What is the Long-term Wear of a 5-Mrad Highly Crosslinked Polyethylene Liner? A 14-year RSA Study.

BACKGROUND: Lower doses of irradiation (≤ 5 Mrad) during the manufacture of highly crosslinked polyethylene acetabular liners may result in less crosslinking and an increased wear rate. Radiostereometric analysis (RSA) studies have found that wear of more highly irradiated highly crosslinked polyethylene liners (7 to 10 Mrad) decreases at longer-term follow-up compared with earlier reports of the same cohorts. Although wear of 5-Mrad irradiated liners appears to increase at midterm follow-up, it is unclear whether that remains true at longer follow-up. QUESTIONS/PURPOSES: We asked: (1) In patients who underwent THA with a 5-Mrad highly crosslinked polyethylene liner, what is the wear rate evaluated with RSA during the first 14 years? (2) Does the wear rate decrease after 6 years in situ? METHODS: This is a brief follow-up of prior RSA studies performed at 2 and 6 years. We prospectively reviewed the longer-term wear rate in 13 patients who underwent primary THAs with the same design of a 5-MRad irradiated crosslinked acetabular liner and a 28-mm cobalt-chromium articulation. Of the initial 30 patients who were enrolled, 13 (43%) were available at the 14-year timepoint; nine patients had died, 1 patient had withdrawn, 1 had an intraoperative fracture, 3 patients were too infirm to have radiographs, 2 had no baseline RSA radiographs, and 1 had poor-quality RSA images. Tantalum markers were inserted during surgery, and all patients had RSA radiographic examinations at 1 week, 6 months, and 1, 2, 6, and 14 years postoperatively. RESULTS: The mean ± standard deviation proximal, 2D, and 3D wear rates calculated between 1 year and 14 years were 0.019 ± 0.013 mm, 0.022 ± 0.015 mm, and 0.025 ± 0.019 mm per year, respectively. No patient had proximal 2D or 3D wear rates exceeding 0.06 mm per year. An increasing wear rate over time was measured for proximal and 2D wear rates between 6 and 14 years (0.024 and 0.030 mm per year) compared with that between 1 and 6 years (0.008 and 0.010 mm per year; p = 0.03). CONCLUSION: The proximal, 2D, and 3D wear of a highly crosslinked polyethylene liner produced using 5-Mrad radiation remains low in the longer-term. With the small numbers available in a long-term RSA study such as this, we confirmed that the wear rate did not decrease at longer-term follow-up, unlike previous RSA studies of more highly irradiated highly crosslinked polyethylene liners. Nevertheless, the wear rate remains very low and below the threshold typically associated with the development of osteolysis (0.1 mm/year of wear). This should provide assurance to orthopaedic surgeons monitoring patients with this 5-Mrad irradiated liner in situ, while providing useful information to manufacturers of future highly crosslinked polyethylene liners. LEVEL OF EVIDENCE: Level IV therapeutic study.

K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin.

Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.

Differential roles and regulation of the protein kinases PAK4, PAK5 and PAK6 in melanoma cells.

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC-PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.

Perception of Perioperative Risk for Arthroplasty Patients: A Poll of Indian Orthopedic Surgeons.

BACKGROUND: A survey of the American Association of Hip and Knee Surgeons (AAHKS) reported that 95% of respondents attempted to modify risk factors prior to arthroplasty. This study investigated Indian arthroplasty surgeons' approach to patients who have modifiable risk factors. METHODS: The AAHKS survey tool was adapted for Indian surgeons and distributed to the membership of the Indian Society of Hip and Knee Surgeons and Indian Arthroplasty Association via a Survey Monkey. A total of 92 survey responses were received, representing a response rate of 12%. RESULTS: Overall, 87% of respondents restricted access to arthroplasty surgery for patients who have modifiable risk factors, but only 51% of respondents reported delays or restricted treatment because of risk factors. Respondents reported that financial implications were more likely to delay or restrict treatment in 97% and social/family reasons in 66%. Poor diabetic control (81%), previous infection (57%), and malnutrition/hypoalbuminemia (47%) were the most frequent modifiable risk factors. There were 82% of surgeons reporting that the patient's socioeconomic status influenced treatment including: 71% of patients who have low socioeconomic status, 57% who do not have insurance, and 45% who have limited social supports. Most surgeons (92%) reported that funding influenced the type of care provided and the choice of implants. CONCLUSION: Over 97% of Indian arthroplasty surgeons thought socioeconomic factors impaired access to orthopaedic treatment. Only half the surgeons restricted access for comorbidities and these were more often related to infection risks and diabetes. These findings contrast dramatically to the practice patterns of American AAHKS members.

Perception of Perioperative Risk for Arthroplasty Patients: A Poll of Australian Orthopedic Surgeons.

BACKGROUND: There is growing interest in the perioperative management of patients who have indications for hip and knee arthroplasty in the setting of modifiable risk factors such as morbid obesity, poorly controlled diabetes, and smoking. A recent survey of the American Association of Hip and Knee Surgeons (AAHKS) found that 95% of respondents address modifiable risk factors prior to surgery. The aim of this study was to poll Australian arthroplasty surgeons regarding their approach to patients who have modifiable risk factors. METHODS: The survey tool used in the AAHKS study was adapted for use in the Australian context and distributed to the membership of the Arthroplasty Society of Australia via SurveyMonkey. There were 77 responses received, representing a response rate of 64%. RESULTS: The majority of respondents were experienced, high volume arthroplasty surgeons. Overall, 91% of respondents restricted access to arthroplasty for patients who have modifiable risk factors. There were 72% restricting access for excessive body mass index, 85% for poor diabetic control, and 46% for smoking. Most respondents made decisions based on personal experience or literature review rather than hospital or departmental pressures. While 49% of surgeons believed that current payment systems did not impair their ability to achieve good outcomes, 58% believed that certain arthroplasty patients would benefit from additional intervention, based on their socioeconomic status. CONCLUSION: Over 90% of surgeons who responded address modifiable risk factors prior to surgery. This finding aligns with the practice patterns of AAHKS members, despite differences in healthcare systems.

Developing conceptually sound items for a clinical courage questionnaire.

INTRODUCTION: Clinical courage can be described as a rural doctor's adaptability and willingness to undertake clinical work at the limits of their training and experience to meet the needs of their patients. This article describes the in-house development of survey items to include in a quantitative measure of clinical courage. METHODS: The questionnaire development involved two key concepts: a second-order latent factor model structure and a nominal group technique, used to develop consensus among the research team members. RESULTS: The steps taken to develop a sound clinical courage questionnaire are described in detail. The resulting initial questionnaire is presented, ready for testing with rural clinicians and refinement. CONCLUSION: This article outlines the psychometric process of questionnaire design and presents the resultant clinical courage questionnaire.

Highly Crosslinked Polyethylene Liners Have Negligible Wear at 10 Years: A Radiostereometric Analysis Study.

BACKGROUND: The introduction of crosslinked ultra-high molecular weight polyethylene (XLPE) acetabular liners has been very successful, with decreased wear and reduction in the rates of revision hip arthroplasties. XLPE is the preferred articulation for most surgeons; however, there are concerns about the long-term performance of XLPE liners created with different manufacturing processes, which may lead to time-dependent failure, including accelerated wear, after several years. QUESTIONS/PURPOSES: (1) What is the amount and rate of wear during the first 10 years using radiostereometric analysis (RSA) measurements of patients who had THAs that included a second-generation XLPE bearing? (2) Does the rate of wear change after 5 years in situ? METHODS: This is a brief follow-up of a previous RSA study. In that study, we prospectively enrolled 21 patients with osteoarthritis who underwent primary cementless THA with an XLPE acetabular liner (three cycles of 3Mrad annealed) and 32-mm articulation. That group represented 44% of the 48 THAs performed by the surgeon at the hospital where RSA was available; 16 had cemented hips, leaving 32 who were invited to participate in this study. Of those, 11 lived rurally and declined to participate, leaving 21 patients who were included in the initial study. Since then, three patients died, one developed dementia and could not participate, and one had revision THA for reasons other than wear, leaving 16 patients available for analysis at 10 years. Tantalum markers were inserted during surgery, and all patients had RSA radiographs taken at 1 week, 6 months, and 1, 2, 5, and 10 years postoperatively. Femoral head penetration into the acetabular component was measured with RSA, including bedding-in during the first year and annual wear thereafter. RESULTS: The median medial, proximal, anterior, two-dimensional (2D), and three-dimensional (3D) wear rates between 1 and 10 years were -0.001, 0.004, -0.012, 0.000, and 0.002 mm/year, respectively. No patient in this cohort had a proximal or 2D wear rate greater than 0.025 mm/year. The median proximal wear rate between 5 and 10 years (0.002 mm/year) was not greater than wear at 1 to 5 years (0.004 mm/year). CONCLUSION: Femoral head penetration in this second-generation XLPE liner remained very low at 10 years and accelerated wear after 5 years in situ did not occur. Concerns about late-onset wear from oxidation of irradiated-annealed XLPE were not observed. The low level of wear remains encouraging for the future clinical performance of this material. LEVEL OF EVIDENCE: Level II, therapeutic study.

Primary Total Knee Arthroplasty Revised for Instability: A Detailed Registry Analysis.

BACKGROUND: Instability after total knee arthroplasty is a common but poorly understood complication. METHODS: Data from a large national registry was used to study patient and prosthesis characteristics of 2605 total knee arthroplasty revisions for instability. The cumulative percent revision was calculated using Kaplan-Meier estimates, and Cox proportional models used to compare revision rates. The rate of further revision was analyzed with regard to prostheses used in the first revision. RESULTS: Instability increased from 6% of all first revision procedures in 2003 to 13% in 2019. The revision risk was lower for minimally stabilized prostheses, males, and patients aged ≥65 years. Polyethylene insert exchange was used for 55% of revision procedures, using a thicker insert in 93% and a change in insert conformity in 24% of cruciate-retaining knees. The increase in either thickness or conformity had no effect on the rate of further revision. After a revision for instability, 24% had a second revision by 14 years. Recurrent instability accounted for 32% of further revisions. A lower second revision rate was seen after revision of both femoral and tibial components, and where constrained components were used. CONCLUSION: Revision for instability is increasing. Revising both femoral and tibial components led to a lower rate of second revision compared to a change in insert alone. Recurrent instability was common.

FAM83D directs protein kinase CK1α to the mitotic spindle for proper spindle positioning.

The concerted action of many protein kinases helps orchestrate the error-free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin-dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α-binding-deficient FAM83DF283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D-/- cells, or artificially delivering CK1α to the spindle in FAM83DF283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.

Acetabular screws do not improve early revision rates in primary total hip arthroplasty. An instrumented registry analysis.

BACKGROUND: Initial stability of uncemented acetabular components in total hip arthroplasty (THA) is important for osseointegration and potentially enhanced by screw fixation. We used Australian Orthopaedic Association National Joint Replacement Registry data to determine whether screw usage influences uncemented acetabular component survival. METHODS: Primary THA with uncemented acetabular components performed for osteoarthritis from 1999 to 2018 was included. Survivorship was calculated using Kaplan-Meier estimates of cumulative percent revision (CPR). Comparisons used Cox proportional hazards method. An instrumental variable analysis adjusted for surgeon preference for screws as a confounding factor was used. RESULTS: Three hundred thirty thousand one hundred ninety-two THAs were included (31.8% with screws, 68.2% without). Two hundred twenty thousand six hundred seven were included in the instrumental variable analysis. Revision rate of acetabular components (all causes) was higher with screws during the first six years (hazard ratio (HR) = 1.45 (95% CI 1.34, 1.57), p < 0.001) and lower thereafter (HR = 0.81 (95% CI 0.67, 0.98), p = 0.027). Revision rate of acetabular components for loosening was higher with screws over the entire study period (HR = 1.73 (95% CI 1.51, 1.98), p < 0.001). Overall THA revision rate was higher with screws during the first six years (HR = 1.20 (95% CI 1.15, 1.26), p < 0.001) but lower thereafter (HR = 0.89 (95% CI 0.81, 0.98), p = 0.020). Revision rate for dislocation was higher with screws over the entire period (HR = 1.16 (95% CI 1.06, 1.26), p < 0.001). Instrumental variable analysis revealed higher revision rates with acetabular screws in the first six years. (HR = 1.18 (95% CI 1.09-1.29), p < 0.001). CONCLUSION: Screws did not confer a protective effect against acetabular loosening and were not associated with long-term negative consequences.

The impact of interpersonal relationships on rural doctors' clinical courage.

INTRODUCTION: Clinical courage occurs when rural doctors push themselves to the limits of their scope of practice to provide the medical care needed by patients in their community. This mental strength to venture, persevere and act out of concern for one's patient, despite a lack of formally recognised expertise, becomes necessary for doctors who work in relative professional isolation. Previous research by the authors suggested that the clinical courage of rural doctors relies on the relationships around them. This article explores in more depth how relationships with others can impact on clinical courage. METHODS: At an international rural medicine conference in 2017, doctors who practised rural/remote medicine were invited to participate in the study. Twenty-seven semistructured interviews were conducted exploring experiences of clinical courage. Initial analysis of the material, using a hermeneutic phenomenological frame, sought to understand the meaning of clinical courage. In the original analysis, an emic question arose: 'How do interpersonal relationships impact on clinical courage'. The material was re-analysed to explore this question, using Wenger's community of practice as a theoretical framework. RESULTS: This study found that clinical courage was affected by the relationships rural doctors had with their communities and patients, with each other, with the local members of their healthcare team and with other colleagues and health leaders outside their immediate community of practice. CONCLUSION: As a collective, rural doctors can learn, use and strengthen clinical courage and support its development in new members of the discipline. Relationships with rural communities, rural patients and urban colleagues can support the clinical courage of rural doctors. When detractors challenge the value of clinical courage, it requires individual rural doctors and their community of practice to champion rural doctors' way of working.

PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP.

Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.

PAWS1 controls Wnt signalling through association with casein kinase 1α.

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.

Does Knee Prosthesis Survivorship Improve When Implant Designs Change? Findings from the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: TKA generally has excellent long-term survivorship. When a new knee system supersedes a previous model, increased survivorship, improved functional performance, or both may be expected, because key areas of design modification are often targeted to address wear, stability, and the patellofemoral articulation. However, not all design changes are beneficial, and to our knowledge, knee arthroplasty has not been systematically evaluated in the context of design changes that occur during the development of new knee arthroplasty systems. QUESTIONS/PURPOSES: Using the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) we performed multiple old-to-new comparisons of frequently used contemporary knee implants to ask: (1) does overall prosthesis survivorship free from revision increase when a new knee prosthesis system is introduced to replace a prior prosthesis system? (2) Has survivorship free from revision improved for the revision indications of wear, instability, and patellofemoral articulation issues, where development efforts have been concentrated? METHODS: Data from the AOANJRR from September 1999 to December 2017 were used to compare the survivorship of prostheses free from revision at a maximum of 17 years in procedures where a new design model was introduced to replace a prior knee system from the same manufacturer. Only prosthesis systems used in a minimum of 2000 primary TKA procedures for osteoarthritis that had a minimum of 5 years of follow-up were included. Varus-valgus constrained and hinge TKA designs were excluded. Cruciate-retaining, posterior-stabilized, and medial pivot-design knees were considered separately. The new and old prosthesis systems were paired for analysis. Survivorship was calculated with Kaplan Meier estimates and comparisons were performed using the Cox proportional hazards method. Subanalyses according to the three main revision indications were performed, and where possible, analyses were performed based on polyethylene types (highly cross-linked polyethylene and ultra-high-molecular-weight polyethylene), combined and separated. Revision was defined as a reoperation of a previous knee arthroplasty in which one or more of the components was removed, replaced, or added. There were 323,955 TKA procedures and 11 new prosthesis system designs that were introduced to replace an earlier knee system from the same manufacturer. Of these prosthesis system pairs, six were cruciate-retaining prostheses, four were posterior-stabilized designs, and one was a medial pivot design. RESULTS: Six of the 11 knee system pairs showed improved survivorship with the new design, three were no different, and in two, the newer prosthesis systems had a higher rate of revision than the old one did. When revision for wear was analyzed, five prosthesis systems showed improvement, five were no different, and one had a higher rate of revision than the previous system did. There was no improvement in the rate of revision for instability; seven new prosthesis systems showed no difference from the previous system and four new prosthesis systems had a higher rate of revision than the previous system did. A subanalysis of revision for patellofemoral complications showed improvement in two comparisons, no difference in six, and a higher revision rate in two; one could not be calculated because of an insufficient number of revisions for this reason. CONCLUSIONS: It is difficult to predict whether a new system will demonstrate better survival than a previous one, and widespread uptake of a new design before a benefit is shown in robust clinical studies is unwise. Similarly, adoption of a new system for which there is no difference in survivorship from a previous model may be premature because a new device may have associated unknown and unintended consequences. Healthcare policy makers and therapeutic device regulators should similarly be guided by results and seek out peer-reviewed evidence before accepting change to established practice. Surgeons must be aware that implant changes may not translate into better survivorship and must seek compelling evidence of improvement in survival and/or function before changing systems. LEVEL OF EVIDENCE: Level III, therapeutic study.

mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival.

Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

A mutant O-GlcNAcase enriches Drosophila developmental regulators.

Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. The Drosophila melanogaster gene supersex combs (sxc), which encodes OGT, is a polycomb gene, whose null mutants display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved and the underlying mechanisms linking these phenotypes to embryonic development are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and by limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, among others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.

Outcomes of a 1-year longitudinal integrated medical clerkship in small rural Victorian communities.

INTRODUCTION: Access to medical services for rural communities is poorer than for metropolitan communities in many parts of the world. One of the strategies to improve rural medical workforce has been rural clinical placements for undergraduate medical students. This study explores the workforce outcomes of one model of such placements - the longitudinal integrated clerkship (LIC) - delivered in year 4, the penultimate year of the medical course, as part of the rural programs delivered by a medical school in Victoria, Australia. The LIC involved student supervision under a parallel consulting model with experienced rural generalist doctors for a whole year in small community rural general practices. METHODS: This study aimed to compare the work locations (regional or more rural), following registration as a medical practitioner, of medical students who had completed 1 year of the LIC, with, first, students who had other types of rural training of comparable duration elsewhere, and second, students who had no rural training. Study participants commenced their medical degree after 2004 and had graduated between 2008 and 2016 and thus were in postgraduate year 1-9 in 2017 when evaluated. Information about the student training location(s), and duration, type and timing of training, was prospectively collected from university administrative systems. The outcome of interest was the main work location in 2017, obtained from the Australian Health Practitioner Regulation Agency's public website. RESULTS: Students who had undertaken the year 4 LIC along with additional rural training in years 3 and/or 5 were more likely than all other groups to be working in smaller regional or rural towns, where workforce need is greatest (relative risk ratio (RRR) 5.62, 95% confidence interval (CI) 2.81-11.20, compared with those having metropolitan training only). Non-LIC training of similar duration in rural areas was also significantly associated, but more weakly, with smaller regional work location (RRR 2.99, 95%CI 1.87-4.77). Students whose only rural training was the year 4 LIC were not significantly associated with smaller regional work location (RRR 1.72, 95%CI 0.59-5.04). Overall, after accounting for both LIC and non-LIC rural training exposure, rural work after graduation was also consistently positively associated with rural background, being an international student and having a return of service obligation under a bonded program as a student. CONCLUSION: This study demonstrates the value of rural LICs, coupled with additional rural training, in contributing to improving Australia's medical workforce distribution. Whilst other evidence has already demonstrated positive educational outcomes for doctors who participate in rural LIC placements, this is the first known study of work location outcomes. The study provides evidence that expanding this model of rural undergraduate education may lead to a better geographically distributed medical workforce.

SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes.

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2­CRTC2­HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP­PKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes.