiexpress arrays. Samples were excluded based on 0.9 and MAF >.01 using logistic regression or an omnibus test for multiallelic markers, including 3 PCs as covariates. Independent associations were identified using forward stepwise logistic regression including previously identified variants as covariates. Comparison of regression models was performed using a likelihood ratio test (LRT).Results:We analysed 7425 markers within the HLA region in 450 JIAU and 2024 JIA cases without uveitis. The most significant association was to amino acid positions 13 of HLA-DRB1 (p=2.9×10-30). Conditional analysis on DRB1 position 13 revealed an independent signal at DRB1 position 67 (p=2.4×10-6). Conditioning on all DRB1 alleles revealed an independent signal at HLA-DPB1 position 69 (p=5.3×10-7). As expected, ILAR subtype was found to be associated with JIAU (p=1.58×10-6). We used LRT to test if genetics provided further information above ILAR subtype alone and found that including residues at DRB1 position 13 significantly improved the fit of a model based on ILAR subtype alone (LRT p = 3.6×10-27). The reciprocal analysis, adding ILAR subtype to a model based on DRB1 position 13 alone, did not significantly improve the fit of a model (LRT p=0.83). Exploring associations in the polygo subgroup (n=1646) we found significant associations to the three previously described amino acids and JIAU (DRB1 position 13 p=3.4×10-20, DRB1 position 67 p=3.3×10-4, DPB1 position 69 p=2.2×10-6).Conclusion:This is largest analysis of HLA markers in JIAU patients to date and we identify two independent associations to amino acids in HLA-DRB1 and a further independent association to HLA-DPB1. This analysis demonstrates that including data on genetic risk factors adds further information to that captured by ILAR subtype alone. It also reveals that the previously validated associations at position 13 of HLA-DRB1 are also correlated with JIAU in the polygo subtype suggesting that genetic risk factors will help refine risk within clinical subtypes. Conditioning on DRB1 alleles reveals that the secondary independent DRB1 position 69 association is also strongly associated in the polygo subset of this cohort. Together these results highlight the potential future use of genetics risk factors for risk classification for uveitis in patients with JIADisclosure of Interests:None declared

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of Pericentrin-Like Protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we present an optimized method for non-invasive imaging of Drosophila melanogaster at high resolution using micro computed tomography (μ-CT). Our method allows for rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use the power of μ-CT imaging to model human diseases through the characterization of microcephaly in the fly. Our work demonstrates that μ-CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.

Background In recent years there have been tremendous strides in determining the genetic component of complex diseases, not least in Juvenile Idiopathic Arthritis (JIA). We now have 17 genetic loci robustly associated with an increased risk for developing JIA.1 In JIA, like all complex diseases, the regions of the genome harbouring genetic changes are found outside protein coding DNA, in regions of the genome shown to control gene expression, often sited some distance from the closest gene. One of these JIA associated regions, on chromosome 21q22, is labelled with the RUNX1 gene, although the associated variants are situated over 450 kb from the gene. We have previously demonstrated, through Capture HiC analysis, that the genomic region containing the JIA associated variants makes a physical contact with the RUNX1 promoter. The next challenge in this region, therefore, is to determine how a risk genetic background effects the expression of the gene, contributing to the susceptibility to JIA. Objectives Determine the function and mechanism of how the gene enhancer region, situated over 450 kb upstream of the RUNX1 gene, works to increase the risk of developing JIA. Methods B-lymphocyte cell lines, containing either a risk or protective JIA genetic background at the ‘RUNX1’ loci were interrogated for potential causative functional mechanism. Allele specific interactions were investigated using chromosome conformation capture (3C), enhancer activity (H3K4me1) with chromatin immunoprecipitation (ChIP), and the resultant expression of the RUNX1 gene analysed by qPCR in both stimulated (IL4/anti CD40) and unstimulated cell lines. Results The enhancer region associated with JIA interacts with the RUNX1 promoter in B-cell lines. A stimulation specific effect is observed in B-cell lines harbouring the protective alleles. In this protective context, prior to stimulation RUNX1 expression is higher than in the risk genotype with enhancer enrichment detected near the proximal promoter. After stimulation the interaction between the enhancer and RUNX1 promoter is much stronger, and whilst no enhancer enrichment was detected, this increased interaction frequency after stimulation corresponds to a sharp downregulation of RUNX1 expression. In contrast, the risk genotype showed no stimulation dependant change, maintaining both interaction and expression levels. Conclusions 3C and ChIP experiments were performed to investigate genotype and stimulation specific DNA-DNA and DNA-protein interactions and identify correlation with gene expression.These results suggest that the ability for B-cells to switch from relatively high expression of the RUNX1 transcript to low expression upon stimulation is crucial to an appropriate immune response. Reference [1] Hinks A, et al. Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis. Nat Genet2013Jun;45(6):664–9. Disclosure of Interest None declared

Whole genome expression profiling, or transcriptomics, is a high throughput technology with the potential for major impacts in both clinical settings and drug discovery and diagnostics. In particular, there is much interest in this technique as a mechanism for predicting treatment response. Gene expression profiling entails the quantitative measurement of messenger RNA levels for thousands of genes simultaneously with the inherent possibility of identifying biomarkers of response to a particular therapy or by singling out those at risk of serious adverse events. This technology should contribute to the era of stratified medicine, in which patient specific populations are matched to potentially beneficial drugs via clinical tests. Indeed, in the oncology field, gene expression testing is already recommended to allow rational use of therapies to treat breast cancer. However, there are still many issues surrounding the use of the various testing platforms available and the statistical analysis associated with the interpretation of results generated. This review will discuss the implications this promising technology has in predicting treatment response and outline the various advantages and pitfalls associated with its use.

The human endometrium is a unique tissue that undergoes dramatic monthly remodeling during the menstrual cycle in preparation for an implanting conceptus. This remodeling involves sequential proliferation and differentiation of endometrial stromal and epithelial cells, coupled with extensive angiogenesis and infiltration of a specific specialized immune cell subset. Increasing evidence points to an essential role for these maternal leukocytes in stimulating the endometrial angiogenesis, and we propose that they also play a key role in the decidual vascular transformation. Aberrant endometrial angiogenesis, decidualisation and vascular transformation is thought to underlie many pathologies of pregnancy, from infertility to the development of preeclampsia and Intra Uterine Growth Restriction. In this chapter we review the cellular processes associated with each stage of endometrial and decidual transformation, detailing the role of the immune cell populations and the angiogenic and chemotactic factors secreted by them.

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation, and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The Adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. While progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called Two-dimensional Difference Gel Electrophoresis (2D-DIGE). We show that loss of APC2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-cat transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by the loss of APC2. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on APC's cellular effects that may lead to a richer understanding of APC's role in development.

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro . Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr -knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

Epigenetic signatures vary between cells (1), and many research groups have moved to analysis of cell subsets rather than mixed cell populations such as whole blood, to simplify the interpretation ...

Background The treatment of rheumatoid arthritis (RA) has changed considerably over the last couple of decades, particularly since the introduction of biological therapies; however, a considerable proportion of patients fail to respond satisfactorily (30–40%). Early and effective treatment is known to correlate with better long-term outcomes and there is an unmet clinical need for predictors of response which can be used to stratify patients prior to treatment; so that those prone to non-response can be offered alternative options. We hypothesis that gene expression profiles of RA patients about to commence treatment with the biological drug etanercept will provide such a biomarker. Objectives To identify gene expression biomarkers which discriminate between good-responders (R) and non-responders (NR) to etanercept in RA patients. Methods 72 RA patients about to commence treatment with etanercept were chosen for analysis from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) longitudinal cohort. Using the established EULAR classification criteria this equated to 34 R (28-joint disease activity score (DAS28) of ≤3.2 and improvement of >1.2) and 38 NR (DAS28 >5.1 and improvement Results Using a fold-change (FC) cut-off of >1.2 between R and NR and p-value IFITM3 (FC 1.53, p-value=0.01), MX1 (FC 1.45, p-value=0.02) and IFI44L (FC 1.28, p-value=0.04) and a type-II IFN, BTN3A2 (FC 1.38, p-value 9.6 x 10 –05 ). Of note, expression of PTPRC , a previously reported treatment response gene, was found to be up-regulated in R as compared to NR prior to treatment (FC 1.24, p-value=0.03). Conclusions The preliminary results suggest that an IFN signature prior to etanercept treatment is indicative of non-response in RA patients. These results are encouraging in that an IFN signature has consistently been associated with RA and clinical outcome to biological therapies. However, to the best of our knowledge this is the first study to observe an IFN signature correlating with response to etanercept. More in-depth analysis is ongoing using packages such as WGCNA (weighted correlation network analysis) and Matrix eQTL to further explore these associations. Acknowledgement SLS was a PhD student funded by an investigator-initiated award to AB from Pfizer (award number WS1940162). Disclosure of Interest None declared

Background Biologic drug therapies represent a huge advance in the treatment of rheumatoid arthritis (RA). However, very good disease control is only achieved in 30% of patients, making identification of biomarkers of response a research priority. We therefore hypothesise that differential DNA methylation patterns may provide biomarkers predictive of response to TNF-inhibitor (TNFi) therapy in patients with RA. Methods An epigenome-wide association study was performed on pre-treatment whole blood DNA from patients with RA. Patients who displayed good response (n = 36) or no response (n = 36) to etanercept therapy at 3-months were selected. Differentially methylated positions (DMPs) were identified using linear regression. Variance of methylation at DMPs was assessed for correlation with cis- acting SNPs. A replication experiment for prioritised SNPs was performed in an independent cohort of 1,204 RA patients. Results Five DMPs between responders groups were identified with a FDR LRPAP1 gene on chromosome 4 (cg04857395, p = 1.39E-08 and cg26401028, 1.96E-08). The A allele of the SNP rs3468 was correlated with higher levels of methylation for both of the top two DMPs (2.63E-07 and 1.05E-06, respectively). Further the A allele of rs3468 was correlated with EULAR non-response in the discovery cohort (n = 56; p = 0.03) and in the independent replication cohort (n = 1.204; p = 0.003). Conclusions We identify DNA methylation as a potential biomarker of TNFi response and report the association between response and the LRPAP1 gene which encodes a chaperone of low-density lipoprotein receptor-related protein-1. Additional replication experiments in independent sample collections are now required.

Background Biologics are an important subset of drugs used to treat rheumatoid arthritis (RA); however, up to 40% of patients have an inadequate response. Precision medicine aims to identify biomarkers that predict sub-populations of patients likely to respond to a given drug in order to optimise patient benefit. MicroRNAs (miRNAs) are small non-coding post-transcriptional regulators of gene expression. Dysregulation of several miRNAs have been implicated in RA and as such have been suggested as potential markers of disease activity. However, little research has been conducted into the potential clinical use of such biomarkers in predicting treatment response. We hypothesise that miRNA expression in pre-treatment serum samples of RA patients will provide such a biomarker. Objectives To identify miRNA biomarkers in pre-treatment serum samples with sufficient power to differentiate between subsequent responder (R) and non-responder (NR) RA patients to the biologic drug, etanercept. Methods Fifteen patients about to undergo treatment with etanercept were selected based on their EULAR response at 3-months from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort. This equated to 6 extreme NR (disease activity score in 28-joints (DAS28) >5.1 and improvement 1.2 at follow-up); all were female and receiving concurrent methotrexate. MicroRNA was extracted from pre-treatment serum and expression of 754 miRNAs was measured on the TaqMan OpenArray Human miRNA Panel on the QuantStudio™ Real-Time qPCR System (Applied Biosystems). Raw data were processed using the Bioconductor package HTqPCR. Briefly, raw cycle threshold (C T ) values were normalised to an endogenous spike-in control miR-ath159a using the deltaCt method and differential expression (DE) was conducted between R and NR. Results Of the 754 miRNAs assayed, 405 were not expressed in any of the samples and were removed from further analysis; miRNAs were also excluded based on an amplification quality score (good amplification indicated by a score >1.1). This left 241 miRNAs for DE analysis. One circulating miRNA, miR-10b, was differentially expressed in pre-treatment samples (p-value =0.004), demonstrating higher relative expression in R vs. NR. A number of validated targets for miR-10b are pertinent to RA, including NOTCH1, a gene with a reported role in TNF induced proliferation of RA synoviocytes; SMRT, a negative regulator of interstitial MMP-1 synthesis and HOXD10, a transcription factor exclusively expressed in RA fibroblast-like synoviocytes (miRTarBase). Conclusions Pilot data from this preliminary study suggests higher expression of miR-10b in pre-treatment RA serum is predictive of subsequent response to etanercept by 3 months. Although the findings require replication, the presence of a miRNA which is known to interact with RA related target genes adds support that circulating miRNAs could be useful predictive markers of response to etanercept in RA patients. Acknowledgements SLS is a PhD student funded by an investigator-initiated award to AB from Pfizer (award WS1940162). Disclosure of Interest None declared

Introduction A growing number of studies have found evidence that anxiety and depressive disorders are associated with atypical amygdala hyperactivation, which decreases with effective treatment. Interest has emerged in this phenomenon as a possible biological marker for individuals who are likely to benefit from tailored treatment approaches. Objective The present study was designed to examine relationships between pretreatment amygdala activity and treatment response in a sample of anxious children and adolescents. Materials and methods Participants, who were diagnosed predominantly with generalized anxiety disorder (GAD), underwent functional magnetic resonance imaging (fMRI) scanning before treatment with fluoxetine or cognitive behavioral therapy (CBT). Results Results indicated significant negative associations between degree of left amygdala activation and measures of posttreatment symptom improvement in the group, as a whole. Discussion Taken together with research on associations between adult amygdala activation and treatment response, these findings suggest that patients whose pretreatment amygdala activity is the strongest may be particularly likely to respond well to such widely used treatments as selective serotonin reuptake inhibitor (SSRI) medications and CBT.