Search

Chi-Han Lin,1,2,&ast; Yung-Chia Kuo,1– 3,&ast; Hsuan-Chih Kuo,1– 3,&ast; Ching-Ting Wang,4 Shi-Ming Lin,1,5 Alan Chao-Wei Lee,1,6 Ming-Chin Yu,1,6,7 Wei-Chen Lee,1,6 Cherry Chiao-Erh Chen,2,3 Jason Chia-Hsun Hsieh1– 3 1College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan; 2Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, 333, Taiwan; 3Division of Hematology-Oncology, Department of Internal Medicine, New Taipei City Municipal TuCheng Hospital, New Taipei City, 236, Taiwan; 4Registered Nurse, Case Manager of Nursing Department, Chang Gung Memorial Hospital, Linkou, Taoyuan, 333, Taiwan; 5Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan; 6Department of Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan, 333, Taiwan; 7Department of Surgery, New Taipei City Municipal TuCheng Hospital, New Taipei City, 236, Taiwan&ast;These authors contributed equally to this workCorrespondence: Jason Chia-Hsun Hsieh, Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, No. 5, Fu-Hsin Street, Gueishan District, Taoyuan City, 333, Taiwan, Tel +886-3-3281200 Ext 8825, Email wisdom5000@gmail.comBackground: Hepatitis often occurs after initiating immune checkpoint inhibitor (ICI) treatment. The time and grade of hepatitis after ICI starts and the prognostic role of immune-related hepatitis in patients with advanced hepatocellular carcinoma (aHCC) remain unclear.Methods: In this real-world analysis, we enrolled aHCC patients receiving ICIs, documented the highest level of liver enzymes during/after ICIs, and analyzed the survival impact of different hepatitis patterns.Results: One hundred and ninety-three aHCC patients receiving ICIs were recruited. During ICIs, 88.6% of patients experienced aspartate transaminase (AST) elevations (Grade III/IV: 7.8%). For alanine transaminase (ALT), 81.3% had elevated levels (Grade III/IV: 3.6%), and 41.5% of patients had elevated bilirubin levels (Grade 3/4: 6.7%). The median AST, ALT, and total bilirubin values significantly increased after ICI treatment initiated (all p < 0.001) and, similarly, after excluding progressive disease (p = 0.014, p = 0.002, p < 0.001). The median time of hepatitis occurrence is from the 4.0th to 15.9th weeks. Multivariable analysis showed that patterns of liver enzyme change of AST and total bilirubin in patients receiving ICIs significantly correlate to overall survival (OS, p = 0.009 and 0.001, respectively). After ICI termination, patients with elevated bilirubin (p = 0.003) and AST (p = 0.005) would indicate poor survival, with adjustment of viral hepatitis and ICI responses.Conclusion: Hepatitis emerges between the 4th and 20th weeks post-ICI initiation. Changes in liver enzymes during ICI therapy do not directly affect OS, implying the safety of ICI use when corticosteroids are promptly administered if clinically indicated.Keywords: hepatitis, immune checkpoint inhibitor, advanced hepatocellular carcinoma, liver enzymes, overall survival

Abstract Background Out-of-hospital cardiac arrest (OHCA) is a prevalent condition with high mortality and poor outcomes even in settings where extensive emergency care resources are available. Interventions to address OHCA have had limited success, with survival rates below 10% in national samples of high-income countries. In resource-limited settings, where scarcity requires careful priority setting, more data is needed to determine the optimal allocation of resources. Objective To establish the cost-effectiveness of OHCA care and assess the affordability of interventions across income settings. Methods The authors conducted a systematic review of economic evaluations on interventions to address OHCA. Six databases (PubMed, EMBASE, Global Health, Cochrane, Global Index Medicus, and Tuft's Cost-Effectiveness Registry) were searched in September 2023. Included studies were (1) economic evaluations (beyond a simple costing exercise); and (2) assessed an intervention in the chain of survival for OHCA. Article quality was assessed using the CHEERs checklist and data summarised. Findings were reported by major themes identified by the reviewers. Based upon the results of the cost-effectiveness analyses we then conduct an analysis for the progressive realization of the OHCA chain of survival from the perspective of decision-makers facing resource constraints. Results Four hundred and sixty-eight unique articles were screened, and 46 articles were included for final data abstraction. Studies predominantly used a healthcare sector perspective, modeled for all patients experiencing non-traumatic cardiac OHCA, were based in the US, and presented results in US Dollars. No studies reported results or used model inputs from low-income settings. Progressive realization of the chain of survival could likely begin with investments in termination of resuscitation protocols, professional prehospital defibrillator use, and CPR training followed by the distribution of AEDs in high-density public locations. Finally, other interventions such as indiscriminate defibrillator placement or adrenaline use, would be the lowest priority for early investment. Conclusion Our review found no high-quality evidence on the cost-effectiveness of treating OHCA in low-resource settings. Existing evidence can be utilized to develop a roadmap for the development of a cost-effective approach to OHCA care, however further economic evaluations using context-specific data are crucial to accurately inform prioritization of scarce resources within emergency care in these settings.

Abstract COVID presented an opportunity to revolutionize the traditional format of Medical Grand Rounds (MGR). In this Commentary, we explore the educational ramifications of shifting MGR virtually with a focus on COVID-related content and its long-term sustainability. This transformation offers an inclusive interdisciplinary approach to sustain learner interest and improve education.

Abstract Microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) tumor status have been demonstrated to predict patient response to immunotherapies. We developed and validated a next-generation sequencing (NGS)-based companion diagnostic (CDx) to detect MSI-H solid tumors via a comprehensive genomic profiling (CGP) assay, FoundationOne®CDx (F1CDx). To determine MSI status, F1CDx calculates the fraction of unstable microsatellite loci across >2000 loci using a fraction-based (FB) analysis. Across solid tumor types, F1CDx demonstrated a high analytical concordance with both PCR (n = 264) and IHC (n = 279) with an overall percent agreement (OPA) of 97.7% and 97.8%, respectively. As part of a retrospective bridging clinical study from KEYNOTE-158 Cohort K and KEYNOTE-164, patients with MSI-H tumors as determined by F1CDx demonstrated an objective response rate (ORR) of 43.0% to pembrolizumab. In real-world cancer patients from a deidentified clinicogenomic database, F1CDx was at least equivalent in assessing clinical outcome following immunotherapy compared with MMR IHC. Demonstrated analytical and clinical performance of F1CDx led to the pan-tumor FDA approval in 2022 of F1CDx to identify MSI-H solid tumor patients for treatment with pembrolizumab. F1CDx is an accurate, reliable, and FDA-approved method for the identification of MSI-H tumors for treatment with pembrolizumab.

Thorough biological safety testing of topical therapeutic compounds and antimicrobials is a critical prerequisite for appropriate cutaneous wound care. Increasing pathogen resistance rates to traditional antibiotics and antifungals are driving the development and registration of novel chemical entities. Although they are notably useful for animal testing reduction, the gold standard in vitro cytotoxicity assays in continuous cell lines (HaCaT keratinocytes, 3T3 fibroblasts) may be discussed from a translational relevance standpoint. The aim of this study was thus to establish and validate a sustainable primary cell banking model with a view to performing optimized in vitro cytotoxicity assay development. Primary dermal fibroblasts and adipose-derived stem cell (ASC) types were established from four infant polydactyly sources. A multi-tiered primary cell banking model was then applied to prepare highly sustainable and standardized dermal fibroblast and ASC working cell banks (WCBs), potentially allowing for millions of biological assays to be performed. The obtained cellular materials were then validated for use in cytotoxicity assays through in vitro biosafety testing of topical antiseptics (chlorhexidine, hypochlorous acid) and an antifungal compound (AR-12) of interest for optimized burn wound care. The experimental results confirmed that IC50 values were comparable between cytotoxicity assays, which were performed with cell lines and with primary cells. The results also showed that hypochlorous acid (HOCl) displayed an enhanced toxicological profile as compared to the gold standard chlorhexidine (CLX). Generally, this study demonstrated that highly sustainable primary cell sources may be established and applied for consistent topical compound biological safety assessments with enhanced translational relevance. Overall, the study underscored the safety-oriented interest of functionally benchmarking the products that are applied on burn patient wounds for the global enhancement of burn care quality.

Abstract Omics-based biomarker technologies, including metabolic profiling (metabolomics/metabonomics) and lipidomics, are making a significant impact on disease understanding, drug development, and translational research. A wide range of patho-physiological processes involve lipids and monitoring changes in lipid abundance can give valuable insights into mechanisms of drug action, off target pharmacology and toxicity. Here we report changes, detected by untargeted LC–MS, in the plasma lipid profiles of male C57Bl/6JRj mice following the PO and IV administration of the epidermal growth factor receptor (EGFR) inhibitor gefitinib. Statistical analysis of the data obtained for both the IV and PO samples showed time-related changes in the amounts of lipids from several different classes. The largest effects were associated with a rapid onset of these changes following gefitinib administration followed by a gradual return by 24 h post dose to the type of lipid profile seen in predose samples. Investigation of the lipids responsible for the variance observed in the data showed that the PI, PC, LPC, PE and TG were subject to the largest disruption with both transient increases and decreases in relative amounts seen in response to administration of the drug. The pattern of the changes in the relative abundances of those lipids subject to variation appeared to be correlated to the pharmacokinetics of gefitinib (and its major metabolites). These observations support the concept of a distinct pharmacolipidodynamic relationship between drug exposure and plasma lipid abundance.

Abstract We conducted a retrospective population-based, matched cohort study using the National Health Insurance Research Database to estimate healthcare resource utilisation (HRU) and costs in patients with newly diagnosed AL amyloidosis in Taiwan. Cases were matched 10:1 by age, sex, and area of residence to patients without AL amyloidosis (comparators) randomly selected from the database during the same time period. Annual all-cause HRU and costs for 3 years were quantified. AL amyloidosis-attributable costs were obtained by subtracting all-cause HRU costs incurred by comparators from cases. The mean age of all patients was 60.78 years and 59.07% were male. Co-morbidities were more frequent in cases than comparators. By 6 months after diagnosis, 12.1% of cases had died versus 0.9% of comparators. In the first year, cases had 103% more outpatient visits, 177% more emergency room visits, were hospitalised 4-times more frequently, and spent 5.5-times more days in hospital than comparators, and total healthcare costs were > sixfold higher. Costs incurred during the first year after diagnosis accounted for 55% of the 3-year cumulative cost. High HRU costs associated with delayed diagnosis and end-organ damage indicate a need for earlier diagnosis and more effective treatments for AL amyloidosis.

Elizabeth Whiting,1– 3 A Curtis Lee,4 Balakrishnan R Nair5,6 1Cross College Examination Review Advisory Group, Royal Australasian College of Physicians, Sydney, NSW, Australia; 2Faculty of Medicine, University of Queensland, Brisbane, Qld, Australia; 3Internal Medicine Services, The Prince Charles Hospital, Brisbane, Qld, Australia; 4School of Medicine, University of Notre Dame, Sydney, NSW, Australia; 5School of Medicine, University of Newcastle, Newcastle, NSW, Australia; 6Centre for Medical Professional Development, Hunter New England Local Health District, Newcastle, NSW, AustraliaCorrespondence: Elizabeth Whiting, Internal Medicine Services, The Prince Charles Hospital, Rode Road, Chermside, Brisbane, Queensland, 4032, Australia, Tel +61 419730908, Email elizabeth.whiting@health.qld.gov.auPurpose: The Royal Australasian College of Physicians (RACP) oversees physician training across Australia and Aotearoa New Zealand. Success in a written examination and clinical skills assessment (known as the clinical examination) at the mid-point of training is a requirement to progress from basic to advanced training. The clinical examination had evolved over many years without a review process. This paper describes the approach taken, the changes made and the evaluation undertaken as part of a formal review.Methods: A working party that included education experts and examiners experienced in the assessment of clinical skills was established. The purpose of the clinical examination and competencies being assessed were clarified and were linked to learning objectives. Significant changes to the marking and scoring approaches resulted in a more holistic approach to the assessment of candidate performance with greater transparency of standards. Evaluation over a 2-year period was undertaken before the adoption of the new approach in 2019.Results: In 2017 testing of a new marking rubric occurred during the annual examination cycle which confirmed feasibility and acceptability. The following year an extensive trial utilising the new marking rubric and a new scoring approach took place involving 1142 examiners, 880 candidates and 5280 scoresheets which led to some minor modifications to the scoring system. The final marking and scoring approaches resulted in unchanged pass rates and improved inter-rater reliability. Feedback from examiners confirmed that the new marking and scoring approaches were easier to use and enabled better feedback on performance for candidates.Conclusion: The refresh of the RACP clinical examination has resulted in an assessment that has clarity of purpose, is linked to learning objectives, has greater transparency of expected standards, has improved inter-rater reliability, is well accepted by examiners and enables feedback on examination performance to candidates.Keywords: physician assessment, reliability, clinical assessment, long case, short case

Abstract Background Understanding how trees develop their root systems is crucial for the comprehension of how wildland and urban forest ecosystems plastically respond to disturbances such as harvest, fire, and climate change. The interplay between the endogenously determined root traits and the response to environmental stimuli results in tree adaptations to biotic and abiotic factors, influencing stability, carbon allocation, and nutrient uptake. Combining the three-dimensional structure of the root system, with root morphological trait information promotes a robust understanding of root function and adaptation plasticity. Low Magnetic Field Digitization coupled with AMAPmod (botAnique et Modelisation de l’Architecture des Plantes) software has been the best-performing method for describing root system architecture and providing reliable measurements of coarse root traits, but the pace and scale of data collection remain difficult. Instrumentation and applications related to Terrestrial Laser Scanning (TLS) have advanced appreciably, and when coupled with Quantitative Structure Models (QSM), have shown some potential toward robust measurements of tree root systems. Here we compare, we believe for the first time, these two methodologies by analyzing the root system of 32-year-old Pinus ponderosa trees. Results In general, at the total root system level and by root-order class, both methods yielded comparable values for the root traits volume, length, and number. QSM for each root trait was highly sensitive to the root size (i.e., input parameter PatchDiam) and models were optimized when discrete PatchDiam ranges were specified for each trait. When examining roots in the four cardinal direction sectors, we observed differences between methodologies for length and number depending on root order but not volume. Conclusions We believe that TLS and QSM could facilitate rapid data collection, perhaps in situ, while providing quantitative accuracy, especially at the total root system level. If more detailed measures of root system architecture are desired, a TLS method would benefit from additional scans at differing perspectives, avoiding gravitational displacement to the extent possible, while subsampling roots by hand to calibrate and validate QSM models. Despite some unresolved logistical challenges, our results suggest that future use of TLS may hold promise for quantifying tree root system architecture in a rapid, replicable manner.

Abstract Background Traumatic brain injury (TBI) is a significant risk factor for Alzheimer’s disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects of antagonizing CLIP in the 5xFAD Alzheimer’s mouse model, with and without TBI. Methods 12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP) or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometric analysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonance diffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over the ensuing 6 months. Results 9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in 5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all of these FPI effects.

Abstract Tumor-Infiltrating Lymphocytes (TILs) have strong prognostic and predictive value in breast cancer, but their visual assessment is subjective. To improve reproducibility, the International Immuno-oncology Working Group recently released recommendations for the computational assessment of TILs that build on visual scoring guidelines. However, existing resources do not adequately address these recommendations due to the lack of annotation datasets that enable joint, panoptic segmentation of tissue regions and cells. Moreover, existing deep-learning methods focus entirely on either tissue segmentation or cell nuclei detection, which complicates the process of TILs assessment by necessitating the use of multiple models and reconciling inconsistent predictions. We introduce PanopTILs, a region and cell-level annotation dataset containing 814,886 nuclei from 151 patients, openly accessible at: sites.google.com/view/panoptils . Using PanopTILs we developed MuTILs, a neural network optimized for assessing TILs in accordance with clinical recommendations. MuTILs is a concept bottleneck model designed to be interpretable and to encourage sensible predictions at multiple resolutions. Using a rigorous internal-external cross-validation procedure, MuTILs achieves an AUROC of 0.93 for lymphocyte detection and a DICE coefficient of 0.81 for tumor-associated stroma segmentation. Our computational score closely matched visual scores from 2 pathologists (Spearman R = 0.58–0.61, p

Abstract Coordination of neuronal differentiation with expansion of the neuroepithelial/neural progenitor cell (NEPC/NPC) pool is essential in early brain development. Our in vitro and in vivo studies identify independent and opposing roles for two neural-specific and differentially expressed non-coding RNAs derived from the same locus: the evolutionarily conserved lncRNA Rncr3 and the embedded microRNA miR124a-1. Rncr3 regulates NEPC/NPC proliferation and controls the biogenesis of miR124a, which determines neuronal differentiation. Rncr3 conserved exons 2/3 are cytosine methylated and bound by methyl-CpG binding protein MeCP2, which restricts expression of miR124a embedded in exon 4 to prevent premature neuronal differentiation, and to orchestrate proper brain growth. MeCP2 directly binds cytosine-methylated Rncr3 through previously unrecognized lysine residues and suppresses miR124a processing by recruiting PTBP1 to block access of DROSHA-DGCR8. Thus, miRNA processing is controlled by lncRNA m5C methylation along with the defined m5C epitranscriptomic RNA reader protein MeCP2 to coordinate brain development.

Summary: Background: CVN424 is a GPR6 inverse agonist that provides selective pharmacological control of the indirect striatopallidal pathway. We assessed the safety and efficacy of CVN424 as an adjunctive treatment to levodopa for reducing OFF-time in individuals with Parkinson's disease (PD) experiencing motor-fluctuations. Methods: This was a randomised, double-blind, placebo-controlled study conducted at 21 sites across the United States to evaluate two doses of CVN424 (NCT04191577). Patients with PD (Hoehn and Yahr stages 2–4) who were on a stable dose of levodopa and experiencing ≥2 h of daily OFF-time were randomised (1:1:1) to receive either once-daily CVN424 (50 mg or 150 mg) or placebo for a 28-day treatment period. The primary endpoints were safety and tolerability. The key secondary endpoint was the change from baseline to Day 27 in OFF-time. Findings: The study was conducted from December 23, 2019, to October 14, 2021. Out of 198 participants screened, 141 eligible participants were randomised to one of the three treatment groups (n = 47 per group), and 127 participants completed the 28-day treatment period. The most common treatment emergent adverse events (TEAEs) were headache (2% with CVN424 50 mg, 9% with CVN424 150 mg, and 2% with placebo) and nausea (4% with CVN424 50 mg, 6% with CVN424 150 mg and 2% with placebo). No serious treatment-related adverse events were reported. On Day 27, the mean ± standard deviation (SD) change from baseline in daily OFF-time was −1.3 ± 3.0 h in the CVN424 50 mg group, −1.6 ± 2.5 h in the CVN424 150 mg group, and −0.5 ± 2.9 h in the placebo group. The placebo-adjusted LS mean ± standard error (SE) treatment difference was significant for the CVN424 150 mg dose (1.3 ± 0.56 h, [95 CI% −2.41 to −0.19], nominal p = 0.02). Interpretation: Treatment with CVN424 was safe and well-tolerated. Despite the short study duration and small sample size, the 150 mg CVN424 dose provided a clinically meaningful reduction in daily OFF-time. This study supports the development of CVN424 for the treatment of PD. Funding: Cerevance.

People who experience a stroke are at a higher risk of recurrent stroke when compared with people who have not had a stroke. Addressing modifiable risk factors like physical inactivity and poor diet has been shown to improve blood pressure, a leading contributor to stroke. However, survivors of stroke often experience challenges with accessing risk reduction services including long wait lists, difficulty with transportation, fatigue, impaired function, and diminished exercise capacity. Providing health interventions via a website can extend the reach when compared with programs that are only offered face to face or via real-time telehealth. Given global challenges of accessing secondary prevention programs, it is important to consider alternative ways that this information can be made available to survivors of stroke worldwide. Using the “design thinking” framework and drawing on principles of the integrated knowledge translation approach, we adapted 2 co-designed telehealth programs called i-REBOUND – Let’s get moving (physical activity intervention) and i-REBOUND – Eat for health (diet Intervention) to create the i-REBOUND after stroke website. The aim of this paper is to describe the systematic process undertaken to adapt resources from the telehealth delivered i-REBOUND – Let’s get moving and i-REBOUND – Eat for health programs to a website prototype with a focus on navigation requirements and accessibility for survivors of stroke. We engaged a variety of key stakeholders with diverse skills and expertise in areas of stroke recovery, research, and digital health. We established a governance structure, formed a consumer advisory group, appointed a diverse project team, and agreed on scope of the project. Our process of adaptation had the following 3 phases: (1) understand, (2) explore, (3) materialize. Our approach considered the survivor of stroke at the center of all decisions, which helped establish guiding principles related to our prototype design. Careful and iterative engagement with survivors of stroke together with the application of design thinking principles allowed us to establish the functional requirements for our website prototype. Through user testing, we were able to confirm the technical requirements needed to build an accessible and easy-to-navigate website catering to the unique needs of survivors of stroke. We describe the process of adapting existing content and co-creating new digital content in partnership with, and featuring, people who have lived experience of stroke. In this paper, we provide a road map for the steps taken to adapt resources from 2 telehealth-delivered programs to a website format that meets specific navigation and accessibility needs of survivors of stroke.

Oliver Davidson,1,2 Anand E Rajesh,1,2 Marian Blazes,1,2 Ashley Batchelor,1,2 Aaron Y Lee,1,2 Cecilia S Lee,1,2,&ast; Laura C Huang1,3,&ast; On behalf of the IRIS® Registry Analytic Center Consortium1Department of Ophthalmology, University of Washington, Seattle, WA, USA; 2Roger and Angie Karalis Johnson Retina Center, Seattle, WA, USA; 3Division of Ophthalmology, Seattle Children’s Hospital, Seattle, WA, USA&ast;These authors contributed equally to this workCorrespondence: Laura C Huang, Department of Ophthalmology, Seattle Children’s Hospital and University of Washington, OA.9.220, 4800 Sand Point Way NE, Seattle, WA, USA, Tel +1 206-987-2177, Email lchuang1@uw.eduPurpose: Understanding sociodemographic factors associated with poor visual outcomes in children with juvenile idiopathic arthritis-associated uveitis may help inform practice patterns.Patients and Methods: Retrospective cohort study on patients < 18 years old who were diagnosed with both juvenile idiopathic arthritis and uveitis based on International Classification of Diseases tenth edition codes in the Intelligent Research in Sight Registry through December 2020. Surgical history was extracted using current procedural terminology codes. The primary outcome was incidence of blindness (20/200 or worse) in at least one eye in association with sociodemographic factors. Secondary outcomes included cataract and glaucoma surgery following uveitis diagnosis. Hazard ratios were calculated using multivariable-adjusted Cox proportional hazards models.Results: Median age of juvenile idiopathic arthritis-associated uveitis diagnosis was 11 (Interquartile Range: 8 to 15). In the Cox models adjusting for sociodemographic and insurance factors, the hazard ratios of best corrected visual acuity 20/200 or worse were higher in males compared to females (HR 2.15; 95% CI: 1.45– 3.18), in Black or African American patients compared to White patients (2.54; 1.44– 4.48), and in Medicaid-insured patients compared to commercially-insured patients (2.23; 1.48– 3.37).Conclusion: Sociodemographic factors and insurance coverage were associated with varying levels of risk for poor visual outcomes in children with juvenile idiopathic arthritis-associated uveitis.Keywords: rheumatology, ophthalmology, uveitis, health inequity, social determinants of health

Alexandria TM Lee,1 Sai-Hong Ignatius Ou2 1Department of Medicine, University of California Irvine School of Medicine, Orange, CA, 92868, USA; 2Chao Family Comprehensive Cancer Center, Orange, CA, 92868, USACorrespondence: Sai-Hong Ignatius Ou, Department of Medicine, Division of Hematology-Oncology, Chao Family Comprehensive Cancer Center, University of California Irvine School of Medicine, 200 South Manchester, Suite 400, Orange, CA, 92868-3298, USA, Email siou@hs.uci.edu; ignatiusou@gmail.comAbstract: Selpercatinib, a potent and highly selective RET kinase inhibitor with significant CNS activity, has recently gained US approval for the treatment of NSCLC harboring RET fusions (RET+) based on a large-scale single-arm study. The LIBRETTO-431 trial was the global pivotal registration phase 3 trial comparing selpercatinib to platinum-based chemotherapy with or without pembrolizumab as the first-line treatment of patients with advanced RET+ NSCLC. Never-smokers constituted 67.4% of the RET+ NSCLC patients enrolled. KIF5B-RET made up the vast majority (77%) of the RET+ fusion variant with known fusion partner. The results of this study demonstrated significant improvement in progression-free survival (PFS) benefit as well as impressive intracranial disease response in participants treated with selpercatinib as compared to those treated with chemotherapy, with a HR [hazard ratio] of 0.46 (95% CI 0.33– 0.70; P < 0.001) for the intention-to-treat (ITT)-pembrolizumab group and HR of 0.46 (95% CI 0.31– 0.70, P < 0.001) for the overall ITT-group of patients. The addition of pembrolizumab to platinum/pemetrexed chemotherapy resulted in numerically identical PFS (11.2 months). These results point to selpercatinib’s superiority to traditional chemotherapy regimens in the treatment of NSCLC harboring RET fusions and add to literature on the salience of targeted precision oncology and lack of efficacy of immune checkpoint inhibitor in NSCLC patients with never-smoker predominant actionable driver mutations. RET+ NSCLC should be added to the list of molecular subtypes (EGFR+, ALK+, ROS1+) of NSCLC to be excluded in chemoimmunotherapy trial.Keywords: LIBRETTO-431, selpercatinib, RET fusion positive NSCLC, pralsetinib, immunotherapy in never-smokers, relative dose intensity

Abstract Background CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources. Methods We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β). Results We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions. Conclusions We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

Objective: The effects of Coronavirus disease 2019 (COVID-19) infection and altered processes of care on nonelective coronary artery bypass grafting (CABG) outcomes remain unknown. We hypothesized that patients with COVID-19 infection would have longer hospital lengths of stay and greater mortality compared with COVID-negative patients, but that these outcomes would not differ between COVID-negative and pre-COVID controls. Methods: The National COVID Cohort Collaborative 2020-2022 was queried for adult patients undergoing CABG. Patients were divided into COVID-negative, COVID-active, and COVID-convalescent groups. Pre-COVID control patients were drawn from the National Surgical Quality Improvement Program database. Adjusted analysis of the 3 COVID groups was performed via generalized linear models. Results: A total of 17,293 patients underwent nonelective CABG, including 16,252 COVID-negative, 127 COVID-active, 367 COVID-convalescent, and 2254 pre-COVID patients. Compared to pre-COVID patients, COVID-negative patients had no difference in mortality, whereas COVID-active patients experienced increased mortality. Mortality and pneumonia were higher in COVID-active patients compared to COVID-negative and COVID-convalescent patients. Adjusted analysis demonstrated that COVID-active patients had higher in-hospital mortality, 30- and 90-day mortality, and pneumonia compared to COVID-negative patients. COVID-convalescent patients had a shorter length of stay but a higher rate of renal impairment. Conclusions: Traditional care processes were altered during the COVID-19 pandemic. Our data show that nonelective CABG in patients with active COVID-19 is associated with significantly increased rates of mortality and pneumonia. The equivalent mortality in COVID-negative and pre-COVID patients suggests that pandemic-associated changes in processes of care did not impact CABG outcomes. Additional research into optimal timing of CABG after COVID infection is warranted.

Background Observational and retrospective studies suggest that people with narcolepsy may have an increased prevalence of cardiovascular and cardiometabolic comorbidities and may be at greater risk for future cardiovascular events. An expert consensus panel was formed to establish agreement on the risk of hypertension and cardiovascular/cardiometabolic disease in people with narcolepsy and to develop strategies to mitigate these risks. Methods and Results Experts in sleep medicine and cardiology were selected to participate in the panel. After reviewing the relevant literature, the experts identified key elements, drafted recommendation statements, and developed discussion points to provide supporting evidence for the recommendations. The draft and final recommendations were rated on a scale from 0 (not at all agree) to 4 (very much agree). All experts had an agreement rating of 4.0 for all 14 revised recommendation statements for patients with narcolepsy. These statements comprised 3 themes: (1) recognize the risk of hypertension and cardiovascular/cardiometabolic disease, (2) reduce the risk of hypertension and cardiovascular/cardiometabolic disease, and (3) reduce sodium intake to lower the risk of hypertension and cardiovascular disease. Conclusions These consensus recommendations are intended to increase awareness of potential cardiovascular/cardiometabolic risks in patients with narcolepsy for all clinicians. Early monitoring for, and prevention of, cardiovascular risks in this population are of great importance, especially as narcolepsy usually develops in adolescents and young adults, who will be exposed to adverse effects of the disease for decades. Prospective systematic studies are needed to determine association and causation of narcolepsy with cardiovascular/cardiometabolic disorders.

Objective To predict birth weight at various potential gestational ages of delivery based on data routinely available at the first antenatal visit.Design Individual participant data meta-analysis.Data sources Individual participant data of four cohorts (237 228 pregnancies) from the International Prediction of Pregnancy Complications (IPPIC) network dataset.Eligibility criteria for selecting studies Studies in the IPPIC network were identified by searching major databases for studies reporting risk factors for adverse pregnancy outcomes, such as pre-eclampsia, fetal growth restriction, and stillbirth, from database inception to August 2019. Data of four IPPIC cohorts (237 228 pregnancies) from the US (National Institute of Child Health and Human Development, 2018; 233 483 pregnancies), UK (Allen et al, 2017; 1045 pregnancies), Norway (STORK Groruddalen research programme, 2010; 823 pregnancies), and Australia (Rumbold et al, 2006; 1877 pregnancies) were included in the development of the model.Results The IPPIC birth weight model was developed with random intercept regression models with backward elimination for variable selection. Internal-external cross validation was performed to assess the study specific and pooled performance of the model, reported as calibration slope, calibration-in-the-large, and observed versus expected average birth weight ratio. Meta-analysis showed that the apparent performance of the model had good calibration (calibration slope 0.99, 95% confidence interval (CI) 0.88 to 1.10; calibration-in-the-large 44.5 g, −18.4 to 107.3) with an observed versus expected average birth weight ratio of 1.02 (95% CI 0.97 to 1.07). The proportion of variation in birth weight explained by the model (R2) was 46.9% (range 32.7-56.1% in each cohort). On internal-external cross validation, the model showed good calibration and predictive performance when validated in three cohorts with a calibration slope of 0.90 (Allen cohort), 1.04 (STORK Groruddalen cohort), and 1.07 (Rumbold cohort), calibration-in-the-large of −22.3 g (Allen cohort), −33.42 (Rumbold cohort), and 86.4 g (STORK Groruddalen cohort), and observed versus expected ratio of 0.99 (Rumbold cohort), 1.00 (Allen cohort), and 1.03 (STORK Groruddalen cohort); respective pooled estimates were 1.00 (95% CI 0.78 to 1.23; calibration slope), 9.7 g (−154.3 to 173.8; calibration-in-the-large), and 1.00 (0.94 to 1.07; observed v expected ratio). The model predictions were more accurate (smaller mean square error) in the lower end of predicted birth weight, which is important in informing clinical decision making.Conclusions The IPPIC birth weight model allowed birth weight predictions for a range of possible gestational ages. The model explained about 50% of individual variation in birth weights, was well calibrated (especially in babies at high risk of fetal growth restriction and its complications), and showed promising performance in four different populations included in the individual participant data meta-analysis. Further research to examine the generalisability of performance in other countries, settings, and subgroups is required.Trial registration PROSPERO CRD42019135045

Summary: Environmental enteric dysfunction (EED) is a subclinical enteropathy challenging to diagnose due to an overlap of tissue features with other inflammatory enteropathies. EED subjects (n = 52) from Pakistan, controls (n = 25), and a validation EED cohort (n = 30) from Zambia were used to develop a machine-learning-based image analysis classification model. We extracted histologic feature representations from the Pakistan EED model and correlated them to transcriptomics and clinical biomarkers. In-silico metabolic network modeling was used to characterize alterations in metabolic flux between EED and controls and validated using untargeted lipidomics. Genes encoding beta-ureidopropionase, CYP4F3, and epoxide hydrolase 1 correlated to numerous tissue feature representations. Fatty acid and glycerophospholipid metabolism-related reactions showed altered flux. Increased phosphatidylcholine, lysophosphatidylcholine (LPC), and ether-linked LPCs, and decreased ester-linked LPCs were observed in the duodenal lipidome of Pakistan EED subjects, while plasma levels of glycine-conjugated bile acids were significantly increased. Together, these findings elucidate a multi-omic signature of EED.

Background: Mobility impairment limits control of posture and body alignment. This leads to altered body shapes, co-occurring problems with pain and sleep, cardiopulmonary concerns, digestive health issues, and emergent health outcomes, which further complicate functions of daily living. 24-hour posture care management was developed to remedy these challenges by restoring body symmetry. Objective: To determine the feasibility of introducing posture care management to a rural-based, medically complex patient population, evaluate response of body symmetry, and examine its impact on pain and sleep quality. Methods: This pilot study employed a longitudinal, quasi-experimental study design from March 2016 to September 2018. The posture care management intervention introduced positioning support for use when lying down, a personalized training workshop for caregiver teams, and in-home initial and follow-up assessments to provide materials and collaboratively develop a personalized care plan. Participants were followed pre-post for 6–9 months. Results: A total of 73 participants enrolled in the study; 55 (75 %) completed. The majority were male (55 %) with a median age of 11. Most caregivers were immediate family members, and most participants had 1+ diagnosis characterized as a neurodevelopmental disorder. A majority of participants improved body symmetry (56–76 %), and 53 % with comparable information saw improvement in body symmetry with no worsening of pain or sleep quality. Conclusion: This study established the feasibility of administering posture care management in North America. These findings provide preliminary evidence of improvements in body symmetry and address concerns that posture care management can interfere with pain and sleep. Future research should consider levels of caregiver engagement and explore remote-monitored options of a posture care management intervention.

Following harvest, monitoring reforestation success is a crucial component of sustainable management. In Alberta, Canada, like other jurisdictions, the efficiency of the current plot-based forest regeneration monitoring regime is challenged by the cost of accessibility and the declining availability of qualified field crews. Fine spatial resolution imagery and deep learning have been proposed as alternative monitoring tools and have proven successful under experimental conditions, yet how successfully models can be applied and transferred between a range of untrained sites and conditions remains unclear.In this research, we repurposed a mask region-based convolutional neural network (Mask R–CNN) model that was previously trained to delineate coniferous tree crowns to instead segment instances of two species of regenerating conifers. We transferred learned parameters by replacing original single-class labels with photo-interpreted species information and retraining a selection of the network's parameters. We assessed the transferability of the new model by testing on five untrained sites, representing a range of forest types and densities typical of reforestation in the region. Results yielded a mean average precision (mAP) of 72% and average class F1 scores of 69% and 78% for lodgepole pine (Pinus contorta) and white spruce (Picea glauca), respectively, demonstrating successful transferability. We then investigated an additional transfer learning scenario by iteratively adding data from four of the five sites to the training set while reserving data from the remaining site for testing. On average, this improved mAP by 5%, lodgepole pine F1 by 7%, and white spruce F1 by 3%, and demonstrated that trained models can be continuously improved as sufficiently representative data becomes available.

Abstract Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by inducing K+ efflux as a key signaling event, suggesting that K+-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K+ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K+ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K+ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K+ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K+-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation. Graphical Abstract

Alexandria TM Lee,1 Sai-Hong Ignatius Ou1,2 1University of California Irvine School of Medicine, Department of Medicine, Orange, CA, USA; 2Chao Family Comprehensive Cancer Center, Orange, CA, USACorrespondence: Sai-Hong Ignatius Ou, Division of Hematology and Oncology, Department of Medicine, University of California Irvine School of Medicine, Chao Family Comprehensive Cancer Center, 200 South Manchester Ave, Suite 400, Orange, CA, 92868, USA, Tel +1 714-456-5153, Email Ignatiusou@gmail.comAbstract: Despite the development and approval of seven anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) spanning over three “generations” since the discovery of ALK fusion positive (ALK+) non-small cell lung cancer (NSCLC), there remains intrinsic and acquired resistances to these approved TKIs. Currently, a fourth-generation (4G) ALK TKI, NVL-655, is being developed to attack some of the unmet needs such as compound resistance mutations in cis. However, EML4-ALK variant 3 and TP53 mutations are intrinsic genomic alterations that negatively modulate efficacy of ALK TKIs. Potentially, in the shifting landscape where lorlatinib should be the first-line ALK TKI of choice based on the CROWN trial, the central β-sheet &num;6 (Cβ 6) mutation ALK L1256F will be the potential acquired resistance mutation to lorlatinib which may be resistant to current ALK TKIs. Here we opine on what additional capacities a putative fifth-generation (5G) ALK TKI will need to possess if it can be achieved in one single molecule. We propose randomized trial schemas targeting some of the intrinsic resistance mechanisms that will lead to approval of a prototypic fifth-generation (5G) ALK TKI and actually be beneficial to ALK+ NSCLC patients rather than just design a positive pivotal superiority trial for the sole purpose of drug approval.Keywords: EML4-ALK variant 3a/b, TP53 mutation, ALK TKIs, fifth generation ALK TKI, ALK+ NSCLC, circulating tumor DNA, next-generation sequencing, Cβ 6 mutation

The hyperdiverse geometrid genus Eois Hübner, estimated to encompass more than 1,000 species, is among the most species-rich genera in all of Lepidoptera. While the genus has attracted considerable attention from ecologists and evolutionary biologists in recent decades, limited progress has been made on its alpha taxonomy. This contribution focuses on the Olivacea clade, whose monophyly has been recognized previously through molecular analyses. We attempt to define the clade from a morphological perspective and recognize the following species based on morphology and genomic data: E. olivacea (Felder & Rogenhofer); E. pseudolivacea Doan, sp. nov.; E. auruda (Dognin), stat. rev.; E. beebei (Fletcher, 1952), stat. rev.; E. boliviensis (Dognin), stat. rev.; and E. parumsimii Doan, sp. nov. Descriptions and illustrations of the immature stages of E. pseudolivacea reared from Piper (Piperaceae) in Ecuador are provided.

Abstract Background c-MYC and BCL2 positivity are important prognostic factors for diffuse large B-cell lymphoma. However, manual quantification is subject to significant intra- and inter-observer variability. We developed an automated method for quantification in whole-slide images of tissue sections where manual quantification requires evaluating large areas of tissue with possibly heterogeneous staining. We train this method using annotations of tumor positivity in smaller tissue microarray cores where expression and staining are more homogeneous and then translate this model to whole-slide images. Methods Our method applies a technique called attention-based multiple instance learning to regress the proportion of c-MYC-positive and BCL2-positive tumor cells from pathologist-scored tissue microarray cores. This technique does not require annotation of individual cell nuclei and is trained instead on core-level annotations of percent tumor positivity. We translate this model to scoring of whole-slide images by tessellating the slide into smaller core-sized tissue regions and calculating an aggregate score. Our method was trained on a public tissue microarray dataset from Stanford and applied to whole-slide images from a geographically diverse multi-center cohort produced by the Lymphoma Epidemiology of Outcomes study. Results In tissue microarrays, the automated method had Pearson correlations of 0.843 and 0.919 with pathologist scores for c-MYC and BCL2, respectively. When utilizing standard clinical thresholds, the sensitivity/specificity of our method was 0.743 / 0.963 for c-MYC and 0.938 / 0.951 for BCL2. For double-expressors, sensitivity and specificity were 0.720 and 0.974. When translated to the external WSI dataset scored by two pathologists, Pearson correlation was 0.753 & 0.883 for c-MYC and 0.749 & 0.765 for BCL2, and sensitivity/specificity was 0.857/0.991 & 0.706/0.930 for c-MYC, 0.856/0.719 & 0.855/0.690 for BCL2, and 0.890/1.00 & 0.598/0.952 for double-expressors. Survival analysis demonstrates that for progression-free survival, model-predicted TMA scores significantly stratify double-expressors and non double-expressors (p = 0.0345), whereas pathologist scores do not (p = 0.128). Conclusions We conclude that proportion of positive stains can be regressed using attention-based multiple instance learning, that these models generalize well to whole slide images, and that our models can provide non-inferior stratification of progression-free survival outcomes.

Abstract Background Low back pain (LBP) is one of the most disabling diseases and a major health issue. Despite the evidence of a link between paraspinal and gluteal muscle dysfunction and LBP, it is unknown whether aquatic exercises can lead to improvements in paraspinal and gluteal muscle morphology and function, and whether improvements in overall muscle health are associated with improvements in patients’ outcomes. The unique properties of water allow a water-based exercise program to be tailored to the needs of those suffering from LBP. This study uses magnetic resonance imaging (MRI) to investigate the effect of an aquatic exercise program versus standard exercise on 1) paraspinal and gluteal muscle size, quality and strength and 2) pain, disability, and psychological factors (pain related fear, depression, anxiety, sleep quality) in chronic LBP. Methods This study will include 34 participants with chronic non-specific LBP and moderate to severe disability, aged between 18 and 65, who will be randomly assigned (1:1) to the aquatic exercise group or land-based standard care exercise group. Both groups will receive 20 supervised sessions, twice per week over 10 weeks. MRIs will be obtained along the lumbosacral spine (L1-L5) and pelvis at the start and end of the intervention to assess the effect of each exercise intervention on paraspinal and gluteal muscle size and quality. Pre- to post-intervention changes in all outcomes between each group will be assessed, and the association between the changes in back muscle quality and clinical outcomes will be examined. Between-subjects repeated measure analysis of variance will be used to examine the changes in paraspinal muscle morphology over the different time points. Linear mixed models will be used to assess whether baseline scores can modify the response to the exercise therapy treatment. Discussion This study will determine if water-based exercises targeting the lower back and gluteal muscles can lead to important changes in muscle quality and function, and their possible relation with patients’ pain and functional improvements. Our findings will have strong clinical implications and provide preliminary data to design a community program to better support individuals with chronic LBP. Trial registration NCT05823857, registered prospectively on April 27th, 2023.

Introduction: Emergency medical service (EMS) resources are limited and should be reserved for incidents of appropriate acuity. Over-triage in dispatching of EMS resources is a global problem. Analysing patients that are not transported to hospital is valuable in contributing to decision-making models/algorithms to better inform dispatching of resources. The aim is to determine variables associated with patients receiving an emergency response but result in non-conveyance to hospital. Methods: A retrospective cross-sectional study was performed on data for the period October 2018 to September 2019. EMS records were reviewed for instances where a patient received an emergency response but the patient was not transported to hospital. Data were subjected to univariate and multivariate regression analysis to determine variables predictive of non-transport to hospital. Results: A total of 245 954 responses were analysed, 240 730 (97.88 %) were patients that were transported to hospital and 5 224 (2.12 %) were not transported. Of all patients that received an emergency response, 203 450 (82.72 %) patients did not receive any medical interventions. Notable variables predictive of non-transport were green (OR 4.33 (95 % CI: 3.55–5.28; p

Introduction: Prehospital care in many low- and middle-income countries is underdeveloped and needs strengthening for improved outcomes. Where formal prehospital care systems are under development, integration of a layperson first responder programme may help improve access for those in need. The World Health Organization recently developed the Community First Aid Responder (CFAR) learning program in support of this system, providing that it may require adaptation to be contextually suitable and sustainably implemented at country level. This study assesses a pilot WHO CFAR course in Kinshasa, Democratic Republic of Congo, to inform future rollouts and related research. Methods: We conducted a 3-day in-person pilot CFAR training with 42 purposively selected community health workers. Data collection involved quantitative and qualitative phases. The first consisted of structured pre- and post-training surveys, and a course evaluation by participants. The second consisted of two focus group discussions involving purposively selected community health workers in one group, and a convenience sample of course instructors and organisers in the other. Perceptions regarding course content, perceived knowledge acquisition and self-confidence gain were analysed using descriptive statistics for the quantitative data and content analysis for qualitative data. Results: Course participants were predominantly male (76.3 %) with a median age of 42 years and most (80.5 %) had no prior first aid training. Most were satisfied that the learning objectives were reached, the logistics were adequate, and that the content and teaching language were appropriately tailored to local context. The majority (94.7 %) found the 3-day duration insufficient. There was a significant self-confidence gain regarding first aid skills (average 17.9 % in pre- to 95.3 % in post-training, p

Knee cartilage has limited natural healing capacity, complicating the development of effective treatment plans. Current non-cell-based therapies (e.g., microfracture) result in poor repair cartilage mechanical properties, low durability, and suboptimal tissue integration. Advanced treatments, such as autologous chondrocyte implantation, face challenges including cell leakage and inhomogeneous distribution. Successful cell therapy relies on prolonged retention of therapeutic biologicals at the implantation site, yet the optimal integration of implanted material into the surrounding healthy tissue remains an unmet need. This study evaluated the effectiveness of a newly developed photo-curable adhesive hydrogel for cartilage repair, focusing on adhesion properties, integration performance, and ability to support tissue regeneration. The proposed hydrogel design exhibited significant adhesion strength, outperforming commercial adhesives such as fibrin-based glues. An in vivo goat model was used to evaluate the hydrogels’ adhesion properties and long-term integration into full-thickness cartilage defects over six months. Results showed that cell-free hydrogel-treated defects achieved superior integration with surrounding tissue and enhanced cartilage repair, with notable lateral integration. In vitro results further demonstrated high cell viability, robust matrix production, and successful cell encapsulation within the hydrogel matrix. These findings highlight the potential of adhesive hydrogel formulations to improve the efficacy of cell-based therapies, offering a potentially superior treatment for knee cartilage defects.

Background/Objectives: Biological products are emerging as therapeutic management options for intervertebral disc (IVD) degenerative affections and lower back pain. Autologous and allogeneic cell therapy protocols have been clinically implemented for IVD repair. Therein, several manufacturing process design considerations were shown to significantly influence clinical outcomes. The primary objective of this study was to preclinically qualify (chondrogenic potential, safety, resistance to hypoxic and inflammatory stimuli) cryopreserved primary progenitor cells (clinical grade FE002-Disc cells) as a potential cell source in IVD repair/regeneration. The secondary objective of this study was to assess the cell source’s delivery potential as cell spheroids (optimization of culture conditions, potential storage solutions). Methods/Results: Safety (soft agar transformation, β-galactosidase, telomerase activity) and functionality-related assays (hypoxic and inflammatory challenge) confirmed that the investigated cellular active substance was highly sustainable in defined cell banking workflows, despite possessing a finite in vitro lifespan. Functionality-related assays confirmed that the retained manufacturing process yielded strong collagen II and glycosaminoglycan (GAG) synthesis in the spheroids in 3-week chondrogenic induction. Then, the impacts of various process parameters (induction medium composition, hypoxic incubation, terminal spheroid lyophilization) were studied to gain insights on their criticality. Finally, an optimal set of technical specifications (use of 10 nM dexamethasone for chondrogenic induction, 2% O2 incubation of spheroids) was set forth, based on specific fine tuning of finished product critical functional attributes. Conclusions: Generally, this study qualified the considered FE002-Disc progenitor cell source for further preclinical investigation based on safety, quality, and functionality datasets. The novelty and significance of this study resided in the establishment of defined processes for preparing fresh, off-the-freezer, or off-the-shelf IVD spheroids using a preclinically qualified allogeneic human cell source. Overall, this study underscored the importance of using robust product components and optimal manufacturing process variants for maximization of finished cell-based formulation quality attributes.

Recent advancements in aesthetic medicine offer innovative cosmetic solutions to enhance patient skin quality and appearance. Advanced treatment options enable practitioners to effectively address skin aging signs, pigmentation imbalance, and loss of elasticity in ambulatory and home-based care regimens. Exosomes (nanoscale cell-derived vesicles) transport a variety of biomolecules and are pivotal in physiological intercellular communication. Importantly, exosomes have recently emerged as key endogenous players in tissular regeneration. More broadly (from an active ingredient purity standpoint), exosomes, stem cell secretomes, and cell culture-conditioned media have been clinically proven to exert multifaceted beneficial topical effects (anti-inflammatory, antioxidant, anti-aging, skin rejuvenation). Therefore, human, animal, and plant-derived exosomes or other refined sub-cellular biological fractions are gaining substantial interest within the aesthetic and cosmetic industries. Notably, such approaches are thought to be among the most promising novel contenders for advanced, biologically inspired skin prejuvenation and rejuvenation care. The present narrative review summarizes the latest clinically oriented research on exosomes and cell culture-conditioned media, highlighting their mechanisms of action in various topical applications. Furthermore, it explores the innovation landscape and currently commercially available products on the global cosmetic market and discusses the potential future applications of advanced, biologically inspired ingredients in the medical aesthetic industry.

Facial hyperpigmentation is a highly prevalent dermatological condition, characterized by dark spots on the skin resulting from excess melanin production. Hyperpigmentation significantly impacts patients’ quality of life and self-esteem. Current treatments often present disadvantages linked to poor product tolerability. A topical cosmetic approach combining three lightening active ingredients (tranexamic acid, niacinamide, vitamin C) offers a new option for treating dark spots on the skin. The present in-use test under dermatological control evaluated the clinical safety and efficacy of a cream and serum containing these three ingredients, formulated with hyaluronic acid for enhanced delivery, stability, and efficacy. A total of 22 Caucasian patients with facial hyperpigmentation, both male and female, aged between 45 and 67 years, applied the cream and serum for 8 weeks. Clinical assessments, colorimetric evaluations, standardized photography, and self-assessment questionnaires were used to measure outcomes. No serious adverse effects were recorded, and the incidence of local adverse effects was low, highlighting good tolerability of the investigated test items. In most participants, significant improvements in hyperpigmented areas were recorded. Clinical scoring by the dermatologist investigator indicated a statistically significant 13% reduction in color intensity and a 6% reduction in the size of dark spots after 8 weeks of treatment. Colorimetric evaluation showed a statistically significant 1% increase in luminosity (L* parameter) and an 8% improvement in the Individual Typological Angle (ITA°) in endpoint, indicating lighter skin spots. Subjective assessments reflected high user satisfaction, with 95% of participants noting improvements in skin hydration and luminosity, and 77% reporting a reduced appearance of dark spots. Overall, the present work supports the use of tranexamic acid, niacinamide, and vitamin C as an effective and well-tolerated combined topical management option for hyperpigmentation. This combination offers a viable alternative to classical whiteners for individuals seeking to reduce facial skin coloration imbalance and improve skin tone.

Cartilage repair remains a major challenge in human orthopedic medicine, necessitating the application of innovative strategies to overcome existing technical and clinical limitations. Adhesive hydrogels have emerged as promising candidates for cartilage repair promotion and tissue engineering, offering key advantages such as enhanced tissue integration and therapeutic potential. This comprehensive review navigates the landscape of adhesive hydrogels in cartilage repair, discussing identified challenges, shortcomings of current treatment options, and unique advantages of adhesive hydrogel products and scaffolds. While emphasizing the critical need for in situ lateral integration with surrounding tissues, we dissect current limitations and outline future perspectives for hydrogel scaffolds in cartilage repair. Moreover, we examine the clinical translation pathway and regulatory considerations specific to adhesive hydrogels. Overall, this review synthesizes the existing insights and knowledge gaps and highlights directions for future research regarding adhesive hydrogel-based devices in advancing cartilage tissue engineering.

Traditional questionnaires do not capture the complexity of how people are viewed by others and grouped into categories on the basis of what is inferred (or not) about them. This is critical in applying an intersectionality framework in research because people are negatively impacted because of “who they are” but also based on “how others see them.” The purpose of this project was to develop and validate a questionnaire, grounded in intersectionality theory and a nuanced understanding of social position, that can be applied in large-scale, population-based surveys and studies.Drawing on 61 existing quantitative surveys collecting identity-based information and 197 qualitative studies on intersectionality describing the complex ways in which people's social positions are constructed and experienced, we created a draft questionnaire comprising five parts: 1) Sex and Gender, 2) Sexuality and Sexual Orientation, 3) Cultural Context, 4) Disability, Health, and Physical Characteristics, and 5) Socioeconomic Status. A draft of the questionnaire was then reviewed by experts via the Delphi process, which gauged the accessibility of the questionnaire (e.g., language used, length) and the relevance of its content using a 5-point scale and open-ended questions. These responses were ranked, analyzed, and synthesized to refine the questionnaire and, ultimately, to obtain ≥75 % consensus on each questionnaire item and response option.The SAFE questionnaire provides an opportunity to take a significant step forward in advancing our understanding of the complex, intersectional nature of social participation and marginalization.

Objectives To evaluate the effectiveness of COVID-19 vaccinations (initial and booster) during pre-Delta, Delta and Omicron dominant periods among pregnant people via (1) COVID-19 incident and severe infections among pregnant people who were vaccinated versus unvaccinated and (2) post-COVID-19 vaccination breakthrough infections and severe infections among vaccinated females who were pregnant versus non-pregnant.Design Retrospective cohort study using nationally sampled electronic health records data from the National COVID Cohort Collaborative, 10 December 2020 –7 June 2022.Participants Cohort 1 included pregnant people (15–55 years) and cohort 2 included vaccinated females of reproductive age (15–55 years).Exposures (1) COVID-19 vaccination and (2) pregnancy.Main outcome measures Adjusted HRs (aHRs) for COVID-19 incident or breakthrough infections and severe infections (ie, COVID-19 infections with related hospitalisations).Results In cohort 1, 301 107 pregnant people were included. Compared with unvaccinated pregnant people, the aHRs for pregnant people with initial vaccinations during pregnancy of incident COVID-19 were 0.77 (95% CI 0.62 to 0.96) and 0.88 (95% CI 0.73 to 1.07) and aHRs of severe COVID-19 infections were 0.65 (95% CI 0.47 to 0.90) and 0.79 (95% CI 0.51 to 1.21) during the Delta and Omicron periods, respectively. Compared with pregnant people with full initial vaccinations, the aHR of incident COVID-19 for pregnant people with booster vaccinations was 0.64 (95% CI 0.58 to 0.71) during the Omicron period. In cohort 2, 934 337 vaccinated people were included. Compared with vaccinated non-pregnant females, the aHRs of severe COVID-19 infections for people with initial vaccinations during pregnancy was 2.71 (95% CI 1.31 to 5.60) during the Omicron periods.Conclusions Pregnant people with initial and booster vaccinations during pregnancy had a lower risk of incident and severe COVID-19 infections compared with unvaccinated pregnant people across the pandemic stages. However, vaccinated pregnant people still had a higher risk of severe infections compared with non-pregnant females.

Abstract Background The gut microbiota has recently attracted attention as a pathogenic factor in Alzheimer’s disease (AD). Microfold (M) cells, which play a crucial role in the gut immune response against external antigens, are also exploited for the entry of pathogenic bacteria and proteins into the body. However, whether changes in M cells can affect the gut environments and consequently change brain pathologies in AD remains unknown. Methods Five familial AD (5xFAD) and 5xFAD-derived fecal microbiota transplanted (5xFAD-FMT) naïve mice were used to investigate the changes of M cells in the AD environment. Next, to establish the effect of M cell depletion on AD environments, 5xFAD mice and Spib knockout mice were bred, and behavioral and histological analyses were performed when M cell-depleted 5xFAD mice were six or nine months of age. Results In this study, we found that M cell numbers were increased in the colons of 5xFAD and 5xFAD-FMT mice compared to those of wild-type (WT) and WT-FMT mice. Moreover, the level of total bacteria infiltrating the colons increased in the AD-mimicked mice. The levels of M cell-related genes and that of infiltrating bacteria showed a significant correlation. The genetic inhibition of M cells (Spib knockout) in 5xFAD mice changed the composition of the gut microbiota, along with decreasing proinflammatory cytokine levels in the colons. M cell depletion ameliorated AD symptoms including amyloid-β accumulation, microglial dysfunction, neuroinflammation, and memory impairment. Similarly, 5xFAD-FMT did not induce AD-like pathologies, such as memory impairment and excessive neuroinflammation in Spib −/− mice. Conclusion Therefore, our findings provide evidence that the inhibiting M cells can prevent AD progression, with therapeutic implications.

Not available.

Although rarely fatal, Chikungunya virus (CHIKV) infection can lead to chronic debilitating sequelae. We describe the outbreak of suspected CHIKV in 93 subjects who presented voluntarily over 2 months to a remote rural Haitian general medical clinic staffed by international healthcare providers. Diagnosis was made on clinical signs and symptoms, as no serum analysis was available in this remote rural site. The subjects were 18.0 ± 16.2 (median±standard deviation) years of age and were of similar gender distribution. The presenting vital signs included a temperature of 102.3°± 0.6F with fever lasting for 3.0 ± 0.7 days. Symptoms mainly consisted of symmetrical polyarthralgia in 82.8%, headache in 28.0%, abdominal pain in 17.2%, cough in 8.6%, maculopapular rash in 30.0%, and extremity bullae in 12.9%. In 84.9% of subjects, symptoms persisted for 7.1 ± 8.3 days with 16.1% having ongoing disability due to persistent pain (?14 day’s duration). There were no deaths. In Haiti, especially in remote, rural regions, the risk for CHIKV spread is high given the shortage of detection methods and treatment in this tropical climate. Preventative efforts are similarly lacking. Implications for a global public health impact are likely with outbreak extension and spread to neighboring countries.

Introduction Gastrointestinal hospitalisations in the USA cause over US$130 billion in expenditures, and acute pancreatitis is a leading cause of these hospitalisations. Adequate pain control is one of the primary treatment goals for acute pancreatitis. Though opioids are commonly used for analgesia in these patients, there have been concerns about short-term and long-term side effects of using opioids. Recently, non-opioid medications have been studied to treat pain in patients with acute pancreatitis. This systematic review and network meta-analysis aims to assess the comparative efficacy of analgesic medication for non-severe, acute pancreatitis.Methods and analysis We will search multiple electronic databases for randomised controlled trials that study pain management in patients with non-severe, acute pancreatitis. The intervention will be any analgesic for acute pancreatitis in the hospital setting. The comparison group will be patients who received a placebo or other active interventions for pain management. The primary outcomes of interest include pain scores and the need for supplementary analgesia. The secondary outcomes will be serious adverse events, local complications, progression to severe pancreatitis, transfer to the intensive care unit, length of hospitalisation, time to start enteral feeds, 30-day all-cause mortality and Quality of Life Scale scores. If sufficient homogeneity exists among included studies, the findings will be pooled using a traditional pairwise and network meta-analysis. The risk of bias in randomised control trials will be evaluated using the Cochrane Risk of Bias Tool 2.0. The Grading of Recommendations, Assessment, Development, and Evaluation approach will be used to report the certainty of evidence.Ethics and dissemination This systematic review will not involve direct contact with human subjects. The findings of this review will be published in a peer-reviewed journal. They will give healthcare providers a better awareness of the optimal analgesic medication for pain treatment in non-severe, acute pancreatitis.

Background: There is little and heterogeneous knowledge on the links between the temperamental predispositions of psychopathology and the contemporary dimensional models of psychopathology, such as the Hierarchical Taxonomy of Psychopathology (HiTOP) classification system, which can be aligned with the five-factor model (FFM) of personality. This meta-analysis seeks to expand the temperamental theoretical basis of the HiTOP model by incorporating associations of temperament traits of two temperamental theories measured, respectively, by the Temperament and Character Inventory (TCI) and the Temperament Evaluation of Memphis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A) with (a) the FFM’s personality domains and (b) HiTOP’s five psychopathological spectra. Methods: A systematic search was done on Web of Science, Scopus, PubMed, ProQuest, Cochrane Database, and Google Scholar for all articles published in English from January 1990 to August 2020. Because of heterogeneity in the results of almost 70% of studies, pooled estimates of correlation coefficients were calculated using the random-effects method. Risk of bias (low-quality studies) and publication bias are reported. Results: The pooled correlations obtained from the analysis of 35 studies showed that the temperamental profile associated with each FFM domain and HiTOP spectra is distinct. Specifically, TCI-harm avoidance (HA) and all TEMPS temperaments were more strongly related to neuroticism/internalizing, extraversion/low detachment, and conscientiousness/disinhibition. In contrast, TCI-novelty seeking was more strongly related to both disinhibited/antagonistic externalizing and thought disorder. Conclusions: A large body of research supports maladaptive variants of all FFM domains and some psychopathological spectra of HiTOP related to the abnormal-range temperaments.

Solid sorbents based on silica and polyethyleneimine (PEI) are intensively investigated in the field of carbon capture and storage (CCS). Pyrolysis was proposed as a thermal process to recover the pure silica from exhausted sorbents and convert PEI into potentially useful products, such as alkylated pyrazines. A GC–MS method based on internal standardisation with 2-methoxypyrazine was developed and evaluated to determine the concentration of six pyrazines in the pyrolysis oils of exhausted silica-PEI sorbent pyrolysed at 400, 500, 600 and 650 °C. The most abundant pyrazines were 2-ethyl and 2,3-dimethyl, occurring at concentrations of 5–28 mg g–1, followed by pyrazine, 2-methyl, 2-ethyl-3-methyl and 2-propylpyrazine. The GC–MS results were compared to those from a HPLC-DAD method using the Welch's test. The 37 % discrepancy of concentrations was attributed to spectral interference in LC-DAD. GC was slightly less precise than HPLC, calibration errors were lower and enabled the identification of highly alkylated pyrazines. Both methods provided comparable values of total pyrazine yields (around 4–7 % by weight).

Declines in biodiversity generated by anthropogenic stressors at both species and population levels can alter emergent processes instrumental to ecosystem function and resilience. As such, understanding the role of biodiversity in ecosystem function and its response to climate perturbation is increasingly important, especially in tropical systems where responses to changes in biodiversity are less predictable and more challenging to assess experimentally. Using large-scale transplant experiments conducted at five neotropical sites, we documented the impacts of changes in intraspecific and interspecific plant richness in the genus Piper on insect herbivory, insect richness, and ecosystem resilience to perturbations in water availability. We found that reductions of both intraspecific and interspecific Piper diversity had measurable and site-specific effects on herbivory, herbivorous insect richness, and plant mortality. The responses of these ecosystem-relevant processes to reduced intraspecific Piper richness were often similar in magnitude to the effects of reduced interspecific richness. Increased water availability reduced herbivory by 4.2% overall, and the response of herbivorous insect richness and herbivory to water availability were altered by both intra- and interspecific richness in a site-dependent manner. Our results underscore the role of intraspecific and interspecific richness as foundations of ecosystem function and the importance of community and location-specific contingencies in controlling function in complex tropical systems.

Abstract Introduction To understand patient preferences around early abortion methods and care‐seeking during the coronavirus disease 2019 pandemic in a state that expanded insurance coverage for abortion care. Methods We conducted phone interviews between July 2021 and February 2022 with 50 Illinois residents (aged 18–45) who had recently sought abortion at 18 clinics throughout Illinois at or before 11 weeks of pregnancy. We used a semistructured interview guide to explore preferences, motivations and decision‐making around the abortion method. We coded transcripts and used code summaries to conduct a thematic content analysis. Results Half of the participants chose medication abortion, and half chose procedural abortion. Some participants relied upon their past abortion experiences to choose a method. Participants' reasons for choosing medication abortion included home setting with support persons, the noninvasive nature, desiring a more ‘natural’ experience or negative perceptions of procedural abortions. Participants choosing procedural abortions valued increased certainty of completion, the option of sedation and the defined timeline of a clinic visit with fewer physical side effects. Some participants without insurance coverage were motivated to select a method based on cost. Around half of the participants expressed interest in considering a telehealth abortion. Conclusions Patients cited complex and personal preferences influencing their method selection; when cost barriers were reduced, preferences centred physical or emotional experiences, setting, effectiveness and timing. As abortion access is increasingly restricted, many patients may still highly value a choice between medication and procedural abortion when possible.

Objectives The extent to which care quality influenced outcomes for patients hospitalised with COVID-19 is unknown. Our objective was to determine if prepandemic hospital quality is associated with mortality among Medicare patients hospitalised with COVID-19.Design This is a retrospective observational study. We calculated hospital-level risk-standardised in-hospital and 30-day mortality rates (risk-standardised mortality rates, RSMRs) for patients hospitalised with COVID-19, and correlation coefficients between RSMRs and pre-COVID-19 hospital quality, overall and stratified by hospital characteristics.Setting Short-term acute care hospitals and critical access hospitals in the USA.Participants Hospitalised Medicare beneficiaries (Fee-For-Service and Medicare Advantage) age 65 and older hospitalised with COVID-19, discharged between 1 April 2020 and 30 September 2021.Intervention/exposure Pre-COVID-19 hospital quality.Outcomes Risk-standardised COVID-19 in-hospital and 30-day mortality rates (RSMRs).Results In-hospital (n=4256) RSMRs for Medicare patients hospitalised with COVID-19 (April 2020–September 2021) ranged from 4.5% to 59.9% (median 18.2%; IQR 14.7%–23.7%); 30-day RSMRs ranged from 12.9% to 56.2% (IQR 24.6%–30.6%). COVID-19 RSMRs were negatively correlated with star rating summary scores (in-hospital correlation coefficient −0.41, p

Objectives The present study aimed to establish appropriate quality standards for emergency departments (EDQS) in Palestine.Methods The study comprised four phases. First, a comprehensive literature review was conducted to develop a framework for assessing healthcare services in EDs. Second, the initial set of EDQS was developed based on the review findings. Third, local experts provided feedback on the EDQS, suggesting additional standards, and giving recommendations. This feedback was analysed to create a preliminary set of EDQS. Finally, an expanded group of local emergency care experts evaluated the preliminary set, providing feedback on content and structure to contribute to the final set of EDQS.Findings We identified quality domains in EDs and categorised them into clinical and administrative pathways. The clinical pathway comprises 39 standards across 7 subdomains: triage, treatment, transportation, medication safety, patient flow and medical diagnostic services. Expert consensus was achieved on 87.5% of these standards. The administrative domain includes 64 consensus-based standards across 9 subdomains: documentation, information management systems, access-location, design, leadership, management, workforce staffing, training, equipment, supplies, capacity-resuscitation rooms, resources for a safe working environment, performance indicators and patient safety-infection prevention and control programmes.Conclusion This study employed a rigorous approach to identify QS for EDs in Palestine. The multiphase consensus process ensured the appropriateness of the developed EDQS. Inclusion of diverse perspectives enriched the content. Future studies will validate and refine the standards based on feedback. The EDQS has potential to enhance emergency care in Palestine and serve as a model for other regions facing similar challenges.

Abstract Invasive species offer insights into rapid adaptation to novel environments. The iconic cane toad (Rhinella marina) is an excellent model for studying rapid adaptation during invasion. Previous research using the mitochondrial NADH dehydrogenase 3 (ND3) gene in Hawai'ian and Australian invasive populations found a single haplotype, indicating an extreme genetic bottleneck following introduction. Nuclear genetic diversity also exhibited reductions across the genome in these two populations. Here, we investigated the mitochondrial genomics of cane toads across this invasion trajectory. We created the first reference mitochondrial genome for this species using long‐read sequence data. We combined whole‐genome resequencing data of 15 toads with published transcriptomic data of 125 individuals to construct nearly complete mitochondrial genomes from the native (French Guiana) and introduced (Hawai'i and Australia) ranges for population genomic analyses. In agreement with previous investigations of these populations, we identified genetic bottlenecks in both Hawai'ian and Australian introduced populations, alongside evidence of population expansion in the invasive ranges. Although mitochondrial genetic diversity in introduced populations was reduced, our results revealed that it had been underestimated: we identified 45 mitochondrial haplotypes in Hawai'ian and Australian samples, none of which were found in the native range. Additionally, we identified two distinct groups of haplotypes from the native range, separated by a minimum of 110 base pairs (0.6%). These findings enhance our understanding of how invasion has shaped the genetic landscape of this species.

Abstract Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either ‘cannibal cues’ from older conspecifics, or ‘alarm cues’ from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific‐derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.