Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1-stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

Ndel1 disfavors dynein-dynactin-adaptor complex formation in two distinct ways.

Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex." The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1-Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.

Predicting employment deterioration with the Processing Speed Test (PST) and SDMT in multiple sclerosis.

BACKGROUND: Employment deterioration is common in people with multiple sclerosis (PwMS). Clinicians often learn of job loss after its occurrence, leaving no opportunity for preventive measures. OBJECTIVES: Identify which neuropsychological measures discriminate between healthy volunteers (HVs) and employed/disabled PwMS at baseline and predict work deterioration over 2 years. METHODS: We examined 198 PwMS with computerized tests such as the Processing Speed Test (PST) and conventional tests such as the Symbol-Digit Modalities Test (SDMT), administered at baseline. Employment was assessed via Buffalo Vocational Monitoring Survey. Univariate and regression analyses identified significant predictors of PwMS categorized as work-stable versus work-deteriorated status. RESULTS: PwMS were impaired on all baseline assessments relative to HVs (p's < 0.001). Post hoc analyses showed that employed PwMS and HVs performed similarly and better than work-disabled PwMS. At the univariate level, both PST and SDMT discriminated between work-deteriorated and work-stable PwMS (p's < 0.01). The logistic regression model accounting for all measures retained PST and the computerized Walking Speed Test. PwMS with increased negative work events had lower PST (p < 0.001), SDMT (p < 0.001), and BVMT-R (p < 0.01) scores than stable PwMS. The related regression model retained PST and BVMT-R (p < 0.001). CONCLUSION: Cognition, as measured by the PST and BVMT-R, are predictive of job deterioration in PwMS and may be a useful screening tool to identify those at high risk of unemployment.

Auditory Test of Processing Speed: Preliminary validation of a smartphone-based test of mental speed.

BACKGROUND: The Symbol Digit Modalities Test (SDMT) is a gold-standard measure of cognitive efficiency and processing speed for people with multiple sclerosis (PwMS) but relies on vision and oculomotor function. OBJECTIVES: To develop and validate a new processing speed test with minimal memory involvement and no eye function requirements. METHODS: We created an Auditory Test of Processing Speed (ATOPS). A total of 122 PwMS, of whom 33 were severely disabled (median Expanded Disability Status Scale 8.0) and 37 healthy volunteers (HVs), were enrolled. We assessed sensitivity to discriminate MS participants from HVs, convergent validity between ATOPS and SDMT, sensitivity to discriminate between cognitively impaired (CI) and cognitively preserved (CP) MS participants, and correlations with MS pathology (overall brain lesion burden). Acceptability was examined with completion rates and participant ratings of ATOPS. RESULTS: ATOPS discriminated PwMS from HVs (d = 0.739-0.856), correlated with SDMT (|r| = 0.528-0.587), discriminated between CI and CP PwMS (d = 0.623-0.776), and correlated with lesion burden (r = 0.332-0.436). All groups indicated high favorability of ATOPS and severely disabled MS patients could be assessed by ATOPS more frequently than by SDMT (100% vs. 72.4% completion). CONCLUSIONS: ATOPS is a novel, accessible, and acceptable cognitive processing speed test that may be useful in clinical and/or research settings.

Half a century of the inverse care law: A comparison of general practitioner job satisfaction and patient satisfaction in deprived and affluent areas of Scotland.

BACKGROUND AND AIMS: The 'inverse care law', first described in 1971, results from a mismatch of healthcare need and healthcare supply in deprived areas. GPs in such areas struggle to cope with the high levels of demand resulting in shorter consultations and poorer patient outcomes. We compare recent national GP and patient satisfaction data to investigate the ongoing existence of this disparity in Scotland. METHODS AND RESULTS: Secondary analysis of cross-sectional national surveys (2017/2018) on upper and lower deprivation quintiles. GP measures; job satisfaction, job stressors, positive and negative job attributes. Patient measures; percentage positive responses per practice on survey questions on access and consultation quality. GPs in high deprivation areas reported lower job satisfaction and positive job attributes, and higher job stressors and negative job attributes compared with GPs in low deprivation areas. Patients living in high deprivation areas reported lower satisfaction with access and consultation quality than patients in low deprivation areas. These differences in GP and patient satisfaction persisted after adjusting for confounding variables. CONCLUSIONS: Lower GP work satisfaction in deprived areas was mirrored by lower patient satisfaction. These findings add to the evidence that the inverse care law persists in Scotland, over 50 years after it was first described.

How do the working lives of general practitioners in rural areas compare with elsewhere in Scotland? Cross-sectional analysis of the Scottish School of Primary Care Survey.

INTRODUCTION: The shortage of GPs in Scotland is concerning, particularly in rural areas. There are many reasons why GPs are leaving general practice; however, satisfaction with working life is an important predictor of GP retention. The aim of this study was to compare the working lives and intentions to reduce work participation of rural GPs and GPs working elsewhere in Scotland. METHODS: Quantitative analysis of responses from a nationally representative survey of GPs in Scotland. GPs were classified as 'non-rural' or 'rural' and these groups were compared using univariate and multivariate statistical analysis on four domains of working lives (job satisfaction, job stressors and positive and negative job attributes) and four intentions to reduce work participation (reducing working hours, working abroad, leaving direct patient care and leaving medical work entirely). RESULTS: There were significant differences in characteristics between rural and non-rural GPs. After controlling for these differences, GP age and gender, rural GPs reported higher job satisfaction, lower job stressors, higher positive job attributes, and lower negative job attributes than GPs elsewhere. A significant interaction between gender and rurality was found for job satisfaction, indicating that it was rural female GPs who were more satisfied. Rural GPs were, however, more likely to intend to work abroad and leave medical work entirely within 5 years than other GPs. DISCUSSION: These findings corroborate research from around the world and have serious implications for the future care of patients in rural areas. Further research is urgently required to understand the drivers of these findings.

How do the working lives of general practitioners in rural areas compare with elsewhere in Scotland? Cross-sectional analysis of the Scottish School of Primary Care National GP Survey.

INTRODUCTION: Like many countries around the world, Scotland faces a shortage of general practitioners (GPs) due to both recruitment and retention issues. Such workforce shortages are of particular concern in rural areas. There are many reasons why GPs are leaving general practice; however, satisfaction with working life is an important predictor of GP retention. It is important, therefore, to understand working life satisfaction of rural GPs. The purpose of this study was to compare the working lives and intentions to reduce work participation of rural GPs and GPs working elsewhere in Scotland. METHODS: This study was a quantitative analysis of survey data from the Scottish School of Primary Care national working lives survey. GPs were classified as working in 'non-rural' or 'rural' practices based on the Scottish Government's rural binary classification system, and were compared using univariate and multivariate statistical analysis on four domains of working lives: job satisfaction, job stressors, positive and negative job attributes, and four intentions to reduce work participation: reducing working hours, working abroad, leaving direct patient care and leaving medical work entirely. RESULTS: A total of 2465 GPs returned the survey, giving a response rate of 56%. Three-hundred and forty seven GPs who returned the survey worked in practices in rural areas (14.1%). Rural GPs were more likely to do out-of-hours work (p<0.001), to have worked in their practice for fewer years (p=0.014), to work in single-GP partnerships (p<0.001), and to work in practices with smaller list sizes (p<0.001), than GPs in non-rural settings. Compared with GPs elsewhere, rural GPs reported higher mean job satisfaction (5.23 v 5.39, respectively; p<0.005), lower mean job stressors (3.58 v 3.29; p<0.001) and lower mean negative job attributes (4.08 vs 3.78; p<0.001). These differences remained highly significant after controlling for potential confounders (age, gender and the differences in work practices shown above). In regression analysis, a significant interaction was found between gender and rurality for job satisfaction (p=0.008), which indicated that rural female GPs' higher job satisfaction mainly accounted for rural GPs' increased job satisfaction. No significant interaction was found between gender and rurality for the other domains of working lives. Compared with GPs elsewhere, however, rural GPs were more likely to intend to work abroad (mean 1.39 v 1.55; p=0.013) and leave medical work entirely within 5 years (mean 2.15 v 2.36; p=0.039). These intentions remained significant after controlling for potential confounders. No significant interaction was found between gender and rurality for variables for intentions to reduce work participation. CONCLUSION: Rural GPs in Scotland are more satisfied with their working lives than GPs working elsewhere in Scotland, which is mainly due to higher job satisfaction in female GPs in rural areas. Despite this, rural GPs as a whole have a higher intention to leave their job in the next 5 years than their non-rural counterparts. Although some of these differences are small, they may signal serious implications for the future care of patients in rural areas and require further research to understand the drivers of this.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Compassion, medical humanities and medical education.

Compassion is central to person centred clinical care. Medical Humanities have developed both as an academic discipline and as a resource for education over the past 30 years. The article will discuss what compassion is in a clinical context, and explore whether compassionate clinical practice can be enhanced by using the humanities in medical education in primary care.

An evaluation of experiences and views of Scottish leadership training opportunities amongst primary care professionals.

AIM: To determine experiences of leadership training of six primary care professions in Scotland and consider future development. METHODS: A questionnaire on previous leadership course attendance and future intentions was distributed to community pharmacists, general dental practitioners, general practitioners, practice nurses, practice managers and optometrists. Analysis comprised descriptive statistics for closed questions and management of textual data. RESULTS: Formal leadership training participation was fairly low except for practice managers. Leadership was perceived to facilitate development of staff, problem-solving and team working. Preference for future delivery was similar across the six professions with e-modules and small group learning being preferred. Time and financial pressures to undertake courses were common barriers for professionals. CONCLUSION: Leadership is key to improve quality, safety and efficiency of care and help deliver innovative services and transformative change. To date, leadership provision for primary care professionals has typically been patchy, uni-disciplinary in focus and undertaken outwith work environments. Future development must reflect needs of busy primary care professionals and the reality of team working to deliver integrated services at local level.

A phase I randomized study to evaluate safety, pharmacokinetics, and pharmacodynamics of SIR2446M, a selective RIPK1 inhibitor, in healthy participants.

Activation of receptor-interacting protein kinase 1 (RIPK1), a broadly expressed serine/threonine protein kinase, by pro-inflammatory cytokines and pathogens can result in apoptosis, necroptosis, or inflammation. RIPK1 inhibition has been shown to reduce inflammation and cell damage in preclinical studies and may have therapeutic potential for degenerative and inflammatory diseases. SIR2446 is a potent and selective novel small molecule RIPK1 kinase inhibitor. This phase I, randomized, double-blind, placebo-controlled study in Australia (ACTRN12621001621808) evaluated the safety (primary objective), pharmacokinetics, and pharmacodynamics of single (3-600 mg) and multiple (5-400 mg for 10 days) ascending oral doses of SIR2446M (SIR2446 magnesium salt form) in healthy adults from Nov 24, 2021, until May 01, 2023. All treatment-emergent adverse events (TEAEs) were mild/moderate. The most reported TEAEs were vascular access site pain, headache, and rash morbilliform. SIR2446M plasma half-lives ranged from 11 to 19 h and there were no major deviations from dose proportionality for maximum concentration and area under the curve across doses. Renal excretion of unchanged SIR2446 was minimal. No marked accumulation was observed (mean accumulation ratio, 1.2-1.6) after multiple daily doses. A high-fat meal mildly reduced the exposure but was not considered clinically significant. SIR2446M had a rapid and sustained inhibitory effect on the activity of RIPK1, with an overall 90% target engagement at repeated doses ranging from 30 to 400 mg in peripheral blood mononuclear cells ex vivo stimulated to undergo necroptosis. The favorable safety, pharmacokinetic, and pharmacodynamic profile of SIR2446M in healthy participants supports its further clinical development in patients with degenerative and inflammatory diseases.

CCSer2 gates dynein activity at the cell periphery.

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dynein's trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

Storylines of family medicine V: ways of thinking-honing the therapeutic self.

Storylines of Family Medicine is a 12-part series of thematically linked essays with accompanying illustrations that explore the many dimensions of family medicine, as interpreted by individual family physicians and medical educators in the USA and elsewhere around the world. In 'V: ways of thinking-honing the therapeutic self', authors present the following sections: 'Reflective practice in action', 'The doctor as drug-Balint groups', 'Cultivating compassion', 'Towards a humanistic approach to doctoring', 'Intimacy in family medicine', 'The many faces of suffering', 'Transcending suffering' and 'The power of listening to stories.' May readers feel a deeper sense of their own therapeutic agency by reflecting on these essays.

Erratum: 210 Preliminary Validation of the Arabic Global Neuropsychological Assessment (GNA) - ERRATUM.

[This corrects the article DOI: 10.1017/cts.2023.283.].

Ndel1 modulates dynein activation in two distinct ways.

Dynein is the primary minus-end-directed microtubule motor [1]. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex" [2, 3]. The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and adaptor [4, 5]. Ndel1 and its orthologue Nde1 are dynein and Lis1 binding proteins that help control where dynein localizes within the cell [6]. Cell-based assays suggest that Ndel1/Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear [6]. Using purified proteins and quantitative binding assays, we found that Ndel1's C-terminal region contributes to binding to dynein and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in Ndel1's C-terminal domain increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.

Evaluating primary care transformation: synthesis of findings from UK pilot project reviews.

BACKGROUND: Pilot 'new models' of primary care have been funded across the UK since 2015, through various national transformation funds. Reflections and syntheses of evaluation findings provide an additional layer of insight into 'what works' in transforming primary care. AIM: To identify good practice in policy design, implementation, and evaluation for primary care transformation. DESIGN & SETTING: A thematic analysis of existing pilot evaluations in England, Wales, and Scotland. METHOD: Ten studies presenting evaluations of three national pilot studies - the Vanguard programme in England, the Pacesetter programme in Wales, and the National Evaluation of New Models of Primary Care in Scotland - were thematically analysed, and findings synthesised in order to identify lessons learnt and good practice. RESULTS: Common themes emerged across studies in all three countries at project and policy level, which can support or inhibit new models of care. At project level, these included the following: working with all stakeholders, including communities and front-line staff; providing the time, space, and support necessary for the project to succeed; agreeing on clear objectives from the outset; and support for data collection, evaluation, and shared learning. At policy level, more fundamental challenges related to the parameters for pilot projects, in particular, the typically short-term nature of funding, with an expectation of results within 2-3 years. Changing expectations about outcome measures or project guidance part-way through project implementation was also identified as a key challenge. CONCLUSION: Primary care transformation requires coproduction and a rich, contextual understanding of local needs and complexities. However, a mismatch between policy objectives (care redesign to better meet patient needs) and policy parameters (short timeframes) is often a significant challenge to success.

Distinct dynein complexes defined by DYNLRB1 and DYNLRB2 regulate mitotic and male meiotic spindle bipolarity.

Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.

Challenges in implementing GP clusters in Scotland: a qualitative study comparing the views of senior primary care stakeholders in 2016 with those in 2021.

BACKGROUND: Formation of GP clusters began in Scotland in April 2016 as part of a new Scottish GP contract. They aim to improve the care quality for local populations (intrinsic role) and the integration of health and social care (extrinsic role). AIM: To compare predicted challenges of cluster implementation in 2016 with reported challenges in 2021. DESIGN & SETTING: Qualitative study of senior national stakeholders in primary care in Scotland. METHOD: Qualitative analysis of semi-structured interviews with 12 senior primary care national stakeholders in 2016 (n = 6) and 2021 (n = 6). RESULTS: Predicted challenges in 2016 included balancing intrinsic and extrinsic roles, providing sufficient support, maintaining motivation and direction, and avoiding variation between clusters. Progress of clusters in 2021 was perceived as suboptimal and was reported to vary significantly across the country, reflecting differences in local infrastructure. Practical facilitation (data, administrative support, training, project improvement support, and funded time) and strategic guidance from the Scottish Government was felt to be lacking. GP engagement with clusters was felt to be hindered by the significant time and workforce pressures facing primary care. These barriers were considered as collectively contributing to cluster lead 'burnout' and loss of momentum, exacerbated by inadequate opportunities for shared learning between clusters across Scotland. Such barriers preceded, but were perpetuated by, the impact of the COVID-19 pandemic. CONCLUSION: Apart from the COVID-19 pandemic, many of the challenges reported by stakeholders in 2021 were predicted in 2016. Accelerating progress in cluster working will require renewed investment and support applied consistently across the country.

HIV-1 binds dynein directly to hijack microtubule transport machinery.

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.