Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

What are the barriers and facilitators to advance care planning with older people in long-term care facilities? A qualitative study.

AIM: To explore the views and preferences for advance care planning from the perspectives of residents, family members and healthcare professionals in long-term care facilities. DESIGN: A qualitative descriptive design. METHODS: We conducted semi-structured interviews with 12 residents of long-term care facilities, 10 family members and 14 healthcare professionals. Data were analysed using reflexive thematic analysis. The social ecological model was used to develop implementation recommendations. RESULTS: We constructed a conceptual model of barriers and facilitators to advance care planning in long-term care facilities, drawing upon four dominant themes from the qualitative analysis: (1) The absence of discourse on end-of-life care: a lack of cultural climate to talk about death, the unspoken agreement to avoid conversations about death, and poor awareness of palliative care may hinder advance care planning initiation; (2) Relational decision-making process is a dual factor affecting advance care planning engagement; (3) Low trust and 'unsafe' cultures: a lack of honest information sharing, risks of violating social expectations and damaging social relationships, and risks of legal consequences may hinder willingness to engage in advance care planning; (4) Meeting and respecting residents' psychosocial needs: these can be addressed by readiness assessment, initiating advance care planning in an informal and equal manner and involving social workers. CONCLUSION: Our findings show that residents' voices were not being heard. It is necessary to identify residents' spontaneous conversation triggers, articulate the value of advance care planning in light of the family's values and preferences, and respect residents' psychosocial needs to promote advance care planning in long-term care facilities. Advance care planning may alleviate the decision-making burden of offspring in nuclear families. IMPLICATIONS FOR CLINICAL PRACTICE: The evidence-based recommendations in this study will inform the implementation of context-specific advance care planning in Asia-Pacific regions. PATIENT AND PUBLIC CONTRIBUTION: Patients and caregivers contributed to the interview pilot and data collection.

Evaluation of a New Model of Care for People with Complications of Diabetic Retinopathy: The EMERALD Study.

PURPOSE: The increasing diabetes prevalence and advent of new treatments for its major visual-threatening complications (diabetic macular edema [DME] and proliferative diabetic retinopathy [PDR]), which require frequent life-long follow-up, have increased hospital demands markedly. Subsequent delays in patient's evaluation and treatment are causing sight loss. Strategies to increase capacity are needed urgently. The retinopathy (EMERALD) study tested diagnostic accuracy, acceptability, and costs of a new health care pathway for people with previously treated DME or PDR. DESIGN: Prospective, multicenter, case-referent, cross-sectional, diagnostic accuracy study undertaken in 13 hospitals in the United Kingdom. PARTICIPANTS: Adults with type 1 or 2 diabetes previously successfully treated DME or PDR who, at the time of enrollment, had active or inactive disease. METHODS: A new health care pathway entailing multimodal imaging (spectral-domain OCT for DME, and 7-field Early Treatment Diabetic Retinopathy Study [ETDRS] and ultra-widefield [UWF] fundus images for PDR) interpreted by trained nonmedical staff (ophthalmic graders) to detect reactivation of disease was compared with the current standard care (face-to-face examination by ophthalmologists). MAIN OUTCOME MEASURES: Primary outcome: sensitivity of the new pathway. SECONDARY OUTCOMES: specificity; agreement between pathways; costs; acceptability; proportions requiring subsequent ophthalmologist assessment, unable to undergo imaging, and with inadequate images or indeterminate findings. RESULTS: The new pathway showed sensitivity of 97% (95% confidence interval [CI], 92%-99%) and specificity of 31% (95% CI, 23%-40%) to detect DME. For PDR, sensitivity and specificity using 7-field ETDRS images (85% [95% CI, 77%-91%] and 48% [95% CI, 41%-56%], respectively) or UWF images (83% [95% CI, 75%-89%] and 54% [95% CI, 46%-61%], respectively) were comparable. For detection of high-risk PDR, sensitivity and specificity were higher when using UWF images (87% [95% CI, 78%-93%] and 49% [95% CI, 42%-56%], respectively, for UWF versus 80% [95% CI, 69-88%] and 40% [95% CI, 34%-47%], respectively, for 7-field ETDRS images). Participants preferred ophthalmologists' assessments; in their absence, they preferred immediate feedback by graders, maintaining periodic ophthalmologist evaluations. When compared with the current standard of care, the new pathway could save £1390 per 100 DME visits and between £461 and £1189 per 100 PDR visits. CONCLUSIONS: The new pathway has acceptable sensitivity and would release resources. Users' suggestions should guide implementation.

The prevalence of personality disorders in the community: a global systematic review and meta-analysis.

BACKGROUND: Personality disorders are now internationally recognised as a mental health priority. Nevertheless, there are no systematic reviews examining the global prevalence of personality disorders. AIMS: To calculate the worldwide prevalence of personality disorders and examine whether rates vary between high-income countries and low- and middle-income countries (LMICs). METHOD: We systematically searched PsycINFO, MEDLINE, EMBASE and PubMed from January 1980 to May 2018 to identify articles reporting personality disorder prevalence rates in community populations (PROSPERO registration number: CRD42017065094). RESULTS: A total of 46 studies (from 21 different countries spanning 6 continents) satisfied inclusion criteria. The worldwide pooled prevalence of any personality disorder was 7.8% (95% CI 6.1-9.5). Rates were greater in high-income countries (9.6%, 95% CI 7.9-11.3%) compared with LMICs (4.3%, 95% CI 2.6-6.1%). In univariate meta-regressions, significant heterogeneity was partly attributable to study design (two-stage v. one-stage assessment), county income (high-income countries v. LMICs) and interview administration (clinician v. trained graduate). In multiple meta-regression analysis, study design remained a significant predictor of heterogeneity. Global rates of cluster A, B and C personality disorders were 3.8% (95% CI 3.2, 4.4%), 2.8% (1.6, 3.7%) and 5.0% (4.2, 5.9%). CONCLUSIONS: Personality disorders are prevalent globally. Nevertheless, pooled prevalence rates should be interpreted with caution due to high levels of heterogeneity. More large-scale studies with standardised methodologies are now needed to increase our understanding of population needs and regional variations.

Quantification of uncertainty in a new network model of pulmonary arterial adventitial fibroblast pro-fibrotic signalling.

Here, we present a novel network model of the pulmonary arterial adventitial fibroblast (PAAF) that represents seven signalling pathways, confirmed to be important in pulmonary arterial fibrosis, as 92 reactions and 64 state variables. Without optimizing parameters, the model correctly predicted 80% of 39 results of input-output and inhibition experiments reported in 20 independent papers not used to formulate the original network. Parameter uncertainty quantification (UQ) showed that this measure of model accuracy is robust to changes in input weights and half-maximal activation levels (EC50), but is more affected by uncertainty in the Hill coefficient (n), which governs the biochemical cooperativity or steepness of the sigmoidal activation function of each state variable. Epistemic uncertainty in model structure, due to the reliance of some network components and interactions on experiments using non-PAAF cell types, suggested that this source of uncertainty had a smaller impact on model accuracy than the alternative of reducing the network to only those interactions reported in PAAFs. UQ highlighted model parameters that can be optimized to improve prediction accuracy and network modules where there is the greatest need for new experiments. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Quantification of model and data uncertainty in a network analysis of cardiac myocyte mechanosignalling.

Cardiac myocytes transduce changes in mechanical loading into cellular responses via interacting cell signalling pathways. We previously reported a logic-based ordinary differential equation model of the myocyte mechanosignalling network that correctly predicts 78% of independent experimental results not used to formulate the original model. Here, we use Monte Carlo and polynomial chaos expansion simulations to examine the effects of uncertainty in parameter values, model logic and experimental validation data on the assessed accuracy of that model. The prediction accuracy of the model was robust to parameter changes over a wide range being least sensitive to uncertainty in time constants and most affected by uncertainty in reaction weights. Quantifying epistemic uncertainty in the reaction logic of the model showed that while replacing 'OR' with 'AND' reactions greatly reduced model accuracy, replacing 'AND' with 'OR' reactions was more likely to maintain or even improve accuracy. Finally, data uncertainty had a modest effect on assessment of model accuracy. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Genetically encoded sensors of protein hydrodynamics and molecular proximity.

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

Mechanisms and contextual influences on the implementation of advance care planning for older people in long-term care facilities: A realist review.

BACKGROUND: Older people in long-term care facilities face clinical uncertainty and unpredictable decline. Advance care planning enables older people to identify preferences and wishes for future treatment and care before any loss of capacity. However, it is unclear how, why and under what circumstances the implementation of advance care planning for older people can be normalised into routine practice within long-term care facilities. OBJECTIVE: To identify and explain mechanisms and contextual factors that underpin the implementation of advance care planning for older people in long-term care facilities. DESIGN: Realist review. SETTING(S): Long-term care facilities. METHODS: Consistent with realist review methodology, we developed the initial programme theory by scoping reviews, engaging UK and China stakeholders and utilising the Normalisation Process Theory. MEDLINE, EMBASE, CINAHL, PsycINFO, Web of Science, and Scopus were subsequently searched from 01/01/1990 to 11/06/2021. Inductive and deductive coding was used to generate context-mechanism-outcome configurations, which were iteratively tested to refine the programme theory. RESULTS: 5459 records were identified, and 48 were retained for final synthesis. Seven context-mechanism-outcome configurations were identified: (1) carry out sensitive conversation gradually in a nonthreatening way; (2) identify 'a window of opportunity'; (3) deliver sustainable and available training; (4) build a collaborative and multidisciplinary network; (5) conduct collaborative negotiation to achieve shared decision-making; (6) secure active leadership buy-in; (7) keep conversation and documentation on track. A logic model was developed to conceptualise the causal pathways between the contexts, mechanisms, and outcomes. CONCLUSIONS: Normalising conversations about death is paramount to mainstreaming advance care planning implementation in long-term care facilities. The key to achieving this is older people, family members and staff have a shared understanding of the aims, values, and potential benefits of advance care planning. Advance care planning should be introduced at a time that is important to older people and families, rather than being process-driven. Nurse facilitators play a vital role in ensuring older people's voices are heard and in building bridges between participants in advance care planning. The findings of this study inform the appropriate development and evaluation of advance care planning interventions for older people in long-term care facilities. Further research is needed to explore mechanisms that underpin the implementation of advance care planning in Asian countries. REGISTRATION: This review is registered with the International Prospective Register of Systematic Reviews (CRD42021214317).

Efficacy of Liposomal Bupivacaine and Bupivacaine Hydrochloride vs Bupivacaine Hydrochloride Alone as a Periarticular Anesthetic for Patients Undergoing Knee Replacement: A Randomized Clinical Trial.

Importance: More than half of patients who undergo knee replacement surgery report substantial acute postoperative pain. Objective: To evaluate the efficacy and cost-effectiveness of periarticular liposomal bupivacaine for recovery and pain management after knee replacement. Design, Setting, and Participants: This multicenter, patient-blinded, pragmatic, randomized clinical superiority trial involved 533 participants at 11 institutions within the National Health Service in England. Adults undergoing primary unilateral knee replacement for symptomatic end-stage osteoarthritis were enrolled between March 29, 2018, and February 29, 2020, and followed up for 1 year after surgery. Follow-up was completed March 1, 2021. A per-protocol analysis for each coprimary outcome was performed in addition to the main intention-to-treat analysis. Interventions: Two hundred sixty-six milligrams of liposomal bupivacaine admixed with 100 mg of bupivacaine hydrochloride compared with 100 mg of bupivacaine hydrochloride alone (control) administered by periarticular injection at the time of surgery. Main Outcome and Measures: The coprimary outcomes were Quality of Recovery 40 (QoR-40) score at 72 hours and pain visual analog scale (VAS) score area under the curve (AUC) from 6 to 72 hours. Secondary outcomes included QoR-40 and mean pain VAS at days 0 (evening of surgery), 1, 2, and 3; cumulative opioid consumption for 72 hours; functional outcomes and quality of life at 6 weeks, 6 months, and 1 year; and cost-effectiveness for 1 year. Adverse events and serious adverse events up to 12 months after randomization were also assessed. Results: Among the 533 participants included in the analysis, the mean (SD) age was 69.0 (9.7) years; 287 patients were women (53.8%) and 246 were men (46.2%). Baseline characteristics were balanced between study groups. There was no difference between the liposomal bupivacaine and control groups in QoR-40 score at 72 hours (adjusted mean difference, 0.54 [97.5% CI, -2.05 to 3.13]; P = .64) or the pain VAS score AUC at 6 to 72 hours (-21.5 [97.5% CI, -46.8 to 3.8]; P = .06). Analyses of pain VAS and QoR-40 scores demonstrated only 1 statistically significant difference, with the liposomal bupivacaine arm having lower pain scores the evening of surgery (adjusted difference -0.54 [97.5% CI, -1.07 to -0.02]; P = .02). No difference in cumulative opioid consumption and functional outcomes was detected. Liposomal bupivacaine was not cost-effective compared with the control treatment. No difference in adverse or serious adverse events was found between the liposomal bupivacaine and control groups. Conclusions and Relevance: This study found no difference in postoperative recovery or pain associated with the use of periarticular liposomal bupivacaine compared with bupivacaine hydrochloride alone in patients who underwent knee replacement surgery. Trial Registration: isrctn.com Identifier: ISRCTN54191675.

Cellular mechanosignaling in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by sustained elevated pulmonary arterial pressures in which the pulmonary vasculature undergoes significant structural and functional remodeling. To better understand disease mechanisms, in this review article we highlight recent insights into the regulation of pulmonary arterial cells by mechanical cues associated with PAH. Specifically, the mechanobiology of pulmonary arterial endothelial cells (PAECs), smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) has been investigated in vivo, in vitro, and in silico. Increased pulmonary arterial pressure increases vessel wall stress and strain and endothelial fluid shear stress. These mechanical cues promote vasoconstriction and fibrosis that contribute further to hypertension and alter the mechanical milieu and regulation of pulmonary arterial cells.

Substrate Stiffness and Stretch Regulate Profibrotic Mechanosignaling in Pulmonary Arterial Adventitial Fibroblasts.

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (Col1a1, Col3a1, Eln) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (Loxl1, Acta2) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA (Fn1) levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin ß3, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-ß (TGF-ß), and syndecan-4, and stretch-regulated integrin ß3, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on Loxl1 and Acta2 expression. Blocking Ang II Receptor Type 1 (AT1R) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of Fn1 expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.

Synergistic Enzyme Mixtures to Realize Near-Complete Depolymerization in Biodegradable Polymer/Additive Blends.

Embedding catalysts inside of plastics affords accelerated chemical modification with programmable latency and pathways. Nanoscopically embedded enzymes can lead to near-complete degradation of polyesters via chain-end mediated processive depolymerization. The overall degradation rate and pathways have a strong dependence on the morphology of semicrystalline polyesters. Yet, most studies to date focus on pristine polymers instead of mixtures that contain additives and other components despite their nearly universal use in plastic production. Here, additives are introduced to purposely change the morphology of polycaprolactone (PCL) by increasing the bending and twisting of crystalline lamellae. These morphological changes immobilize chain ends preferentially at the crystalline/amorphous interfaces and limit chain-end accessibility by the embedded processive enzyme. This chain-end redistribution reduces the polymer-to-monomer conversion from >95% to less than 50%, causing formation of highly crystalline plastic pieces, including microplastics. By synergizing both random chain scission and processive depolymerization, it is feasible to navigate morphological changes in polymer/additive blends and to achieve near-complete depolymerization. The random scission enzymes in the amorphous domains create new chain ends that are subsequently bound and depolymerized by processive enzymes. Present studies further highlight the importance to consider how the host polymer's morphologies affect the reactions catalyzed by embedded catalytic species.

Study of Peri-Articular Anaesthetic for Replacement of the Knee (SPAARK): statistical analysis plan for a randomised controlled trial assessing the effectiveness of peri-articular liposomal bupivacaine plus bupivacaine hydrochloride compared with bupivacaine hydrochloride alone.

BACKGROUND: Up to three quarters of surgical patients receive inadequate pain relief, with 40% of patients reporting severe pain following knee replacement, which may indicate the current pain relief strategies using opiate-based analgesia cannot achieve patient satisfaction. Liposomal bupivacaine is liposome-encapsulated bupivacaine which has been reported to be effective for up to 72 h. The study of Peri-Articular Anaesthetic for Replacement of the Knee (SPAARK) trial has been designed to assess the effectiveness of peri-articular liposomal bupivacaine and bupivacaine hydrochloride compared with peri-articular bupivacaine hydrochloride alone in the management of post-operative pain following knee replacement. METHODS/DESIGN: The SPAARK trial is a multi-centre, patient-blinded, randomised controlled trial. The co-primary outcomes are post-operative recovery assessed by global QoR-40 scores at 72 h and cumulative pain VAS score from 6 to 72 h following surgery. Longer-term measures of the co-primary outcomes are collected at 6 weeks and 6 and 12 months post randomisation, together with secondary outcomes, i.e. the Oxford Knee Score, and the American Knee Society Score. Cumulative opiate use and fitness for discharge are measured up to 72 h post-surgery. The analysis approaches for the primary and secondary outcomes are described here, as are the descriptive statistics which will be reported. The full SPAARK protocol has already been published. RESULTS: The co-primary outcomes will be analysed using multivariate linear regression adjusting for stratification factors and other important prognostic variables, including baseline scores in the case of the QoR-40. The adjusted mean difference between the two groups together with 97.5% confidence intervals will be reported for each of the primary outcomes. Other continuous variables will be assessed using the same method. Binary outcomes will be assessed using chi-squared tests. DISCUSSION: The paper provides details of the planned statistical analyses for the SPAARK trial and aims to reduce the risk of outcome reporting bias from prior data knowledge. Any changes or deviations from this statistical analysis plan will be described and justified in the final study report. TRIAL REGISTRATION: ISRCTN54191675 . Registered on 13 November 2017.

Multimodal imaging interpreted by graders to detect re-activation of diabetic eye disease in previously treated patients: the EMERALD diagnostic accuracy study.

BACKGROUND: Owing to the increasing prevalence of diabetes, the workload related to diabetic macular oedema and proliferative diabetic retinopathy is rising, making it difficult for hospital eye services to meet demands. OBJECTIVE: The objective was to evaluate the diagnostic performance, cost-effectiveness and acceptability of a new pathway using multimodal imaging interpreted by ophthalmic graders to detect reactivation of diabetic macular oedema/proliferative diabetic retinopathy in previously treated patients. DESIGN: This was a prospective, case-referent, cross-sectional diagnostic study. SETTING: The setting was ophthalmic clinics in 13 NHS hospitals. PARTICIPANTS: Adults with type 1 or type 2 diabetes with previously successfully treated diabetic macular oedema/proliferative diabetic retinopathy in one/both eyes in whom, at the time of enrolment, diabetic macular oedema/proliferative diabetic retinopathy could be active or inactive. METHODS: For the ophthalmic grader pathway, review of the spectral domain optical coherence tomography scans to detect diabetic macular oedema, and seven-field Early Treatment Diabetic Retinopathy Study/ultra-wide field fundus images to detect proliferative diabetic retinopathy, by trained ophthalmic graders. For the current standard care pathway (reference standard), ophthalmologists examined patients face to face by slit-lamp biomicroscopy for proliferative diabetic retinopathy and, in addition, spectral domain optical coherence tomography imaging for diabetic macular oedema. OUTCOME MEASURES: The primary outcome measure was sensitivity of the ophthalmic grader pathway to detect active diabetic macular oedema/proliferative diabetic retinopathy. The secondary outcomes were specificity, agreement between pathways, cost-consequences, acceptability and the proportion of patients requiring subsequent ophthalmologist assessment, unable to undergo imaging and with inadequate quality images/indeterminate findings. It was assumed for the main analysis that all patients in whom graders diagnosed active disease or were 'unsure' or images were 'ungradable' required examination by an ophthalmologist. RESULTS: Eligible participants with active and inactive diabetic macular oedema (152 and 120 participants, respectively) and active and inactive proliferative diabetic retinopathy (111 and 170 participants, respectively) were recruited. Under the main analysis, graders had a sensitivity of 97% (142/147) (95% confidence interval 92% to 99%) and specificity of 31% (35/113) (95% confidence interval 23% to 40%) to detect diabetic macular oedema. For proliferative diabetic retinopathy, graders had a similar sensitivity and specificity using seven-field Early Treatment Diabetic Retinopathy Study [sensitivity 85% (87/102), 95% confidence interval 77% to 91%; specificity 48% (77/160), 95% confidence interval 41% to 56%] or ultra-wide field imaging [sensitivity 83% (87/105), 95% confidence interval 75% to 89%; specificity 54% (86/160), 95% confidence interval 46% to 61%]. Participants attending focus groups expressed preference for face-to-face evaluations by ophthalmologists. In the ophthalmologists' absence, patients voiced the need for immediate feedback following grader's assessments, maintaining periodic evaluations by ophthalmologists. Graders and ophthalmologists were supportive of the new pathway. When compared with the reference standard (current standard pathway), the new grader pathway could save £1390 per 100 patients in the review of people with diabetic macular oedema and, depending on the imaging modality used, between £461 and £1189 per 100 patients in the review of people with proliferative diabetic retinopathy. CONCLUSIONS: For people with diabetic macular oedema, the ophthalmic grader pathway appears safe and cost saving. The sensitivity of the new pathway to detect active proliferative diabetic retinopathy was lower, but may still be considered acceptable for patients with proliferative diabetic retinopathy previously treated with laser. Suggestions from focus group discussions should be taken into consideration if the new pathway is introduced to ensure its acceptability to users. LIMITATIONS: Lack of fundus fluorescein angiography to confirm diagnosis of active proliferative diabetic retinopathy. FUTURE WORK: Could refinement of the new pathway increase its sensitivity to detect proliferative diabetic retinopathy? Could artificial intelligence be used for automated reading of images in this previously treated population? TRIAL REGISTRATION: Current Controlled Trials ISRCTN10856638 and ClinicalTrials.gov NCT03490318. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology AssessmentVol. 25, No. 32. See the NIHR Journals Library website for further project information.

More and more people are developing diabetes. Diabetic macular oedema and proliferative diabetic retinopathy are complications of diabetes, which could cause blindness. Thus, people with diabetic macular oedema and proliferative diabetic retinopathy need to be treated in a timely manner and reviewed in clinic for life. The population in the world is ageing. As a result, there are more people with eye diseases. There are also more treatments now for people with eye diseases. The workload in hospitals is increasing, making it difficult for the NHS to cope with the demand. There are not enough ophthalmologists (eye doctors) to look after patients. Delayed appointments and treatment mean that patients may lose sight. The goal of EMERALD (Effectiveness of Multimodal imaging for the Evaluation of Retinal oedema And new vesseLs in Diabetic retinopathy) was to see if patients with treated and stable diabetic macular oedema or proliferative diabetic retinopathy could be followed by 'ophthalmic graders', who are not doctors but are trained to diagnose diabetic macular oedema and proliferative diabetic retinopathy. In EMERALD, trained ophthalmic graders examined photographs of the back of the eye of people with diabetic macular oedema and proliferative diabetic retinopathy. They checked if diabetic macular oedema and proliferative diabetic retinopathy remain inactive. If so, patients could continue follow-up with the ophthalmic graders. If diabetic macular oedema or proliferative diabetic retinopathy were active, graders would immediately refer patients to ophthalmologists. EMERALD found that graders were excellent at detecting diabetic macular oedema, and this could give ophthalmologists time to see other patients. Graders were not quite as good at detecting active proliferative diabetic retinopathy. However, considering that patients had already had treatment, this may still be safe. Patients participating in focus group discussions mentioned that they would prefer to see ophthalmologists, so they could ask questions about their eye condition. If this was not possible, they would like to have immediate results from graders and still see the ophthalmologist from time to time.

Azithromycin versus standard care in patients with mild-to-moderate COVID-19 (ATOMIC2): an open-label, randomised trial.

BACKGROUND: The antibacterial, anti-inflammatory, and antiviral properties of azithromycin suggest therapeutic potential against COVID-19. Randomised data in mild-to-moderate disease are not available. We assessed whether azithromycin is effective in reducing hospital admission in patients with mild-to-moderate COVID-19. METHODS: This prospective, open-label, randomised superiority trial was done at 19 hospitals in the UK. We enrolled adults aged at least 18 years presenting to hospitals with clinically diagnosed, highly probable or confirmed COVID-19 infection, with fewer than 14 days of symptoms, who were considered suitable for initial ambulatory management. Patients were randomly assigned (1:1) to azithromycin (500 mg once daily orally for 14 days) plus standard care or to standard care alone. The primary outcome was death or hospital admission from any cause over the 28 days from randomisation. The primary and safety outcomes were assessed according to the intention-to-treat principle. This trial is registered at ClinicalTrials.gov (NCT04381962) and recruitment is closed. FINDINGS: 298 participants were enrolled from June 3, 2020, to Jan 29, 2021. Three participants withdrew consent and requested removal of all data, and three further participants withdrew consent after randomisation, thus, the primary outcome was assessed in 292 participants (145 in the azithromycin group and 147 in the standard care group). The mean age of the participants was 45·9 years (SD 14·9). 15 (10%) participants in the azithromycin group and 17 (12%) in the standard care group were admitted to hospital or died during the study (adjusted OR 0·91 [95% CI 0·43-1·92], p=0·80). No serious adverse events were reported. INTERPRETATION: In patients with mild-to-moderate COVID-19 managed without hospital admission, adding azithromycin to standard care treatment did not reduce the risk of subsequent hospital admission or death. Our findings do not support the use of azithromycin in patients with mild-to-moderate COVID-19. FUNDING: National Institute for Health Research Oxford Biomedical Research Centre, University of Oxford and Pfizer.

Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin.

Background Pressure overload of the heart occurs in patients with hypertension or valvular stenosis and induces cardiac fibrosis because of excessive production of extracellular matrix by activated cardiac fibroblasts. This initially provides essential mechanical support to the heart, but eventually compromises function. Osteopontin is associated with fibrosis; however, the underlying signaling mechanisms are not well understood. Herein, we examine the effect of thrombin-cleaved osteopontin on fibrosis in the heart and explore the role of syndecan-4 in regulating cleavage of osteopontin. Methods and Results Osteopontin was upregulated and cleaved by thrombin in the pressure-overloaded heart of mice subjected to aortic banding. Cleaved osteopontin was higher in plasma from patients with aortic stenosis receiving crystalloid compared with blood cardioplegia, likely because of less heparin-induced inhibition of thrombin. Cleaved osteopontin and the specific osteopontin peptide sequence RGDSLAYGLR that is exposed after thrombin cleavage both induced collagen production in cardiac fibroblasts. Like osteopontin, the heparan sulfate proteoglycan syndecan-4 was upregulated after aortic banding. Consistent with a heparan sulfate binding domain in the osteopontin cleavage site, syndecan-4 was found to bind to osteopontin in left ventricles and cardiac fibroblasts and protected osteopontin from cleavage by thrombin. Shedding of the extracellular part of syndecan-4 was more prominent at later remodeling phases, at which time levels of cleaved osteopontin were increased. Conclusions Thrombin-cleaved osteopontin induces collagen production by cardiac fibroblasts. Syndecan-4 protects osteopontin from cleavage by thrombin, but this protection is lost when syndecan-4 is shed in later phases of remodeling, contributing to progression of cardiac fibrosis.

Identification of European starling GnRH-I precursor mRNA and its seasonal regulation.

Songbirds show dynamic seasonal changes in their reproductive activities during the year. Gonadotropin-releasing hormone-I (GnRH-I) is critical for the control of reproduction in vertebrates. The molecular mechanisms controlling reproduction are not well understood in songbirds, largely because the GnRH-I precursor polypeptide gene was unknown until now. Here, we report the complete sequence and seasonal regulation of GnRH-I precursor polypeptide mRNA in a songbird, European starling (Sturnus vulgaris). The translated starling GnRH-I precursor polypeptide contained an amino acid sequence that can be processed into chicken GnRH-I peptide (pEHWSYGLQPG-NH(2)). However, the overall homology of GnRH-I precursor polypeptide (including a 23 amino acid signal peptide, the decapeptide hormone and Gly-Lys-Arg cleavage site followed by 55 amino acid GnRH-associated peptide sequences) between starling and chicken was only 58%. GnRH-I mRNA and GnRH-I peptide were observed to be co-localized in the preoptic area of sexually mature birds using in situ hybridization and immunocytochemistry. GnRH-I mRNA exhibited large variance in photosensitive birds, and converged to a high level in photostimulated birds. Subsequently, GnRH-I mRNA decreased to below detectability in most of the photorefractory birds. Changes were also observed in GnRH-I peptide levels, although changes in GnRH-I peptide were not as marked. Our data indicate that GnRH-I mRNA synthesis commences but is variable in photosensitive birds, stabilizes in photostimulated birds, then ceases when birds become photorefractory. Finer-scale investigation into temporal regulation of GnRH-I precursor polypeptide mRNA will provide insight into its regulation by environmental, social and physiological cues.

RNA interference of gonadotropin-inhibitory hormone gene induces arousal in songbirds.

Gonadotropin-inhibitory hormone (GnIH) was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi) of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts.

Gonadotropin-inhibitory hormone identification, cDNA cloning, and distribution in rhesus macaque brain.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that modulates the reproductive physiology of birds and mammals by inhibiting gonadotropin secretion from the anterior pituitary gland. GnIH can also directly inhibit reproductive behaviors, possibly via action within the brain. Identification of the distribution of GnIH neurons and fibers may provide us with clues to how the brain controls reproductive activities of the animal. Here, we characterized the location and connectivity of GnIH neurons in the rhesus macaque (Macaca mulatta) brain. We determined the macaque GnIH precursor mRNA, and further identified a mature GnIH peptide (SGRNMEVSLVRQVLNLPQRF-NH(2)) by mass spectrometry combined with immunoaffinity purification. The majority of GnIH precursor mRNA-positive and GnIH-immunoreactive (GnIH-ir) cell bodies were localized in the intermediate periventricular nucleus (IPe) in the hypothalamus, as determined by in situ hybridization and immunocytochemistry, respectively. Abundant GnIH-ir fibers were observed in the nucleus of the stria terminalis in the telencephalon; habenular nucleus, paraventricular nucleus of the thalamus, preoptic area, paraventricular nucleus of the hypothalamus, IPe, arcuate nucleus of hypothalamus, median eminence and dorsal hypothalamic area in the diencephalon; medial region of the superior colliculus, central gray substance of the midbrain and dorsal raphe nucleus in the midbrain; and parabrachial nucleus in the pons. GnIH-ir fibers were observed in close proximity to gonadotropin-releasing hormone-I, dopamine, beta-endorphin, and gonadotropin-releasing hormone-II neurons in the preoptic area, IPe, arcuate nucleus of hypothalamus, and central gray substance of midbrain, respectively. GnIH neurons might thus regulate several neural systems in addition to pituitary gonadotropin release.