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Dominique A Cadilhac,1,2 Doreen Busingye,1 Jonathan C Li,3 Nadine E Andrew,1,4 Monique F Kilkenny,1,2 Amanda G Thrift,1 Vincent Thijs,2,5 Maree L Hackett,6,7 Ian Kneebone,8 Natasha A Lannin,9,10 Alana Stewart,11 Ida Dempsey,12 Jan Cameron,1,13 1Stroke and Ageing Research, Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Melbourne, VIC, Australia; 2Stroke Division, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC, Australia; 3Department of Electrical and Computer Systems Engineering, Monash University, Melbourne, VIC, Australia; 4Department of Medicine, Central Clinical School, Monash University, Melbourne, VIC, Australia; 5Department of Neurology, Austin Health, Melbourne, VIC, Australia; 6The George Institute for Global Health, University of New South Wales, Sydney, NSW, Australia; 7Faculty of Health and Wellbeing, University of Central Lancashire, Preston, UK; 8Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia; 9College of Science, Health and Engineering, School of Allied Health, La Trobe University, Melbourne, VIC, Australia; 10Occupational Therapy Department, Alfred Health, Melbourne, VIC, Australia; 11Stroke Foundation, Melbourne, VIC, Australia; 12Consumer Representative, Melbourne, VIC, Australia; 13Australian Centre for Heart Health, Royal Melbourne Hospital, Melbourne, VIC, Australia Purpose: Worldwide, stroke is a leading cause of disease burden. Many survivors have unmet needs after discharge from hospital. Electronic communication technology to support post-discharge care has not been used for patients with stroke. In this paper, we describe the development of a novel electronic messaging system designed for survivors of stroke to support their goals of recovery and secondary prevention after hospital discharge. Participants and methods: This was a formative evaluation study. The design was informed by a literature search, existing data from survivors of stroke, and behavior change theories. We established two working groups; one for developing the electronic infrastructure and the other (comprising researchers, clinical experts and consumer representatives) for establishing the patient-centered program. Following agreement on the categories for the goal-setting menu, we drafted relevant messages to support and educate patients. These messages were then independently reviewed by multiple topic experts. Concurrently, we established an online database to capture participant characteristics and then integrated this database with a purpose-built messaging system. We conducted alpha testing of the approach using the first 60 messages. Results: The initial goal-setting menu comprised 26 subcategories. Following expert review, another 8 goal subcategories were added to the secondary prevention category: managing cholesterol; smoking; physical activity; alcohol consumption; weight management; medication management; access to health professionals, and self-care. Initially, 455 health messages were created by members of working group 2. Following refinement and mapping to different goals by the project team, 980 health messages across the health goals and 69 general motivational messages were formulated. Seventeen independent reviewers assessed the messages and suggested adding 73 messages and removing 16 (2%). Overall, 1,233 messages (18 administrative, 69 general motivation and 1,146 health-related) were created. Conclusion: This novel electronic self-management support system is ready to be pilot tested in a randomized controlled trial in patients with stroke. Keywords: stroke, e-health, self-management, health promotion, secondary prevention

Nadine E Andrew,1 Monique F Kilkenny,1,2 Rebecca Naylor,3 Tara Purvis,1 Dominique A Cadilhac1,2 1Translational Public Health and Evaluation Division, Stroke and Ageing Research, School of Clinical Sciences at Monash Health, Monash University, Clayton, 2Florey Institute of Neuroscience and Mental Health, Heidelberg, 3National Stroke Foundation, Melbourne, VIC, Australia Background: Caregivers play a crucial role in meeting the needs of survivors of stroke. Yet, little is known about how they are impacted by their caregiving role. Objectives: To describe the relationship between survivor long-term unmet needs (>12 months) and caregiver impacts, and identify characteristics that are associated with reported moderate to severe impacts on caregivers. Method: This was a cross-sectional survey using data from the Australian Stroke Survivor and Carer Needs Survey. Community dwelling adults 12+ months poststroke and their caregivers participated. Caregivers and survivors were asked about the extent to which the domains of work, leisure and family, and friend and spousal relationships had been impacted using a Likert scale of responses. The extent to which survivor needs were being met was measured over the domains of health, everyday living, work, leisure, and finances, and the total number of unmet needs was calculated. The association between survivor unmet needs and caregiver impacts was assessed using multivariable logistic regression adjusted for caregiver and survivor characteristics. Results: Of the 738 completed survivor surveys, 369 contained matched caregiver data (survivors: median age, 71 years; 67% male) (caregivers: median age, 64 years; 26% male). For caregivers, the domains of work, leisure, and friendships were most impacted. The odds of a caregiver experiencing moderate to extreme impacts increased with the number of reported survivor unmet needs. This was greatest for spousal (aOR [adjusted odds ratio]: 1.14; 95% CI [confidence interval]: 1.07, 1.21; P

People with communication disabilities post-stroke have poor quality-of-life. We aimed to explore the association of self-reported communication disabilities with different dimensions of quality-of-life between 90 and 180 days post-stroke. Cross-sectional survey data were obtained between 90 and 180 days post-stroke from registrants in the Australian Stroke Clinical Registry recruited from three hospitals in Queensland. The usual follow-up survey included the EQ5D-3L. Responses to the Hospital Anxiety and Depression Scale, and extra questions (e.g. communication disabilities) were also collected. We used χ2 statistics to determine differences. Overall, 244/647 survivors completed the survey. Respondents with communication disabilities (n = 72) more often reported moderate to extreme problems in all EQ5D-3L dimensions, than those without communication disabilities (n = 172): anxiety or depression (74% vs 40%, p <.001), pain or discomfort (58% vs 39%, p =.006), self-care (46% vs 18%, p <.001), usual activities (77% vs 49%, p <.001), and mobility (68% vs 35%, p <.001). Respondents with communication disabilities reported less fatigue (66% vs 89%, p <.001), poorer cognitive skills (thinking) (16% vs 1%, p <.001) and lower social participation (31% vs 6%, p <.001) than those without communication disabilities. Survivors of stroke with communication disabilities are more negatively impacted across different dimensions of quality-of-life (as reported between 90 and 180 days post-stroke) compared to those without communication disabilities. This highlights the need for timely and on-going comprehensive multidisciplinary person-centered support. [ABSTRACT FROM AUTHOR]

Abstract Objectives To determine predictors of successful ictal single photon emission computed tomography (SPECT) injections during Epilepsy Monitoring Unit (EMU) admissions for patients undergoing presurgical evaluation for drug‐resistant focal epilepsy. Methods In this retrospective study, consecutive EMU admissions were analyzed at a single center between 2019 and 2021. All seizures that occurred during the admission were reviewed. “Injectable seizures” occurred during hours when the radiotracer was available. EMU‐level data were analyzed to identify factors predictive of an EMU admission with a successful SPECT injection (successful admission). Seizure‐level data were analyzed to identify factors predictive of an injectable seizure receiving a SPECT injection during the ictal phase (successful injection). A multivariate generalized linear model was used to identify predictive variables. Results 125 EMU admissions involving 103 patients (median 37 years, IQR 27.0–45.5) were analyzed. 38.8% of seizures that were eligible for SPECT (n = 134) were successfully injected; this represented 17.4% of all seizures (n = 298) that occurred during admission. Unsuccessful admissions were most commonly due to a lack of seizures during EMU‐SPECT (19.3%) or no injectable seizures (62.3%). Successful EMU‐SPECT was associated with baseline seizure frequency >1 per week (95% CI 2.1–3.0, P 1 per week, a PET hypometabolic focus, and a patient's ability to indicate seizure onset were identified as predictors of success. These findings may assist EMUs in optimizing their SPECT protocols, patient selection, and resource allocation.

Abstract Long intergenic non-coding RNAs (lincRNAs) are emerging as regulators of protein-coding genes (PCGs) in many plant and animal developmental processes and stress responses. In this study, we characterize the genome-wide lincRNAs in potatoes responsive to a vascular bacterial disease presumably caused by Candidatus Liberibacter solanacearum (CLso). Approximately 4397 lincRNAs were detected in healthy and infected potato plants at various stages of zebra chip (ZC) disease progression. Of them, ~65% (2844) were novel lincRNAs, and less than 1% (9) were orthologs of Arabidopsis and rice based on reciprocal BLAST analysis, suggesting species-specific expansion. Among the proximal lincRNAs within 50 kbp from a PCG, ~49% were transcribed from the same strand, while ~39% and ~15% followed convergent (head-to-head) and divergent (tail-to-tail) orientations, respectively. Approximately 30% (1308) were differentially expressed following CLso infection, with substantial changes occurring 21 days after infection (DAI). Weighted Gene Co-expression Network Analysis (WGCNA) of lincRNAs and PCGs identified 46 highly correlated lincRNA–PCG pairs exhibiting co-up or co-downregulation. Furthermore, overexpression of selected lincRNAs in transgenic potato hairy roots resulted in perturbation of neighboring PCG expression and conferred tolerance to CLso infection. Our results provide novel insights into potato lincRNAs’ identity, expression dynamics, and functional relevance to CLso infection.

Abstract Bacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognise and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognises cognate motifs has not been fully understood. In this study we characterise BREX from pathogenic Salmonella and present X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that both methyltransferase activity and defence require larger BREX complexes. Through rational engineering of PglX we broaden both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is used to recognise specific DNA sequences for BREX activity, contributing to motif recognition for both phage defence and host methylation.

Background Barth syndrome is a rare, life-threatening X-linked recessive mitochondrial disorder of lipid metabolism primarily affecting males. Previous research suggests that bezafibrate may ameliorate cellular lipid abnormalities and reduce cardiac dysfunction in an animal model. Objectives Estimate the effect of bezafibrate on clinical, biochemical, and quality-of-life outcomes. Investigate whether within-participant clinical changes parallel in vitro changes in cardiolipin ratio/profile and mitochondrial morphology when each participant’s cells are cultured with bezafibrate. Investigate as for objective 2, culturing each participant’s cells with resveratrol. Describe the most feasible methods and standardised outcome measures to optimise the conduct of future trials and evaluations in Barth syndrome. Describe features of the research infrastructure which optimised recruitment, retention and communication with families and people with Barth syndrome. Describe the perceptions of participants and their families about the research and any important potential barriers to participation. Design Randomised, placebo-controlled, crossover trial of bezafibrate versus placebo. Setting NHS hospital providing UK-wide Barth Syndrome Service. Participants Males aged ≥ 6 years with a confirmed diagnosis of Barth syndrome with stable cardiac status, able to swallow tablets of bezafibrate/placebo. Exclusions were: hypersensitivity or allergy to bezafibrate or any component of bezafibrate; hepatic, liver or renal dysfunction; gallbladder disease; or recent deterioration in general health. Interventions Fifteen weeks of bezafibrate in one period and placebo in a second period, or vice versa (randomly allocated), with at least a 1-month washout between periods. Main outcome measures The primary outcome was peak VO2; secondary outcomes were cardiac function rest and exercise echocardiography and magnetic resonance imaging, cardiolipin ratio, quality of life, dynamic skeletal muscle P-magnetic resonance spectroscopy, mitochondrial studies and neutrophil counts, and adverse events. Outcomes were measured at baseline and the end of each period. Results Eleven males were studied; all attended all three assessments. There was no difference in peak VO2 between periods (0.66 ml/kg/min lower with bezafibrate than placebo, 95% confidence interval 2.34 to 1.03; p = 0.43). There was a trend towards a higher left ventricular ejection fraction with bezafibrate when measured by echocardiography but not magnetic resonance imaging, and better echocardiography-derived rest longitudinal and circumferential strain with bezafibrate. There was no difference in quality of life or cardiolipin ratio between periods. Skeletal muscle 31P magnetic resonance spectroscopy was performed cross-sectionally and showed a trend to higher Tau and lower Qmax indices in the bazafibrate group. Two participants had serious, expected adverse reactions when taking bezafibrate; otherwise, bezafibrate was well tolerated. Limitations The sample size was very small; the bezafibrate dose may have been too low or 15 weeks too short to observe an effect; measurements of mitochondrial content and membrane potential were highly variable; P-magnetic resonance spectroscopy was available only at the final assessment. Conclusions This study did not show significant improvement in the primary and secondary outcomes with bezafibrate treatment. Future work Elamipretide, studied in a small crossover trial in the USA, is another potential intervention which may be worth evaluating in an international study. Trial registration This trial is registered as ISRCTN58006579. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 12/205/56) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 13. See the NIHR Funding and Awards website for further award information. Plain language summary Barth syndrome is a rare genetic syndrome with mutations in the tafazzin gene causing long-term effects in several organ systems, leading to significant lifelong comorbidities and a reduced quality of life. Barth syndrome shows an X-linked inheritance, hence it is almost exclusively present in males. Tafazzin gene mutations impair overall cell energy metabolism, and affected individuals show compromise of the immune system with a high risk for life-threatening infections, as well as cardiac dysfunction and cardiomyopathy, muscle and general fatigue, and generally reduced mobility also leading to psychological challenges and dysfunction. Currently, specific medical therapies are limited to symptomatic treatment, and drug therapies targeting the causative mitochondrial energy disturbance are urgently needed. This randomised controlled trial explored the use of oral bezafibrate, a licensed drug that can improve cell energy metabolism. Eleven male patients received either bezafibrate or a placebo for 4 months. Exercise capacity by cardiopulmonary exercise testing on a cycle ergometer, as the primary outcome measurement, as well as cardiac function by echocardiography and magnetic resonance imaging, and cellular anatomy and function by in vitro assays and microscopy, were measured before and after this 4-month period. Bezafibrate did not significantly improve exercise capacity, cardiac function or the cellular metabolism, although several cardiac function parameters showed slight improvements with bezafibrate treatment. No significant adverse effects were recorded. The limitations of the trial were its low participant number and the relatively short duration of the drug treatment phase. The trial did prove that complex randomised controlled drug trials are feasible in people with rare diseases. Adherence was good, with parents reporting few difficulties with pill swallowing. The qualitative interviews also found that the study was acceptable to participants and the burden of participating not too onerous. We hope the research can provide a template for future studies. Scientific summary Introduction Barth syndrome is a very rare, life-threatening, X-linked recessive genetic disease almost exclusively affecting young males, caused by abnormal lipids in mitochondria. The tafazzin gene (TAZ) encodes tafazzin, a phospholipid acyltransferase. Tafazzin transfers unsaturated fatty acids with acyl chains from phospholipids to monolysocardiolipin (MLCL) and regulates remodelling and maturation of cardiolipin, a phospholipid located exclusively in the inner mitochondrial membrane. Mature tetralinoleoyl-cardiolipin (L4-CL) is essential in the mitochondrial membrane for maintaining mitochondrial membrane potential, structural integrity of electron transport chain complexes and mitochondrial cristae architecture. Cardiolipin is a major constituent of inner mitochondrial membranes, and the major effects of the aberrant gene are on muscular tissues. Cardiac and skeletal manifestations of Barth syndrome are thought to result from impaired formation of respiratory chain super-complexes. TAZ mutations decrease L4-CL and increase intermediate species of MLCL, resulting in a marked increase in the MLCL : L4-CL ratio, which provides a highly sensitive and specific biochemical test for Barth syndrome. Barth syndrome is characterised by many gene mutations and profound phenotypic variability between affected patients within families and those in different families. The disease carries many risks and problems for its sufferers, as well as major healthcare costs. Almost one-third (30%) of living UK patients have undergone cardiac transplantation. Several patients who have died due to cardiac rejection or post-transplant lymphoproliferative disease had previously undergone cardiac transplantation. Other patients who have died deteriorated too fast to undergo transplantation or died of complications while awaiting transplantation. Neutropenia is another life-threatening issue. Two-thirds of UK patients are treated with granulocyte colony-stimulating factor by subcutaneous injection. Managing neutropenia in this disease is challenging, since patients have highly variable neutrophil counts, which prevents administration of a consistent daily dose and requires repeated blood counts and physician oversight. Exercise intolerance, lethargy and fatigue are universal, interfering with daily life, schoolwork and play and often necessitating use of a wheelchair. Patients cannot perform strenuous or demanding jobs. Many have major feeding problems from infancy through to adult life; for example, requiring feeding via gastrostomies. There can be rapid deterioration during periods of stable health despite expert medical care. Ventricular arrhythmia (tachycardia or fibrillation) affects 10% of adolescents and can cause sudden cardiac death at any time. There are no specific treatments for Barth syndrome. There is no evidence that standard medications for cardiomyopathy ameliorate long-term poor outcomes. Treatment is multidisciplinary and currently limited to treating intercurrent bacterial infections, bone marrow support, physiotherapy, nutritional support, management of heart failure and arrhythmia, and consideration for heart transplantation. Experiments using lymphoblasts from patients with Barth syndrome show that either bezafibrate or resveratrol can partially normalise the deranged cardiolipin ratio. This may be important, since some patients with Barth syndrome who lack neutropenia and tend to have good exercise tolerance have cardiolipin ratios intermediate between those of typical patients and normal individuals. Bezafibrate also improved left ventricular function and showed other benefits at supraphysiological doses in a TAZ knockdown mouse model. This trial was motivated by the need for disease-specific therapy to prevent morbidity, mortality, psychological distress, and disruption of quality of life (QoL) in affected patients and their families. An effective therapy also has the potential to generate savings for the NHS. Bezafibrate is a lipid-lowering drug used in adults and children, with a good safety record in long-term use. These characteristics made it a candidate for investigation. Bezafibrate has been reported to significantly ameliorate a mitochondrial myopathy in adults and shown promising results in several animal/human cellular models of mitochondrial disease. Several of the laboratory assays and clinical investigations proposed in this study were also considered potentially important beyond the scope of this specific study, to assess associations between genotype and phenotype and for future evaluations of therapies in Barth syndrome. Objectives Estimate the effect on clinical, biochemical and QoL outcome measures of bezafibrate compared to placebo. Investigate whether clinical improvements parallel in vitro changes in cardiolipin ratio and mitochondrial morphology in each participant’s cells when cultured with bezafibrate. Investigate whether clinical improvements and culture findings with bezafibrate parallel in vitro changes in each participant’s cells when exposed to resveratrol in laboratory culture. Describe the most feasible methods and standardised outcome measures for future trials. Describe features of the research infrastructure which optimised recruitment, retention and communication with participants and families. Describe participants’ and their families’ perceptions of research and important potential barriers to participation. Methods Study design and participants CARDIOlipin MANipulation trial (CARDIOMAN) was a double-blind, placebo-controlled crossover randomised trial. Treatment was given in two 15-week periods with a minimum of 1 month’s washout period between these when no treatment was given. Twenty-six males and one female with the disease were alive in the UK when the trial was designed. Males aged ≥ 6 years in the UK diagnosed with Barth syndrome were eligible. Exclusion criteria were: hypersensitivity to bezafibrate or component of the investigational medicinal product (IMP), photoallergic or phototoxic reaction to fibrate, unstable cardiac condition, cardiac chamber shortening fraction < 25% or significant decrease in shortening fraction in the preceding year, atrial or ventricular arrhythmia not stabilised by treatment, inability to swallow the IMP, hepatic dysfunction, renal impairment (creatinine clearance < 90 ml/min), gallbladder disease, recent unspecified significant deterioration in general health, prisoners and adults lacking capacity to provide informed consent. Randomisation and masking Participants were randomised to receive either bezafibrate then placebo, or the reverse order. Allocations were generated in advance by the Bristol Trials Centre and provided to the trial pharmacy which dispensed the IMP. All investigators, members of the research team (apart from pharmacy) and participants remained blind to treatment allocation. Intervention and procedures The intervention IMP was bezafibrate taken orally as 100-mg tablets. Children aged 6–9 years started on 100 mg once daily for the first 4 weeks and, if well tolerated, increased to 100 mg twice daily for the remaining 11 weeks. Children aged 10–17 years started on 200 mg once daily for the first 4 weeks and, if well tolerated, increased to 200 mg twice daily for the remaining 11 weeks. Adults (≥ 18 years) took 200 mg twice daily throughout. The comparator IMP was placebo taken orally as visually identical tablets and manufactured to be as similar as possible in taste and smell to the intervention. The IMP was prescribed at the start of the first period and again at the end of the first period for the second period. Outcomes All in vivo outcomes except magnetic resonance spectroscopy (MRS) were assessed three times: at baseline and in the final week of each treatment phase. The primary outcome measure was peak oxygen consumption on bicycle ergometry [peak volume of oxygen (VO2)]. Secondary outcomes were: MLCL/L4-CL ratio and cardiolipin profile in blood cells; phosphocreatine (PCr)/adenosine 5’ triphosphate (ATP) ratio in cardiac muscle on 31P MRS; skeletal muscle oxidative function/on 31P MRS; QoL [age-appropriate Pediatric Quality of Life Inventory (PedsQLTM) questionnaires]; absolute neutrophil count; amino acid expression (serum arginine and cysteine); cardiac function [left and right ventricular function: left ventricular ejection fraction (LVEF%), right ventricular ejection fraction (RVEF%); and 2-D longitudinal and circumferential strain at rest, peak exercise and after recovery]; mitochondrial size and number in lymphocytes; mitochondrial area in lymphocytes and as a proportion of cytoplasm area; mitochondrial function and cristae organisation in lymphocytes/neutrophils; arrhythmia profile from 12-lead electrocardiogram at rest and during exercise. Qualitative research methods explored participants’ and families’ experiences of the different interventions. Adverse events Serious adverse events (SAEs) and non-serious adverse events (AEs) were collected from participants from the time of consent until 1 month after the final period. Participants’ general practitioners were asked to inform the research team of suspected AEs. Participants were asked questions about SAEs/AEs at each research clinic visit and in monthly phone calls, including during the washout period. SAEs were reviewed by the Chief Investigator, who decided whether the SAE was related to bezafibrate. Statistical analysis A total of 20 males aged ≥ 6 years were potentially eligible. The sample size was dictated by the number of eligible boys willing to take part. The primary analysis estimated the difference in mean peak VO2 between placebo and bezafibrate periods (two-tailed 5% significance level). A sample size of 12 participants allowed the trial to detect a difference of 0.90 (within subject) standard deviations with 80% power. The statistical analysis plan (SAP) prespecified that all analyses should be conducted according to the intention-to-treat (ITT) principle. The ITT population comprised all participants and periods, according to the randomised allocations. The safety population comprised all randomised participants, according to the treatment received in each period, who received at least one dose of IMP. All statistical tests were two-sided and used a 5% significance level, except for interactions that used a 10% significance level. Treatment effects were estimated with 95% confidence intervals (CIs). No formal adjustment was made for multiple testing. The treatment effect for the primary outcome was analysed using mixed linear regression models, adjusting for period (fixed effect; exploring interactions where necessary) and participants (random effects). Model assumptions were tested using standard methods, and the carry-over effect was estimated by including treatment order. Treatment effects for continuously scaled secondary outcomes (prespecified in the SAP) were estimated and tested using the same methods. Results There was no statistically significant difference between bezafibrate and placebo in peak VO2, which was 0.66 ml/kg/min lower (95% CI −2.34 to 1.03; p = 0.43) with bezafibrate. There was a borderline statistically significant increase in LVEF% using echocardiography (3.72%, 95% CI −0.26% to 7.69%; p = 0.065) but no evidence of a difference when using magnetic resonance imaging (MRI) (0.15%, 95% CI −3.21% to 3.51%; p = 0.926). There was also no difference for RVEF% between bezafibrate and placebo (0.32, 95% CI −2.32 to 2.96; p = 0.801). Longitudinal and circumferential strain at rest were better (lower) with bezafibrate than with placebo; differences were –1.67 (95% CI −3.11 to −0.22; p = 0.026) and −2.72 (95% CI −5.03 to −0.40; p = 0.024) respectively. There was no difference in diastolic ratio at rest between bezafibrate and placebo, and no differences in strain or diastolic ratio at peak exercise or after 2 minutes’ recovery. All participants had sinus rhythm at rest and during exercise for all three time points. Magnetic resonance spectroscopy was only performed after the second period for 10 participants. PCr/ATP ratio in cardiac muscle was not collected. Cross-sectionally (five vs. five participants), MRS appeared to increase Tau and reduce Qmax parameters with bezafibrate compared to placebo. Monolysocardiolipin/L4-CL ratio did not differ between bezafibrate and placebo but deteriorated in the second period (20% higher, 95% CI 3% to 40%; p = 0.024). Neutrophil count also showed no difference between bezafibrate and placebo but increased in the second period (2.26, 95% CI 0.08 to 4.45; p = 0.043). Plasma cysteine increased with bezafibrate (32% higher, 95% CI 8% to 62%; p = 0.001) but plasma arginine did not (25% higher, 95% CI −10% to 72%; p = 0.180). Mitochondrial outcomes on blood samples did not show marked differences and were not formally compared. Quality-of-life scores were transformed to a 0–100 scale (higher being better). Bezafibrate had no effect on core (−3.41, 95% CI −8.67 to 1.86; p = 0.192) or fatigue (1.71, 95% CI −6.28 to 9.71; p = 0.659) QoL scores. Emerging themes identified in qualitative interviews were classified as overarching, related to lived experience with Barth syndrome, and trial participation and conduct. Participants and families were pleased to take part in the trial, viewing participation as ‘giving back’ to the medical community. No participant withdrew, and the trial was not perceived to be too onerous for families. Participating in the research gave both individuals and families a sense of responsibility for their condition, partly driven by appreciation for the Barth Syndrome Service and staff expertise. Monolysocardiolipin/L4-CL ratio in vitro was estimated from samples of cells incubated with control, bezafibrate or resveratrol. Each drug reduced the MLCL/L4-CL ratio, largely by increasing intermediate cardiolipin species, and more so with resveratrol than with bezafibrate. Ratios were nevertheless profoundly abnormal in comparison to patients with intermediate Barth phenotypes. Discussion Main findings: study results There was no clinically important or statistically significant improvement in peak VO2 with bezafibrate compared to placebo. There were inconsistent findings for LVEF% by method of measurement; echocardiography showed a borderline improvement that cardiac MRI did not demonstrate. Longitudinal and circumferential strain measurements were better at rest with bezafibrate, but not at peak exercise. There were no clinically important or statistically significant improvements in core or fatigue QoL domains. Plasma cysteine was significantly improved with bezafibrate treatment. Plasma arginine also increased but not significantly. Neutrophil count was unaffected and there was no significant change in cardiolipin ratio in participants’ blood with bezafibrate. There were no significant period effects except for MLCL/L4-CL ratio and neutrophil count, both increased in the second period. In vitro incubation of Epstein–Barr virus-transformed patient lymphoblasts with either bezafibrate or resveratrol resulted in improved MLCL/L4-CL ratios, an effect that was more profound with resveratrol. Treatment with this drug also resulted in a more obvious increase in intermediate MLCL species. Strengths and limitations Strengths of the trial are: randomisation and placebo blinding; crossover design; excellent retention; completeness of the data, good adherence and IMP tolerance. There were several limitations. The sample size (outside our control) was very small, and interpretation of effects for many secondary outcomes was challenging. Longer treatment duration or a higher bezafibrate dose might have produced better outcomes. Bezafibrate may have induced adverse reactions in some participants, inadvertently causing unblinding. The group of participants randomised to receive bezafibrate first was older than the group randomised to the opposite order, which may explain period effects. The electron microscopic and mitochondrial assessments proved problematic and were not completed in vitro. Measurements of mitochondrial content and membrane potential were highly variable. Amino acid levels were not assayed after fasting. Echocardiography images during exercise testing were often suboptimal. Lessons for the future Magnetic resonance spectroscopy in children is still exploratory, and protocols need to be better validated, but it is considered very promising as a potential objective functional outcome. Cardiolipin profile is not a good outcome for research, although it is appropriate for diagnosis because the Barth cardiolipin profile is so extremely different from a normal profile. Electron microscopy measurements of mitochondria should not be considered as outcomes in future trials. Peak VO2 should be the preferred current primary outcome because it is resistant to bias compared to other functional outcomes. Conclusion The trial did not show a significant treatment effect for the primary outcome, peak VO2. Some secondary outcomes such as systolic function measured by echocardiography and amino acid levels provided some evidence of benefit. The trial provided insights about trial methods and conduct in the context of a very rare paediatric disease that should inform future studies. Trial registration This trial is registered as ISRCTN58006579. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 12/205/56) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 13. See the NIHR Funding and Awards website for further award information.

Introduction Epilepsy surgery is the only curative treatment for patients with drug-resistant focal epilepsy. Stereoelectroencephalography (SEEG) is the gold standard to delineate the seizure-onset zone (SOZ). However, up to 40% of patients are subsequently not operated as no focal non-eloquent SOZ can be identified. The 5-SENSE Score is a 5-point score to predict whether a focal SOZ is likely to be identified by SEEG. This study aims to validate the 5-SENSE Score, improve score performance by incorporating auxiliary diagnostic methods and evaluate its concordance with expert decisions.Methods and analysis Non-interventional, observational, multicentre, prospective study including 200 patients with drug-resistant epilepsy aged ≥15 years undergoing SEEG for identification of a focal SOZ and 200 controls at 22 epilepsy surgery centres worldwide. The primary objective is to assess the diagnostic accuracy and generalisability of the 5-SENSE in predicting focality in SEEG in a prospective cohort. Secondary objectives are to optimise score performance by incorporating auxiliary diagnostic methods and to analyse concordance of the 5-SENSE Score with the expert decisions made in the multidisciplinary team discussion.Ethics and dissemination Prospective multicentre validation of the 5-SENSE score may lead to its implementation into clinical practice to assist clinicians in the difficult decision of whether to proceed with implantation. This study will be conducted in accordance with the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans (2014). We plan to publish the study results in a peer-reviewed full-length original article and present its findings at scientific conferences.Trial registration number NCT06138808.

Accurate resection cavity segmentation on MRI is important for neuroimaging research involving epilepsy surgical outcomes. Manual segmentation, the gold standard, is highly labour intensive. Automated pipelines are an efficient potential solution; however, most have been developed for use following temporal epilepsy surgery. Our aim was to compare the accuracy of four automated segmentation pipelines following surgical resection in a mixed cohort of subjects following temporal or extra temporal epilepsy surgery. We identified 4 open-source automated segmentation pipelines. Epic-CHOP and ResectVol utilise SPM-12 within MATLAB, while Resseg and Deep Resection utilise 3D U-net convolutional neural networks. We manually segmented the resection cavity of 50 consecutive subjects who underwent epilepsy surgery (30 temporal, 20 extratemporal). We calculated Dice similarity coefficient (DSC) for each algorithm compared to the manual segmentation. No algorithm identified all resection cavities. ResectVol (n = 44, 88 %) and Epic-CHOP (n = 42, 84 %) were able to detect more resection cavities than Resseg (n = 22, 44 %, P < 0.001) and Deep Resection (n = 23, 46 %, P < 0.001). The SPM-based pipelines (Epic-CHOP and ResectVol) performed better than the deep learning-based pipelines in the overall and extratemporal surgery cohorts. In the temporal cohort, the SPM-based pipelines had higher detection rates, however there was no difference in the accuracy between methods. These pipelines could be applied to machine learning studies of outcome prediction to improve efficiency in pre-processing data, however human quality control is still required.

Background: Cardiovascular disease is a leading cause of illness and death globally, primarily due to atherosclerosis. This disease reduces blood flow and oxygen delivery to organs, and when it affects the carotid arteries, it can lead to cognitive impairment and dementia. In a population of 104 individuals, comprising both healthy controls and individuals at elevated risk for developing cardiovascular diseases (CVD) due to identified risk factors, we used PET imaging with 18F-fluorodeoxyglucose (FDG) to assess cerebral glucose metabolism and 18F-sodium fluoride (NaF) to detect atherosclerotic calcification. Our statistical analysis revealed significant differences in metabolic activity between healthy and at-risk individuals in specific brain regions. 18F-FDG uptake in the brain varied inversely with respect to the clinical assessment of cardiovascular risk in regions such as the cuneus (β = −0.030, SE = 0.014, p = 0.035), middle occipital gyrus (β = −0.032, SE = 0.011, p = 0.005), and posterior cingulate gyrus (β = −0.032, SE = 0.015, p = 0.044). In contrast, areas including the basis pontis (β = 0.025, SE = 0.012, p = 0.038) and the pons (β = 0.034, SE = 0.013, p = 0.008) exhibited direct correlations. Notably, carotid 18F-NaF uptake had inverse associations with 18F-FDG uptake in the cerebellum (β = −0.825, SE = 0.354, p = 0.021), medulla (β = −0.888, SE = 0.405, p = 0.029), and posterior cingulate gyrus (β = −1.253, SE = 0.567, p = 0.028), while increased carotid calcification influenced metabolic activity in the fusiform gyrus (β = 1.660, SE = 0.498, p = 0.001) and globus pallidus (β = 1.505, SE = 0.571, p = 0.009). We observed that atherosclerotic plaque accumulation, especially in the carotid arteries, has potential implications for metabolic changes in brain regions governing cognition, emotion, sensory perception, and motor activities. Our findings underscore the possible early interventions that can be used to preempt or delay cognitive deterioration linked with cardiovascular ailments.

Abstract Objective Epilepsy surgery is known to be underutilized. Machine learning‐natural language processing (ML‐NLP) may be able to assist with identifying patients suitable for referral for epilepsy surgery evaluation. Methods Data were collected from two tertiary hospitals for patients seen in neurology outpatients for whom the diagnosis of “epilepsy” was mentioned. Individual case note review was undertaken to characterize the nature of the diagnoses discussed in these notes, and whether those with epilepsy fulfilled prespecified criteria for epilepsy surgery workup (namely focal drug refractory epilepsy without contraindications). ML‐NLP algorithms were then developed using fivefold cross‐validation on the first free‐text clinic note for each patient to identify these criteria. Results There were 457 notes included in the study, of which 250 patients had epilepsy. There were 37 (14.8%) individuals who fulfilled the prespecified criteria for epilepsy surgery referral without described contraindications, 32 (12.8%) of whom were not referred for epilepsy surgical evaluation in the given clinic visit. In the prediction of suitability for epilepsy surgery workup using the prespecified criteria, the tested models performed similarly. For example, the random forest model returned an area under the receiver operator characteristic curve of 0.97 (95% confidence interval 0.93–1.0) for this task, sensitivity of 1.0, and specificity of 0.93. Significance This study has shown that there are patients in tertiary hospitals in South Australia who fulfill prespecified criteria for epilepsy surgery evaluation who may not have been referred for such evaluation. ML‐NLP may assist with the identification of patients suitable for such referral. Plain Language Summary Epilepsy surgery is a beneficial treatment for selected individuals with drug‐resistant epilepsy. However, it is vastly underutilized. One reason for this underutilization is a lack of prompt referral of possible epilepsy surgery candidates to comprehensive epilepsy centers. Natural language processing, coupled with machine learning, may be able to identify possible epilepsy surgery candidates through the analysis of unstructured clinic notes. This study, conducted in two tertiary hospitals in South Australia, demonstrated that there are individuals who fulfill criteria for epilepsy surgery evaluation referral but have not yet been referred. Machine learning‐natural language processing demonstrates promising results in assisting with the identification of such suitable candidates in Australia.

Abstract Background Patagonian toothfish (Dissostichus eleginoides) is an economically and ecologically important fish species in the family Nototheniidae. Juveniles occupy progressively deeper waters as they mature and grow, and adults have been caught as deep as 2500 m, living on or in just above the southern shelves and slopes around the sub-Antarctic islands of the Southern Ocean. As apex predators, they are a key part of the food web, feeding on a variety of prey, including krill, squid, and other fish. Despite its importance, genomic sequence data, which could be used for more accurate dating of the divergence between Patagonian and Antarctic toothfish, or establish whether it shares adaptations to temperature with fish living in more polar or equatorial climes, has so far been limited. Results A high-quality D. eleginoides genome was generated using a combination of Illumina, PacBio and Omni-C sequencing technologies. To aid the genome annotation, the transcriptome derived from a variety of toothfish tissues was also generated using both short and long read sequencing methods. The final genome assembly was 797.8 Mb with a N50 scaffold length of 3.5 Mb. Approximately 31.7% of the genome consisted of repetitive elements. A total of 35,543 putative protein-coding regions were identified, of which 50% have been functionally annotated. Transcriptomics analysis showed that approximately 64% of the predicted genes (22,617 genes) were found to be expressed in the tissues sampled. Comparative genomics analysis revealed that the anti-freeze glycoprotein (AFGP) locus of D. eleginoides does not contain any AFGP proteins compared to the same locus in the Antarctic toothfish (Dissostichus mawsoni). This is in agreement with previously published results looking at hybridization signals and confirms that Patagonian toothfish do not possess AFGP coding sequences in their genome. Conclusions We have assembled and annotated the Patagonian toothfish genome, which will provide a valuable genetic resource for ecological and evolutionary studies on this and other closely related species.

Torn and damaged menisci resulting from trauma are very common knee injuries, which can cause pain and mobility limitations and lead to osteoarthritis. Meniscal injuries can require surgery to repair the tissue damage and restore mobility. Here we describe the biomechanical testing of a 3D-printed meniscus to illustrate methods to determine if it has the strength and durability to effectively repair meniscal tears and restore knee biomechanics. This work was designed to demonstrate the steps needed to test novel meniscus repair devices prior to moving toward animal testing. The first testing step determined the ability of the 3D-printed meniscus to withstand surgical fixation by measuring the suture pull-out force. We show that vertical 2/0 silk or Fiberwire sutures need an average of 1.4 or 1.8 N, respectively, to pull through the meniscus, while horizontal sutures need only 0.7 and 1.2 N, respectively. The next step measured the compressive strength of normal, damaged, and repaired porcine meniscus tissue. Here, we show that meniscectomy decreased the stiffness of meniscus tissue from 26.7 ± 0.85 N to 7.43 ± 0.81 N at 25% strain. Menisci repaired with the 3D-printed tissue restored 66% of the measured force at 25% strain. The final step measured the contact pressures and areas in an ex vivo porcine knee before and after meniscal repair was made with the 3D-printed meniscus tissue. The example 3D-printed meniscus was successfully sutured into the porcine knee joint but failed to restore normal knee contact pressures. This work demonstrates the need for an iterative biomechanical testing process of biomaterial development, 3D-printing optimization, and knee kinematics to develop a durable and functional meniscus repair device. In summary, the methods described here serve as a guide for the functional evaluation of novel meniscus repair devices.

The Romance Inter-Views are short, multiple Q&A pairs that address key issues, definitions and ideas regarding Romance linguistics or general linguistics from a Romance viewpoint. Prominent exponents of different approaches to the study of Romance linguistics are asked to answer some general questions. The answers are then assembled so that readers can get a comparative picture of what’s going on in the field. This is the third Inter-view. The first Inter-view, on Syntax, can be found here. The second Inter-view, on Cartography, can be found here.

Crop yield forecasts allow policy makers to anticipate market behaviour and regulate prices. Annual updates on which crops are grown where can improve crop yield forecast accuracy. Existing efforts to map crops across the European Union resulted in late-season map availability or short time series that do not meet forecasting requirements. We propose a new approach to retrieve annual winter crop maps and improve forecasting efforts by identifying pixels with dominant winter crop signals using moderate resolution imagery. These pixels are distinguished from summer crop signals based on their senescence date. When this date precedes the theoretical maturity date of a winter crop, expressed in GDD, the pixel is labelled as having a dominant winter crop signal. Our 2018 map accurately identified 77% and 83% of dominantly winter-crop area, when compared to farmers’ declaration data and a high-resolution crop map for Europe, respectively. While the resulting annual winter crop maps underestimated winter crop area, derived region-specific annual NDVI profiles better described winter crop phenology as compared to the use of static maps. Regression analysis between these regional NDVI profiles and statistical wheat yield data indicates that our annual maps help explain more yield variability than static maps, with an RMSE reduction of 3% for the EU27 as whole. The proposed approach is applicable to long historical timeseries and provides maps before the end of the agricultural season. Those maps positively impact crop yield description, notably in eastern, northern, and northeastern European regions.

Cropping patterns, including intercropping, are recognized as sustainable agricultural practices in many parts of Africa. However, there is a lack of data and information regarding their extent and specific locations. This study aims to understand the dynamics of these cropping patterns using hyperspectral satellite data. We examined the spectral reflectance of maize and intercropped maize (imaize) fields using DESIS hyperspectral satellite data during the flowering growth phase for discrimination and classification. We used mean reflectance spectra, coefficient of variation, and the Mann–Whitney U-test to characterize the cropping patterns and identify optimal spectral bands. Principal component analysis (PCA) was used to reduce the dimensionality of the hyperspectral data, followed by RF classification. Our findings reveal that the optimal spectral bands for classification fell within the 730–1000 nm range, with the spectral band at 849.8 nm being the most prominent. The RF algorithm performed well, achieving an overall accuracy of 80%. The maize class was distinguished with high precision (0.9) and recall (0.8), resulting in an F1-score of 0.9, indicating a robust ability to accurately identify and classify most of the maize class. In comparison, the imaize class exhibited lower precision (0.6) but a reasonable recall (0.7), leading to an F1-score of 0.6. These findings highlight the potential of using DESIS hyperspectral satellite data in conjunction with PCA and RF for the classification of maize cropping patterns. Additionally, our findings suggest avenues for further research using full waveform hyperspectral satellite data, such as EnMAP and PRISMA, to enhance our understanding of spectral dynamics among different cropping patterns.

Optical fiber Bragg grating (FBG) technology offers many advantages over conventional electrical sensors for the measurement of fluid-structure interaction (FSI). In this work, FBGs are used to measure the dynamic response of 3 mm, 2 mm, and 1 mm thick aluminum cantilever panels under the influence of Mach 2 reflected shock impingement in the UNSW Canberra supersonic blowdown facility. Results compared during start-up and steady-state with high-speed digital video schlieren and laser Doppler vibrometry indicate that FBGs can successfully capture the temporal and spectral characteristics of high-speed FSI. The torsional mode of the 1 mm plate is spectrally captured by the FBG with only a single sensor. Furthermore, it is shown that error in the FBG signal is comparable to that of a laser Doppler vibrometer. The greatest challenge remains the efficient decoupling of thermal effects.

Many wetlands in East Africa are farmed and wetland reservoirs are used for irrigation, livestock, and fishing. Water quality and agriculture have a mutual influence on each other. Turbidity is a principal indicator of water quality and can be used for, otherwise, unmonitored water sources. Low-cost turbidity sensors improve in situ coverage and enable community engagement. The availability of high spatial resolution satellite images from the Sentinel-2 multispectral instrument and of bio-optical models, such as the Case 2 Regional CoastColor (C2RCC) processor, has fostered turbidity modeling. However, these models need local adjustment, and the quality of low-cost sensor measurements is debated. We tested the combination of both technologies to monitor turbidity in small wetland reservoirs in Kenya. We sampled ten reservoirs with low-cost sensors and a turbidimeter during five Sentinel-2 overpasses. Low-cost sensor calibration resulted in an R2 of 0.71. The models using the C2RCC C2X-COMPLEX (C2XC) neural nets with turbidimeter measurements (R2 = 0.83) and with low-cost measurements (R2 = 0.62) performed better than the turbidimeter-based C2X model. The C2XC models showed similar patterns for a one-year time series, particularly around the turbidity limit set by Kenyan authorities. This shows that both the data from the commercial turbidimeter and the low-cost sensor setup, despite sensor uncertainties, could be used to validate the applicability of C2RCC in the study area, select the better-performing neural nets, and adapt the model to the study site. We conclude that combined monitoring with low-cost sensors and remote sensing can support wetland and water management while strengthening community-centered approaches.

Abstract Here, we report the development of a novel polymer composite (PC) purification column and kit. The performance of the PC columns was compared to conventional silica gel (SG) columns for the purification of nucleic acids from coronaviruses, including SARS-CoV-2, in 82 clinical samples. The results shows that PC-based purification outperforms silica gel (SG)-based purification by enabling a higher sensitivity (94%), accuracy (97%), and by eliminating false positives (100% specificity). The high specificity is critical for efficient patient triage and resource management during pandemics. Furthermore, PC-based purification exhibits three times higher analytical precision than a commonly used SG-based nucleic acid purification thereby enabling a more accurate quantification of viral loads and higher reproducibility.

Crop yield estimates are an important data output of agricultural monitoring systems. In sub-Saharan Africa, large input requirements of crop growth models, fragmented agricultural systems, and small field sizes are substantial challenges to accurately estimate crop yield. Multisensor data fusion can be a valuable source of high spatial and temporal resolution data to meet the requirements of crop growth models in Africa. In this study, we estimated crop yield in smallholder agricultural systems of Ethiopia by assimilating Landsat and MODIS fused data in the simple algorithm for crop yield estimation (SAFY) model. Enhanced spatial and temporal adaptive reflectance fusion model (ESTARFM) adapted for fragmented agricultural landscapes was used for data fusion. We assimilated LAI and phenology information derived from Landsat–MODIS fusion, MODIS and field data for comparison. The model was validated with in situ LAI and yield measured in rice and maize fields during the 2019 growing season. Data fusion minimized the yield estimation error (rRMSE = 16% for maize and rRMSE = 23% in rice) more than MODIS (rRMSE = 20% for maize and rRMSE = 35% in rice) because of its higher LAI and phenology estimation accuracy. Data fusion improved the calibration accuracy of the field and crop-specific model parameters and better captured the spatial variability of yield, which is vital for crop production monitoring and food security in smallholder agricultural systems in Africa. Considering the promising results, further investigation into the transferability of the approach to other smallholder agricultural landscapes and hybridization with machine learning is needed for large-area applications.

Abstract Background Given limited data regarding the involvement of disadvantaged groups in paediatric diabetes clinical trials, this study aimed to evaluate the socioeconomic representativeness of participants recruited into a multinational clinical trial in relation to regional and national type 1 diabetes reference populations. Methods Retrospective, cross-sectional evaluation of a subset of adolescent type 1 diabetes cardiorenal intervention trial (AdDIT) participants from Australia (n = 144), Canada (n = 312) and the UK (n = 173). Validated national measures of deprivation were used: the Index of Relative Socioeconomic Disadvantage (IRSD) 2016 (Australia), the Material Resources (MR) dimension of the Canadian Marginalisation index 2016 (Canada) and the Index of Multiple Deprivation (IMD) 2015 (UK). Representativeness was assessed by comparing the AdDIT cohort’s distribution of deprivation quintiles with that of the local paediatric type 1 diabetes population (regional), and the broader type 1 diabetes population for which the trial’s intervention was targeted (national). Results Recruited study cohorts from each country had higher proportions of participants with higher SES, and significant underrepresentation of lower SES, in relation to their national references. The socioeconomic make-up in Australia mirrored that of the regional population (p = 0.99). For Canada, the 2nd least deprived (p = 0.001) and the most deprived quintiles (p

Abstract Teleost fishes, which are the largest and most diverse group of living vertebrates, have a rich history of ancient and recent polyploidy. Previous studies of allotetraploid common carp and goldfish (cyprinids) reported a dominant subgenome, which is more expressed and exhibits biased gene retention. However, the underlying mechanisms contributing to observed ‘subgenome dominance’ remains poorly understood. Here we report high-quality genomes of twenty-one cyprinids to investigate the origin and subsequent subgenome evolution patterns following three independent allopolyploidy events. We identify the closest extant relatives of the diploid progenitor species, investigate genetic and epigenetic differences among subgenomes, and conclude that observed subgenome dominance patterns are likely due to a combination of maternal dominance and transposable element densities in each polyploid. These findings provide an important foundation to understanding subgenome dominance patterns observed in teleost fishes, and ultimately the role of polyploidy in contributing to evolutionary innovations.

Abstract Short‐range functional connectivity in the limbic network is increased in patients with temporal lobe epilepsy (TLE), and recent studies have shown that cortical myelin content correlates with fMRI connectivity. We thus hypothesized that myelin may increase progressively in the epileptic network. We compared T1w/T2w gray matter myelin maps between TLE patients and age‐matched controls and assessed relationships between myelin and aging. While both TLE patients and healthy controls exhibited increased T1w/T2w intensity with age, we found no evidence for significant group‐level aberrations in overall myelin content or myelin changes through time in TLE.