RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study

Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2 + channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad−/− calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, + 11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity

EXOGEN ultrasound bone healing system for long bone fractures with non-union or delayed healing: a NICE medical technology guidance

Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.This article has been made available through the Brunel Open Access Publishing Fund.A routine part of the process for developing National Institute for Health and Care Excellence (NICE) medical technologies guidance is a submission of clinical and economic evidence by the technology manufacturer. The Birmingham and Brunel Consortium External Assessment Centre (EAC; a consortium of the University of Birmingham and Brunel University) independently appraised the submission on the EXOGEN bone healing system for long bone fractures with non-union or delayed healing. This article is an overview of the original evidence submitted, the EAC’s findings, and the final NICE guidance issued.The Birmingham and Brunel Consortium is funded by NICE to act as an External Assessment Centre for the Medical Technologies Evaluation Programme

Changes in physical activity following total hip or knee arthroplasty: a matched case-control study from the EPIC-Norfolk cohort

Objective: To assess self-reported physical activity changes pre- compared to post-operatively in patients undergoing total hip or knee arthroplasty, and to compare this to an age- and gender-matched cohort of people who have not undergone arthroplasty.  Design: Population-based prospective cohort study.  Setting: Norfolk, UK  Subjects: People who had undergone hip or knee arthroplasty, compared to an age- and gender-matched non-arthroplasty cohort.  Intervention: Primary total hip or knee arthroplasty.  Main measures: Physical activity, measured using the EPIC Physical Activity Questionnaire (EPAQ2).  Results: 400 people from the EPIC-Norfolk community cohort were identified who had undergone hip or knee arthroplasty. 767 people were identified to form an age- and gender-matched non-arthroplasty cohort. Mean post-operative follow-up was 43 months post-total hip and 41 months post-total knee arthroplasty. There was a statistically significant reduction from pre- to post-arthroplasty in the number of flights of stairs climbed weekly (hip: mean difference (MD): 6.8; p<0.01; knee: MD: 10.2; p<0.01); duration of walking (hip: MD: 1.4 hours/week; p=0.02; knee: MD: 2.2 hours/week; p<0.01) and duration of total recreational activity (hip: MD: 1.1 hours/week; p=0.02). Compared to the non-arthroplasty cohort, duration of physical activity was lower post-total hip arthroplasty (MD: 1.8 hours/week; p=0.01). The number of flights of stairs climbed weekly (MD: 12.0; p<0.01), total recreational activity (MD: 1.7 hours/week; p=0.04) and physical activity energy expenditure (MD: 5.7 Mets-hours/week; p=0.05) was lower for people post-total knee arthroplasty compared to the matched controls.  Conclusions: Physical activity did not increase, and in instances decreased, following total hip or knee arthroplasty

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1

Is there a difference in physical activity levels in patients before and up to one year after unilateral total hip replacement? A systematic review and meta-analysis

Objective: To determine if there is a difference in physical activity levels before and up to one year after unilateral primary total hip replacement. Data sources: A search was performed on 13th July 2016. Studies were eligible for inclusion if they presented pre-operative and up to one year post-operative measures of physical activity for patients who had undergone unilateral primary total hip replacement. Review methods: Any paper that used a measure of physical activity pre and up to one year post unilateral primary total hip replacement. Data was synthesised using a meta-analysis with 95% confidence intervals (CI), if appropriate. The Critical Appraisal Skills Programme cohort study checklist was used to assess the quality of evidence. Results: From 6024 citations, nine studies were analysed in a meta-analysis and eight studies were analysed qualitatively. The quality of the evidence was ‘low’ to ‘moderate’. There was no statistically significant difference in physical activity pre- to post-total hip replacement when assessed using: movement-related activity (mean difference (MD): -0.08; 95% CI: 1.60, 1.44; I2=0%; n=77), percentage of 24-hours spent walking (MD: -0.21; 95% CI: -1.36, 0.93; I2=12%; n=65), six-minute walk test (MD: -60.85; 95% CI: -122.41, 0.72; I2=84%; n=113) or the cardiopulmonary exercise test (MD: -0.24; 95% CI: -1.36, 0.87; I2=0%;n=76). Conclusion: There is no statistically significant difference in physical activity levels before and up to one year after unilateral primary total hip replacement. However the low to moderate methodological quality of the included papers should be taken into consideration when drawing conclusions

The Debrisoft ® monofilament debridement pad for use in acute or chronic wounds: A NICE medical technology guidance

As part of its Medical Technology Evaluation Programme, the National Institute for Health and Care Excellence (NICE) invited a manufacturer to provide clinical and economic evidence for the evaluation of the Debrisoft ® monofilament debridement pad for use in acute or chronic wounds. The University of Birmingham and Brunel University, acting as a consortium, was commissioned to act as an External Assessment Centre (EAC) for NICE, independently appraising the submission. This article is an overview of the original evidence submitted, the EAC’s findings and the final NICE guidance issued. The sponsor submitted a simple cost analysis to estimate the costs of using Debrisoft® to debride wounds compared with saline and gauze, hydrogel and larvae. Separate analyses were conducted for applications in home and applications in a clinic setting. The analysis took an UK National Health Service (NHS) perspective. It incorporated the costs of the technologies and supplementary technologies (such as dressings) and the costs of their application by a district nurse. The sponsor concluded that Debrisoft® was cost saving relative to the comparators. The EAC made amendments to the sponsor analysis to correct for errors and to reflect alternative assumptions. Debrisoft® remained cost saving in most analyses and savings ranged from £77 to £222 per patient compared with hydrogel, from £97 to £347 compared with saline and gauze, and from £180 to £484 compared with larvae depending on the assumptions included in the analysis and whether debridement took place in a home or clinic setting. All analyses were severely limited by the available data on effectiveness, in particular a lack of comparative studies and that the effectiveness data for the comparators came from studies reporting different clinical endpoints compared with Debrisoft®. The Medical Technologies Advisory Committee made a positive recommendation for adoption of Debrisoft® and this has been published as a NICE medical technology guidance (MTG17).The Birmingham and Brunel Consortium is funded by NICE to act as an External Assessment Centre for the Medical Technologies Evaluation Programme

Rad GTPase Deletion Atenuates Post-Ischemic Cardiac Dysfunction and Remodeling

The protein Rad interacts with the L-type calcium channel complex to modulate trigger Ca2+ and hence to govern contractility. Reducing Rad levels increases cardiac output. Ablation of Rad also attenuated the inflammatory response following acute myocardial infarction. Future studies to target deletion of Rad in the heart could be conducted to establish a novel treatment paradigm whereby pathologically stressed hearts would be given safe, stable positive inotropic support without arrhythmias and without pathological structural remodeling. Future investigations will also focus on establishing inhibitors of Rad and testing the efficacy of Rad deletion in cardioprotection relative to the time of onset of acute myocardial infarction

Association of resilience and psychological flexibility with surgeons' mental wellbeing.

BACKGROUND: Existing research highlights the link between certain personality traits and mental health in surgeons. However, little research has explored the important role of psychological skills and qualities in potentially explaining this link. A cross-sectional survey of UK-based surgeons was used to examine whether two such skills (psychological flexibility and resilience) helped to explain why certain personality traits might be linked to mental health in surgeons. METHOD: An online survey comprising measures of personality (neuroticism, extraversion and conscientiousness), psychological skills/qualities (psychological flexibility and resilience) and mental health (depression, anxiety, stress and burnout) was sent to surgeons practising in the UK. Mediation analyses were used to examine the potential mediating role of psychological flexibility and resilience in explaining the relationship between personality factors and mental health. RESULTS: A total of 348 surgeons completed the survey. In all 12 mediation models, psychological flexibility and/or resilience played a significant role in explaining the relationship between personality traits (neuroticism, extraversion and conscientiousness) and mental health (depression, anxiety and burnout). CONCLUSION: Findings suggest that it is not only a surgeon's personality that is associated with their mental health, but the extent to which a surgeon demonstrates specific psychological qualities and skills (psychological flexibility and resilience). This has important implications for improving surgeons' mental wellbeing, because psychological flexibility and resilience are malleable, and can be successfully targeted with interventions in a way that personality traits cannot