DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS.

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3-MAVS and TRAF3-IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling.

The Staphylococcus aureus Cell Wall-Anchored Protein Clumping Factor A Is an Important T Cell Antigen.

Staphylococcus aureus has become increasingly resistant to antibiotics, and vaccines offer a potential solution to this epidemic of antimicrobial resistance. Targeting of specific T cell subsets is now considered crucial for next-generation anti-S. aureus vaccines; however, there is a paucity of information regarding T cell antigens of S. aureus This study highlights the importance of cell wall-anchored proteins as human CD4+ T cell activators capable of driving antigen-specific Th1 and Th17 cell activation. Clumping factor A (ClfA), which contains N1, N2, and N3 binding domains, was found to be a potent human T cell activator. We further investigated which subdomains of ClfA were involved in T cell activation and found that the full-length ClfA N123 and N23 were potent Th1 and Th17 activators. Interestingly, the N1 subdomain was capable of exclusively activating Th1 cells. Furthermore, when these subdomains were used in a model vaccine, N23 and N1 offered Th1- and Th17-mediated systemic protection in mice upon intraperitoneal challenge. Overall, however, full-length ClfA N123 is required for maximal protection both locally and systemically.

A short sequence within subdomain N1 of region A of the Staphylococcus aureus MSCRAMM clumping factor A is required for export and surface display.

Clumping factor A (ClfA) is the archetypal fibrinogen-binding surface protein of Staphylococcus aureus and a member of the microbial surface component recognizing adhesive matrix molecules (MSCRAMM) family. An N-terminal signal sequence directs export of the MSCRAMM by the Sec pathway and the C-terminal cell wall-anchoring domain allows covalent attachment of ClfA to peptidoglycan by sortase. Region A of ClfA comprises three independently folded subdomains N1, N2 and N3. Subdomains N2N3 comprise IgG-like folds and promote fibrinogen binding. Nothing is known about the structure or function of subdomain N1. Here we demonstrate an unexpected role for N1 in the export and surface localization of ClfA. Attempted expression of a ClfA variant lacking subdomain N1 resulted in impaired growth of S. aureus and accumulation of ClfA protein in the cytoplasm and cytoplasmic membrane. The presence of residues 211-228 of N1 was required to allow display of ClfA on the bacterial surface. The importance of this region was confirmed when a ClfA variant lacking residues 211-220 was also mislocalized to the cytoplasm and cytoplasmic membrane. However, these residues were not required for export of ClfA lacking the Ser-Asp repeats that link region A to the wall-anchoring domain. Similarly, subdomain N1 of a related MSCRAMM fibronectin-binding protein B was required for export and surface display of the full-length protein, but not a derivative lacking fibronectin-binding repeats. In summary, we demonstrate that residues in the N1 subdomain are required for export and cell wall localization of S. aureus MSCRAMM proteins.

Undergraduate research involving human subjects should not be granted ethical approval unless it is likely to be of publishable quality.

Small-scale research projects involving human subjects have been identified as being effective in developing critical appraisal skills in undergraduate students. In deciding whether to grant ethical approval to such projects, university research ethics committees must weigh the benefits of the research against the risk of harm or discomfort to the participants. As the learning objectives associated with student research can be met without the need for human subjects, the benefit associated with training new healthcare professionals cannot, in itself, justify such risks. The outputs of research must be shared with the wider scientific community if it is to influence future practice. Our survey of 19 UK universities indicates that undergraduate dissertations associated with the disciplines of medicine, dentistry and pharmacy are not routinely retained in their library catalogues, thus closing a major avenue to the dissemination of their findings. If such research is unlikely to be published in a peer-reviewed journal, presented at a conference, or otherwise made available to other researchers, then the risks of harm, discomfort or inconvenience to participants are unlikely to be offset by societal benefits. Ethics committees should be satisfied that undergraduate research will be funnelled into further research that is likely to inform clinical practice before granting ethical approval.

DDX3X functionally and physically interacts with Estrogen Receptor-alpha.

DEAD-box protein 3X (DDX3X) is a human DEAD-box protein with conventional roles in RNA metabolism and unconventional functions in signalling pathways that do not require its enzymatic activity. For example, DDX3X acts as a multifunctional adaptor molecule in anti-viral innate immune signalling pathways, where it interacts with and regulates the kinase IKB-kinase-epsilon (IIKKε). Interestingly, both DDX3X and IKKɛ have also independently been shown to act as breast cancer oncogenes. IKKɛ's oncogenic functions are likely multifactorial, but it was suggested to phosphorylate the transcription factor Estrogen receptor alpha (ERα) at Serine 167, which drives expression of Erα target genes in an estrogen-independent manner. In this study, we identified a novel physical interaction between DDX3X and ERα that positively regulates ERα activation. DDX3X knockdown in ER+ breast cancer cell lines resulted in reduced ERα phosphorylation, reduced Estrogen Response Element (ERE)-controlled reporter gene expression, decreased expression of ERα target genes, and decreased cell proliferation. Vice versa, overexpression of DDX3X resulted in enhanced ERα phosphorylation and activity. Furthermore, we provide evidence that DDX3X physically binds to ERα from co-immunoprecipitation and pulldown experiments. Based on our data, we propose that DDX3X acts as an adaptor to facilitate IKKε-mediated ERα activation, akin to the mechanism we previously elucidated for IKKε-mediated Interferon Regulatory factor 3 (IRF3) activation in innate immune signalling. In conclusion, our research provides a novel molecular mechanism that might contribute to the oncogenic effect of DDX3X in breast cancer, potentially linking it to the development of resistance against endocrine therapy.

Fibronectin-binding protein B variation in Staphylococcus aureus.

BACKGROUND: Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA. RESULTS: Seven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnbB variant. The phylogeny of fnbB alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnbB allelic variants encode seven distinct isotypes of the FnBPB A domain that are 61 to 85% identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism. CONCLUSIONS: Seven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism. All seven isotypes of FnBPB are capable of binding fibronectin though none contain any known fibronectin-binding motifs. These results have implications for the development of vaccines or immunotherapeutics that target FnBPB.

A pilot study to monitor changes in spirometry and lung volume, following an exacerbation of Chronic Obstructive Pulmonary Disease (COPD), as part of a supported discharge program.

BACKGROUND: One-third of patients with an exacerbation of Chronic Obstructive Pulmonary Disease(COPD) are re-hospitalised at 90 days. Exacerbation recovery is associated with reductions in lung hyperinflation and improvements in symptoms and physical activity. We assessed the feasibility of monitoring these clinical parameters in the home. We hypothesised that the degree of change in spirometry and lung volumes differs between those who had an uneventful recovery and those who experienced a further exacerbation. METHODS: Hospitalised patients with an acute exacerbation of COPD referred for a supported discharge program participated in the study. Spirometry and Inspiratory Vital Capacity(IVC) were measured in the home at Days 1, 14 and 42 post-discharge. Patients also completed Medical Research Council(MRC), Borg and COPD Assessment Test(CAT) scores and were provided with a tri-axial accelerometer. Any new exacerbation events were recorded. RESULTS: Sixty-five patients with 72 exacerbation episodes were recruited. Fifty percent experienced a second exacerbation. Adequate IVC measurements were achieved by 90%, while only 70% completed spirometry. Uneventful recovery was accompanied by significant improvements in physiological measurements at day14, improved symptom scores and step count, p < 0.05. Failure of MRC to improve was predictive of re-exacerbation(Area Under Receiver Operating Curve(AUROC) 0.6713) with improvements in FEV1≥100 ml(AUROC 0.6613) and mean daily step count ≥396 steps(AUROC 0.6381) predictive of recovery. CONCLUSION: Monitoring the pattern of improvement in spirometry, lung volumes, symptoms and step count following a COPD exacerbation may help to identify patients at risk of re-exacerbation. It is feasible to carry out these assessments in the home as part of a supported discharge programme.

Use of a care bundle in the emergency department for acute exacerbations of chronic obstructive pulmonary disease: a feasibility study.

AIM: To determine the efficacy and usefulness of a chronic obstructive pulmonary disease (COPD) care bundle designed for the initial management of acute exacerbations of COPD and to assess whether it improves quality of care and provides better outcomes. INTRODUCTION: The level of care provided in the emergency department (ED) for COPD exacerbations varies greatly, and there is a need for a more systematic, consistent, evidence-based quality improvement approach to improve outcomes and costs. METHODS: A prospective before and after study was carried out in a university teaching hospital. Fifty consecutive patients were identified in the ED with COPD exacerbations and their management was reviewed. Following the education of ED staff and the implementation of a COPD care bundle, the outcome for 51 consecutive patients was analyzed. This COPD care bundle consisted of ten elements considered essential to the management of COPD exacerbations and was scored 0-10 according to the number of items on the checklist implemented correctly. RESULTS: Following implementation, the mean bundle score out of 10 improved from 4.6 to 7 (P<0.001). There was a significant decrease in the unnecessary use of intravenous corticosteroids from 60% to 32% (P=0.003) and also a marked improvement in the use of oxygen therapy, with appropriate treatment increasing from 76% to 96% (P=0.003). Prophylaxis for venous thromboembolism also improved from 54% to 73% (P=0.054). The 30-day readmission rate did not significantly improve. CONCLUSION: The use of a bundle improves the delivery of care for COPD exacerbations in the ED. There is more appropriate use of therapeutic interventions, especially oxygen therapy and intravenous corticosteroids.

Short term and long term effects of pulmonary rehabilitation on physical activity in COPD.

UNLABELLED: The central purpose of pulmonary rehabilitation is to reduce morbidity by improving functional capacity through exercise. It is still unknown if improvements in functional capacity are maintained in the long-term and if this leads to increased physical activity levels as measured by a free-living activity monitor. The hypothesis of this study was that pulmonary rehabilitation would lead to a sustained increase in standard outcome measures and in daily physical activity. METHODS: A prospective study of 47 subjects with COPD was performed, registered at ClinicalTrials.gov (Clinical Trial Number NCT 0112943). The primary outcome was a maintained improvement in standard outcome measures with a secondary aim of an increase in daily physical activity. A convenient sample of the cohort (n = 17) was re-evaluated at a third time point at 1 year. RESULTS: A seven week hospital based outpatient pulmonary rehabilitation program led to a significant reduction in total energy expenditure (p < 0.044) and breathlessness (Borg, p < 0.011) and improved exercise capacity (ISWT, p > 0.001, 6MWT, p > 0.002) PiMax (p > 0.007) and quality of life scores (SGRQ, p > 0.001, EQ5D, 0.025). However, pulmonary rehabilitation did not significantly change the average number of daily steps taken, time spent sedentary activity, METs consumed or daily physical activity. Indeed, all of the standard and freeliving values had returned towards the baseline value at 1 year. DISCUSSION: These findings show that while pulmonary rehabilitation increased exercise capacity this was not transmitted into increased daily physical activity. Hence, alternative methods to alter/affect behavioural change need to be addressed.

Acupuncture as an adjunct to pulmonary rehabilitation.

PURPOSE: Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation and by both systemic and airway inflammation. In COPD, acupuncture has been shown to improve quality-of-life scores and decrease breathlessness; similar findings have also been reported after pulmonary rehabilitation (PR). The hypothesis of this study was that acupuncture in conjunction with pulmonary rehabilitation would improve COPD outcome measures compared to pulmonary rehabilitation alone. METHODS: The design was a randomized prospective study; all subjects had COPD. There were 19 controls, 25 who underwent PR, and 16 who had both acupuncture and PR. The primary outcome measure was a change in measures of systemic inflammation at the end of PR and at 3 month followup. Lung function, including maximum inspiratory pressure (PiMax), quality-of-life scores, functional capacity including steps taken, dyspnea scores, and exercise capacity, were secondary endpoints. RESULTS: After PR, both groups had significantly improved quality-of-life scores, reduced dyspnea scores, improved exercise capacity, and PiMax, but no change in measures of systemic inflammation compared with the controls. There were no differences in most of the outcome measures between the 2 treatment groups except that subjects who had both acupuncture and PR remained less breathless for a longer period. CONCLUSION: The addition of acupuncture to PR did not add significant benefit in most of the outcomes measured.