De novo variants in GABRA4 are associated with a neurological phenotype including developmental delay, behavioral abnormalities and epilepsy.

Nine out of 19 genes encoding GABAA receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders. Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. However, no new cases have been reported since then. Through an international collaboration, we collected molecular and phenotype data of individuals carrying de novo variants in GABRA4. Patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. All variants within the transmembrane domain, including the previously reported p.(Thr300Ile) variant, were characterized in silico and analyzed by molecular dynamics (MD) simulation studies. We identified three novel de novo missense variants in GABRA4 (NM_000809.4): c.797 C > T, p.(Pro266Leu), c.899 C > A, p.(Thr300Asn), and c.634 G > A, p.(Val212Ile). The p.(Thr300Asn) variant impacts the same codon as the previously reported variant p.(Thr300Ile) and likely arose post-zygotically as evidenced by sequencing oral mucosal cells. Overlapping phenotypes among affected individuals included developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. Taken together, our findings further corroborate an association between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.

Health and illness beliefs in adults with tuberculosis infection during the COVID-19 pandemic in the UK.

Background: COVID-19 disrupted the TB prevention programme in the UK, especially for TB infection (TBI) care. We explore whether experience of the COVID-19 pandemic impacted on patients' perceptions of TBI and its treatment. Methods: Semi-structured interviews were conducted as part of the Research to Improve Detection and Treatment of TBI (RID-TB) programme, exploring perceptual and practical barriers to TBI treatment. Nineteen people diagnosed with TBI were interviewed between August 2020 and April 2021. Recordings were transcribed and analysed using a constant comparative approach, allowing for a dynamic and iterative exploration of themes. Themes are organised using the Perceptions and Practicalities Approach. Findings: Some participants perceived TBI as a risk factor for increased susceptibility to COVID-19, while some thought that treatment for TBI might protect against COVID-19 or mitigate its effects. Adaptations to TB services (e.g., remote follow-up) and integrated practices during the COVID-19 restrictions (e.g., medication being posted) addressed some practical barriers to TBI treatment. However, we identified beliefs about TBI and COVID-19 that are likely to act as barriers to engagement with TBI treatment, including: interpreting service delays as an indication of TBI not being serious enough for treatment and concerns about contracting COVID-19 in TB clinics. Interpretation: COVID-19 and TBI service delays influence people's perceptions and practical barriers to TBI treatment adherence. Failure to address these beliefs may lead to people's concerns about their treatment not being fully addressed. Utilised service adaptations like remote consultations to address practical barriers may be relevant beyond COVID-19. Funding: NIHR RID-TB Program (RP-PG-0217-20009).

Free energy profile of the substrate-induced occlusion of the human serotonin transporter.

The serotonin transporter (SERT) is a member of the Solute Carrier 6 (SLC6) family and is responsible for maintaining the appropriate level of serotonin in the brain. Dysfunction of SERT has been linked to several neuropsychiatric disorders, including depression, anxiety and obsessive-compulsive disorder. Therefore, an in-depth understanding of the mechanism on an atomistic level, coupled with a quantification of transporter dynamics and the associated free energies is required. Here, we constructed Markov state models (MSMs) from extensive unbiased molecular dynamics simulations to quantify the free energy profile of serotonin (5HT) triggered SERT occlusion and explored the driving forces of the mechanism of occlusion. Our results reveal that SERT occludes via multiple intermediate conformations and show that the motion of occlusion is energetically downhill for the 5HT-bound transporter. Force distribution analyses show that the interactions of 5HT with the bundle domain are crucial. During occlusion, attractive forces steadily increase and pull on the bundle domain, which leads to SERT occlusion. Some interactions become repulsive upon full occlusion, suggesting that SERT creates pressure on 5HT to promote its movement towards the cytosol.

A Practical Guide to Identify Patients With Multifocal Motor Neuropathy, a Treatable Immune-Mediated Neuropathy.

Multifocal motor neuropathy (MMN) is a rare immune-mediated motor neuropathy characterized by asymmetric weakness that preferentially affects distal upper limb muscles. The clinical features of MMN may be difficult to differentiate from motor neuron disease. Other conditions that may be mistaken for MMN include inclusion body myositis, chronic inflammatory demyelinating polyradiculoneuropathy, hereditary neuropathy with liability to pressure palsy, focal neuropathies, and radiculopathies. A key distinguishing electrophysiologic feature of MMN is the motor nerve conduction block located at noncompressible sites. MMN is a treatable neuropathy; therefore it is important that primary care physicians are aware of the features of the disease to identify potential patients and make referrals to a neuromuscular specialist in a timely manner. This review provides an overview of the disease, highlights key differential diagnoses, and describes available treatment options for patients with MMN.

Probing binding and occlusion of substrate in the human creatine transporter-1 by computation and mutagenesis.

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane-embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148-creatine-Na+ is essential to the process of occlusion, which occurs via a "hold-and-pull" mechanism. The model should be useful to study the impact of disease-associated point mutations on the folding of CRT1 and identify approaches which correct folding-deficient mutants.

Perceptions and Experiences of High-Intensity Interval Training in Kidney Transplant Recipients: A Big HIIT?

High-intensity interval training (HIIT) is considered a novel and time-efficient method to reduce cardiovascular disease risk, a leading cause of mortality in kidney transplant recipients. However, research in this population is severely limited. The aim of this study was to understand kidney transplant recipients' perceptions and experiences of HIIT and their readiness to participate in HIIT. Individual, semi-structured interviews were conducted in adults with a kidney transplant (n = 13; 53±13 years). Interviews were audiorecorded, transcribed verbatim, and subjected to framework analysis. Overall, participants had a good knowledge of HIIT and were open to participation. Acknowledgment of the superior benefits to cardiovascular, mental, and general health, as well as the lower time commitment, were all motivators for participation. There were some heightened concerns around damaging the kidney and 'knowing your limits. Personalization, physician's approval, and supervision were all important factors in participation. This study provides evidence that HIIT would be, in principle, largely accepted by recipients of a kidney transplant. However, several considerations are also identified in the present study, which would be essential to the success of any future efficacy trial or rehabilitation program.

Microstructural Pattern Formation during Far-from-Equilibrium Alloy Solidification.

We introduce a new phase-field formulation of rapid alloy solidification that quantitatively incorporates nonequilibrium effects at the solid-liquid interface over a very wide range of interface velocities. Simulations identify a new dynamical instability of dendrite tip growth driven by solute trapping at velocities approaching the absolute stability limit. They also reproduce the formation of the widely observed banded microstructures, revealing how this instability triggers transitions between dendritic and microsegregation-free solidification. Predicted band spacings agree quantitatively with observations in rapidly solidified Al-Cu thin films.

Exploring TRPC3 Interaction with Cholesterol through Coarse-Grained Molecular Dynamics Simulations.

Transient receptor potential canonical 3 (TRPC3) channel belongs to the superfamily of transient receptor potential (TRP) channels which mediate Ca2+ influx into the cell. These channels constitute essential elements of cellular signalling and have been implicated in a wide range of diseases. TRPC3 is primarily gated by lipids and its surface expression has been shown to be dependent on cholesterol, yet a comprehensive exploration of its interaction with this lipid has thus far not emerged. Here, through 80 µs of coarse-grained molecular dynamics simulations, we show that cholesterol interacts with multiple elements of the transmembrane machinery of TRPC3. Through our approach, we identify an annular binding site for cholesterol on the pre-S1 helix and a non-annular site at the interface between the voltage-sensor-like domain and pore domains. Here, cholesterol interacts with exposed polar residues and possibly acts to stabilise the domain interface.

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state.

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca2+ signaling function. Single particle cryo-EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure-guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG-probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C-DAG pathway.

A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus.

Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly silenced by the host genome, they have been found to be activated in multiple disease states, such as auto-inflammatory disorders and neurological diseases. However, the numerous copies in mammalian genomes and the lack of tools to study them make defining their role in health and diseases challenging. In this study, we identified eight copies of the zebrafish endogenous retrovirus zferv. We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single-cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases.

Perceived barriers and facilitators to exercise in kidney transplant recipients: A qualitative study.

BACKGROUND: Exercise has the potential to attenuate the high levels of cardiovascular morbidity and mortality present in kidney transplant recipients (KTRs). Despite this, activity levels in KTRs remain low. The aim of this qualitative study was to explore the barriers and facilitators of exercise in KTRs. METHODS: Thirteen KTRs (eight males; mean ± SD; age 53 ± 13 years; estimated glomerular filtration rate 53 ± 21 ml/min/1.73 m2 ) were recruited and completed semistructured one-to-one interviews at University Hospitals of Leicester NHS Trust. All KTRs were eligible if their kidney transplant was completed >12 weeks before interview and their consultant considered them to have no major contraindications to exercise. All interviews were audio recorded, transcribed verbatim and subject to framework analysis to identify and report themes. RESULTS: Themes were organized into personal, behavioural and environmental factors based on social cognitive theory. Facilitators of exercise were largely internal: enjoyment, exercise for general health and health of the transplanted kidney and desire to maintain normality. Social interaction, support and guidance of healthcare professionals and goal setting were perceived as motivational. Harming the kidney, a lack of guidance, self-motivation and accessibility were barriers to exercise. CONCLUSION: These results provide detailed insight into the development of interventions designed to increase physical activity in KTRs. They provide strong evidence that specific exercise guidelines are required for this population and that the healthcare system could have a key role in supporting KTRs to become more physically active. Interventions need to be multifaceted to appeal to the differing levels of support desired by KTRs. PATIENT OR PUBLIC CONTRIBUTION: KTRs were involved in the development of the interview topic guide to ensure all relevant topics were explored.

Evaluating the effect of short-course rifapentine-based regimens with or without enhanced behaviour-targeted treatment support on adherence and completion of treatment for latent tuberculosis infection among adults in the UK (RID-TB: Treat): protocol for an open-label, multicentre, randomised controlled trial.

INTRODUCTION: The successful scale-up of a latent tuberculosis (TB) infection testing and treatment programme is essential to achieve TB elimination. However, poor adherence compromises its therapeutic effectiveness. Novel rifapentine-based regimens and treatment support based on behavioural science theory may improve treatment adherence and completion. METHODS AND ANALYSIS: A pragmatic multicentre, open-label, randomised controlled trial assessing the effect of novel short-course rifapentine-based regimens for TB prevention and additional theory-based treatment support on treatment adherence against standard-of-care. Participants aged between 16 and 65 who are eligible to start TB preventive therapy will be recruited in England. 920 participants will be randomised to one of six arms with allocation ratio of 5:5:6:6:6:6: daily isoniazid +rifampicin for 3 months (3HR), routine treatment support (control); 3HR, additional treatment support; weekly isoniazid +rifapentine for 3 months (3HP), routine treatment support; weekly 3HP, additional treatment support ; daily isoniazid +rifapentine for 1 month (1HP), routine treatment support; daily 1HP, additional treatment support. Additional treatment support comprises reminders using an electronic pillbox, a short animation, and leaflets based on the perceptions and practicalities approach. The primary outcome is adequate treatment adherence, defined as taking ≥90% of allocated doses within the pre-specified treatment period, measured by electronic pillboxes. Secondary outcomes include safety and TB incidence within 12 months. We will conduct process evaluation of the trial interventions and assess intervention acceptability and fidelity and mechanisms for effect and estimate the cost-effectiveness of novel regimens. The protocol was developed with patient and public involvement, which will continue throughout the trial. ETHICS AND DISSEMINATION: Ethics approval has been obtained from The National Health Service Health Research Authority (20/LO/1097). All participants will be required to provide written informed consent. We will share the results in peer-reviewed journals. TRIAL REGISTRATION NUMBER: EudraCT 2020-004444-29.

Microstructure and Thickness Effects on Impact Behavior and Separation Formation in X70 Pipeline Steel.

In an effort to optimize the transportation of oil and gas, the pipeline industry is developing large-diameter, thick-walled pipelines that can withstand low temperatures and high pressures. In this study, three X70 steel plates of similar chemistry, ranging in thickness from 13.5 mm to 22 mm, were subjected to drop-weight tear and Charpy V-notch tests to determine the effects of plate thickness and microstructure on the formation of separations and impact behavior. Constraint induced by specimen thickness appears to dictate the location of separations, the three microstructures exhibited different separation behaviors, and microstructural banding was not found to promote separation formation. Separations were most frequent when the primary fracture plane was parallel to the rolling direction. This study also found that standardized empirical relationships between Charpy V-notch and drop-weight tear tests do not estimate to the advanced high-strength and -toughness steels investigated.

Feasibility, acceptability and effect of the Mindful Practice curriculum in postgraduate training of general practitioners.

BACKGROUND: Early career general practitioners are known to be at high risk of burnout. There is a need for widely applicable, cost-effective evidence-based interventions to develop trainees' protective skills and strategies. RESULTS: Of 120 eligible trainees, 23 (19.2%) expressed interest in participating, 17 subsequently started the course, and 15 completed at least 5 out of its 6 sessions. All psychological measures were stable for the six-week period prior to commencing the course. Following the course, there were statistically significant (p < 0.05) improvements in wellbeing, resilience, mindfulness, emotional exhaustion, disengagement, and stress scores. Participants described numerous benefits, and most stated that they would recommend it to colleagues. CONCLUSION: Including mindful practice within general practice vocational training is feasible, and in this study it benefited the psychological wellbeing of participants. Further research is needed to explore ways of increasing uptake and course completion, the sustainability of its effects, and the wider applicability of this approach.

"You have to change your whole life": A qualitative study of the dynamics of treatment adherence among adults with tuberculosis in the United Kingdom.

Maintaining adherence to treatment for tuberculosis (TB) is essential if the disease is to be eliminated. As part of formative research to develop an intervention to improve adherence, we documented the lived experiences of adults receiving anti-TB treatment (ATT) in three UK cities and examined how personal, social, and structural circumstances interacted to impact on individuals' adherence to treatment. Using a topic guide that explored social circumstances and experiences of TB care, we conducted in-depth interviews with 18 adults (six women) who were being or had been treated for TB (patients) and four adults (all women) who were caring for a friend, relative, or partner being treated for TB (caregivers). We analysed transcripts using an adapted framework method that classified factors affecting adherence as personal, social, structural, health systems, or treatment-related. Eleven of 18 patients were born outside the UK (in South, Central, and East Asia, and Eastern and Southern Africa); among the seven who were UK-born, four were Black, Asian, or Minority Ethnic and three were White British. TB and its treatment were often disruptive: in addition to debilitating symptoms and side effects of ATT, participants faced job insecurity, unstable housing, stigma, social isolation, worsening mental health, and damaged relationships. Those who had a strong support network, stable employment, a routine that could easily be adapted, a trusting relationship with their TB team, and clear understanding of the need for treatment reported finding it easier to adhere to ATT. Changes in circumstances sometimes had dramatic effects on an individual's ability to take ATT; participants described how the impact of certain acute events (e.g., the onset of side effects or fatigue, episodes of stigmatisation, loss of income) were amplified by their timing or through their interaction with other elements of the individual's life. We suggest that the dynamic and fluctuating nature of these factors necessitates comprehensive and regular review of needs and potential problems, conducted before and during ATT; this, coupled with supportive measures that consider (and seek to mitigate) the influence of social and structural factors, may help improve adherence.

Texture evolution as a function of scan strategy and build height in electron beam melted Ti-6Al-4V.

Metal additive manufacturing (AM) enables customizable, on-demand parts, allowing for new designs and improved engineering performance. Yet, the ability to control AM metal alloy microstructures (i.e., grain morphology, crystallographic texture, and phase content) is lacking. This work performs corroborative neutron diffraction and large-scale electron backscatter diffraction (EBSD) measurements to assess crystallographic texture in electron beam melted (EBM) Ti-6Al-4V as a function of scan strategy and build height. Texture components for one raster and two spot melt scan strategies were evaluated using a triclinic specimen symmetry to capture all possible texture components, which were found to be considerably different than previously reported values from studies employing orthotropic specimen symmetry. This finding highlights the importance of a standard method and best practice for assessing textures produced by AM. Texture was found to vary between scan strategies, but changed minimally as a function of build height. Parent phase ß-Ti reconstructions obtained from as-built crystallographic orientations revealed spot melt scan strategies produced finer equiaxed/columnar grains with clear 001 ß build direction fiber textures, whereas the raster scan strategy produced large columnar grains and a weaker 001 ß build direction fiber texture. The observed grain morphologies agree with those predicted by solidification theory for the thermal gradients and solidification velocities experienced during the build process. The presence of a strong 001 ß fiber orientation (typical of cubic solidification) produced by spot melting was found to correlate with a previously unreported 01 1 ¯ 2 α fiber texture in the as-built condition and colony microstructures. The 01 1 ¯ 2 α fiber texture was weakly observed for the raster scan strategy, and 001 ß oriented grains preferentially transformed into α' martensite with orientations between 1 1 ¯ 00 α and 11 2 ¯ 0 α . This shift in product α-Ti orientations has not yet been reported, and further work is recommended to understand these crystallographic signatures in the context of solid-state phase transformations. The presence of the 01 1 ¯ 2 α fiber texture is proposed as a useful diagnostic for evaluating the solidification or transformed microstructure condition (e.g., grain morphology and texture) of Ti-6Al-4V AM builds via accessible techniques like laboratory X-ray diffraction.

MAUD Rietveld Refinement Software for Neutron Diffraction Texture Studies of Single­ and Dual­Phase Materials.

This work presents a detailed instructional demonstration using the Rietveld refinement software MAUD for evaluating the crystallographic texture of single- and dual-phase materials, as applied to High-Pressure-Preferred-Orientation (HIPPO) neutron diffraction data obtained at Los Alamos National Laboratory (LANL) and electron backscatter diffraction (EBSD) pole figures on Ti-6Al-4V produced by additive manufacturing. This work addresses a number of hidden challenges intrinsic to Rietveld refinement and operation of the software to improve users' experiences when using MAUD. A systematic evaluation of each step in the MAUD refinement process is described, focusing on devising a consistent refinement process for any version of MAUD and any material system, while also calling out required updates to previously developed processes. A number of possible issues users may encounter are documented and explained, along with a multilayered assessment for validating when a MAUD refinement procedure is finished for any dataset. A brief discussion on appropriate sample symmetries is also included to highlight possible oversimplifications of the texture data extracted from MAUD. Included in the appendix of this work are two systematic walkthroughs applying the process described. Files for these walkthroughs can be found at the data repository located at: https://doi.org/10.18434/mds2-2400.

Prevalence and correlates of physical activity across kidney disease stages: an observational multicentre study.

BACKGROUND: People with chronic kidney disease (CKD) report high levels of physical inactivity, a major modifiable risk factor for morbidity and mortality. Understanding the biological, psychosocial and demographic causes of physical activity behaviour is essential for the development and improvement of potential health interventions and promotional initiatives. This study investigated the prevalence of physical inactivity and determined individual correlates of this behaviour in a large sample of patients across the spectrum of kidney disease. METHODS: A total of 5656 people across all stages of CKD (1-2, 3, 4-5, haemodialysis, peritoneal dialysis and renal transplant recipients) were recruited from 17 sites in England from July 2012 to October 2018. Physical activity was evaluated using the General Practice Physical Activity Questionnaire. Self-reported cardiorespiratory fitness, self-efficacy and stage of change were also assessed. Binominal generalized linear mutually adjusted models were conducted to explore the associations between physical activity and correlate variables. This cross-sectional observational multi-centre study was registered retrospectively as ISRCTN87066351 (October 2015). RESULTS: The prevalence of physical activity (6-34%) was low and worsened with disease progression. Being older, female and having a greater number of comorbidities were associated with greater odds of being physically inactive. Higher haemoglobin, cardiorespiratory fitness and self-efficacy levels were associated with increased odds of being active. Neither ethnicity nor smoking history had any effect on physical activity. CONCLUSIONS: Levels of physical inactivity are high across all stages of CKD. The identification of stage-specific correlates of physical activity may help to prioritize factors in target groups of kidney patients and improve the development and improvement of public health interventions.