MethodsX

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Objectives: To determine the impact of discharge home on physical activity and sedentary behaviour following orthopaedic trauma. Design: Observational study. Setting: Acute hospital. Participants: Between October 2022 and January 2024, 31 adult orthopaedic trauma patients were recruited during hospital admission. Participants had either an isolated hip fracture or multi-trauma (i.e., a lower limb fracture, with an upper limb and/or spinal fracture). Interventions: Participants wore two activity monitors (activPAL3 and ActiGraphGT3x) during the final days of an acute hospital admission and the first five days at home. An interrupted time series analysis evaluated changes physical activity variables during the hospital to home transition. Participants were analysed individually using mixed-effects linear regression allowing the intercept to vary by participant. Main outcome measures: Primary outcome was daily steps; secondary outcomes included sedentary time and other activity measures. Results: Daily steps (mean +- SD) were higher at home (4552.4 +- 2639.5) compared to hospital (2597.8 +- 1450.8). Modelled results indicated a 27% increase in daily steps following hospital discharge (exp(beta946;): 1.27, 95% CI: 1.01,1.59, p=0.039) and a sustained improvement at home. No significant differences were observed between hip fracture and multi-trauma participants. Conclusion: Participants recovering from orthopaedic trauma showed a significant increase in daily step count upon discharge home from hospital, highlighting the positive impact of the home environment on activity levels. Further research is warranted to assess the effectiveness of interventions to improve activity levels in hospital (e.g., early intensive therapy) and at home (e.g., immediate home-based physiotherapy) in individuals following orthopaedic trauma.

Aims: Explainable advanced electrocardiography (A-ECG) can be used to estimate heart age from the standard 12-lead ECG. A-ECG heart age gap (HAG) represents the difference between A-ECG heart age and chronological age. Increased A-ECG HAG is associated with cardiovascular outcomes and can be used to communicate risk. The aim was to investigate whether A-ECG heart age demonstrates acceptable within- and between-session reproducibility. Methods: Healthy adults (n=42, age 23+/-4 years, 52% male) attended up to two sessions ~14 days apart, with 36 participants completing both sessions. During each session, five standard resting 12-lead ECGs were obtained while lying in the supine position with unchanged electrode positions. A-ECG heart age was extracted using dedicated software. Within-session reproducibility was assessed using all five recorded ECGs with coefficient of variation (CV) and a two-way random effects intraclass correlation coefficient (ICC). Between-session reproducibility was assessed using the first recorded ECG of each session with a paired t-test, CV and ICC. A further analysis assessed the reproducibility of the parameters used in the A-ECG heart age regression model. Results: A-ECG heart age showed excellent within-session reproducibility in session one and two (both CV 5.8%, ICC 0.99). A-ECG heart age was slightly lower in session one than two (24.0+/-7.5 vs. 25.5+/-7.8 years, p=0.04) and showed good between-session reproducibility (CV 8.3%, ICC 0.84). All but one parameter used to estimate A-ECG heart age showed acceptable within- and between-session reproducibility (CV<10%). Conclusion: A-ECG heart age demonstrates excellent within-session reproducibility and good between-session reproducibility in healthy young adults.

Global healthcare is targeting patient-centred care, as it leads to better health outcomes and higher level of patient satisfaction. Patient-centred communication, is an important part of patient-centred care because it focuses on involving patients in their care. Recent surveys both nationally and globally have shown that patients are not involved enough in their own healthcare decisions. This problem is especially common among the elderly with chronic conditions. This study aimed to describe patient-healthcare professional interactions, expectations, and satisfaction in physiotherapy within an understudied context, thereby providing important, specific data on ICE dynamics and satisfaction in the specific setting. Cross-sectional study of participants in scheduled consultations was conducted. Two government physiotherapy centres, seven private physiotherapy centres and two trust centres with physiotherapy facilities in Gujarat, India. 232 patients (from various public and private physiotherapy clinics) participated in the study. Patients' ideas, concerns, expectations (ICE) and satisfaction were explored. Almost 88% of patients reported their thoughts and explanations about their symptoms during the consultation. Most patients described not having any concerns about the diagnosis/treatment, and more than two-third of patients consulting PTs expected explanation for their symptoms. Almost 90% patients were satisfied with the consultation. The study revealed that while most patients conveyed their thoughts during consultations, very few expressed their concerns. Overall, patients were satisfied with their consultations.

Recent progress in mathematical kinship modelling has allowed one to predict the probable numbers of kin for a typical population member. In the models, kin may be structured by age and sex, both in static or time-variant demographies. Knowing the probable numbers of kin in different stages - such as parity, health status, or geographic location - however, remains an open challenge in Kinship Demography. Knowing how population structure delimits kin to distinct stages is an advance - for instance, the probability of having one sister at home and one sister away has different social implications from the probability of having two sisters. We present a novel analytical framework, grounded in branching process theory, that provides kin-number distributions jointly structured by age and stage. Using recursive compositions of probability generating functions (PGFs), we derive the joint age, stage, and age x stage kin-number distributions. All marginal distributions over either dimension naturally emerge. Simple extensions of the PGF approach additionally yield: the joint distribution of an individuals own stage and their kins stage; the probable numbers of kin deaths, both in total and by generation number; and the probabilities of being kinless and/or orphaned. We demonstrate the framework through novel results in an application using UK parity-specific fertility and mortality data. HighlightsO_LIA new method calculates probability generating functions for the number of kin structured by age and stage C_LIO_LIThe model allows predicting the probable numbers of kin organised by age and stage C_LIO_LIRecursive nesting of probability generating functions in branching processes is used C_LIO_LIAn application is presented highlighting the novel results C_LI

Objective: A questionnaire survey was conducted on the willingness and demand for acupuncture treatment in patients with malignant tumors, and the possible factors affecting patients' willingness and demand for acupuncture treatment were explored. Methods: A voluntary, anonymous survey was conducted between February and May 2025 among patients with malignant tumors aged 18 years and older who visited Beijing Cancer Hospital. The questionnaire included 16 questions addressing three dimensions:current medical purposes,Traditional Chinese Medicine(TCM) literacy, and acupuncture treatment needs.The questionnaire was posted online and completed by respondents using a smartphone interface. Results: A total of 511 valid questionnaires were retrieved in the survey, and 481 patients(94.1%) are willing to receive acupuncture treatment. Among the 481 patients willing to receive acupuncture treatment, the top five symptoms they hoped to improve with acupuncture were: disturbed sleep (245 participants, 50.9%); pain (229 participants, 47.6%); fatigue (177 participants, 36.8%); numbness (165 participants, 34.3%); and poor appetite (144 participants, 29.9%). Among patients who chose to "explicitly accept" acupuncture treatment and those who "accepted acupuncture treatment upon doctor's recommendation", 55% and 56% respectively had good knowledge of traditional Chinese medicine (TCM) culture. In contrast, this proportion was only 36.7% among patients who refused acupuncture treatment, and the difference was statistically significant (P<0.05). The survey results also show that Female patients reported significantly higher demands for pain relief and improved sleep than male patients, with statistically significant differences (P<0.05). Furthermore, those aged 18-45 and with better TCM literacy were more likely to desire acupuncture to improve sleep, with statistically significant differences (P<0.05). Conclusion: Differences in TCM literacy can influence patients' willingness to choose acupuncture treatment. Strengthening patient health education and improving TCM literacy will help increase cancer patients' willingness to choose TCM acupuncture treatment, thereby enabling them to benefit from acupuncture. For patients aged 18-45, those with good TCM literacy female with high acupuncture needs, acupuncture treatment may be recommended as a priority.

Tox21 assays compile extensive chemical bioactivity data across diverse biological targets, making them widely utilized resources for in silico model development. Nuclear receptor-specific assays within this dataset are particularly valuable for screening potential endocrine disrupting chemicals. This study presents a comprehensive benchmarking of diverse machine learning (ML), deep learning (DL), and transformer-based architectures with varied chemical feature representations across nuclear receptor assays. First, 43 datasets associated with 18 nuclear receptors within Tox21 assays were systematically curated from ToxCast invitrodb v4.3. Upon testing across these datasets, model performance was found to be dependent on the degree of class imbalance. Tree-based ML models such as random forest (RF) and extreme gradient boosting (XGBoost) trained on descriptors, or combination of descriptors and fingerprints, consistently outperformed in datasets with higher proportions of active chemicals (>10%), while DL models showed greater robustness for those with moderate proportions (5-10%). Further analysis revealed that approximately 40% of misclassified active chemicals occupied structurally isolated regions of the chemical space, suggesting absence of close structural analogues in the training set potentially contributed to their misclassification. External validation using in vitro and in vivo androgen and estrogen receptor bioactivity data showed generally good concordance. Finally, a systematic literature review revealed that the models in this study span wider range of architectures, feature representations, and assay endpoints, and are broadly comparable to or better than existing work. Overall, insights from this study can inform the development of more reliable in silico tools supporting new approach methodologies for nuclear receptor bioactivity predictions.

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

Transcriptomic analyses are widely used to elucidate the molecular mechanisms driving gametogenesis and reproduction in fish, yet their accuracy depends heavily on appropriate normalization of gene expression data. Conventional approaches that rely on single or multiple reference genes are problematic during teleost oogenesis, as profound structural and physiological remodeling of the ovary challenges the assumption that commonly used reference transcripts remain stable. In this study, we assessed by qPCR the transcriptional variability of four widely used reference genes (actb, ef-1, gapdh, and 18S rRNA) throughout the oogenic cycle of the thicklip grey mullet (Chelon labrosus), using geNorm and NormFinder analyses, and we additionally evaluated total cDNA concentration as an alternative normalization factor. To examine the performance and interpretive consequences of each normalization strategy, we compared expression patterns of key steroidogenic genes (star, cyp19a1a, and cyp11b) normalized by individual reference genes, combinations of reference genes, or total cDNA concentration. All evaluated reference genes displayed notable transcriptional variability across oogenesis, confirming their limited suitability as sole internal controls. In contrast, normalization approaches integrating multiple reference genes and/or total cDNA concentration consistently provided greater stability and more reliable biological interpretation. These results support a refined and more robust normalization framework for transcriptional analyses in fish ovaries, particularly during stages of extensive tissue remodeling. Our findings demonstrate cDNA-based normalization is straightforward, rapid, and easy to implement across laboratories, providing a practical alternative for achieving accurate, reproducible transcript quantification in fish ovary studies.

Background/Objective: Common measures of physical activity (PA) based on duration and intensity do not fully capture its complexity. Adding additional PA components of muscle strength, mechanical strain, and turning actions, can provide a more complete view of activity behavior. Furthermore, PA behaviors differ between men and women. Therefore, the goal of this study is to identify and cluster similar long-term PA patterns over time for each PA component, examined separately for men and women. Methods: We used data from 4963 participants (52% women; mean age 66 years, SD = 8.6) of the Longitudinal Aging Study Amsterdam (1992 to 2019). PA component scores were assigned to self-reported activities, and Sequence Analysis with Optimal Matching was used to identify and cluster similar activity patterns over a period of 10 years, separately for each component and stratified by sex. Results: PA components varied by sex and displayed a unique mix of trajectories, including predominately low, medium, or high activity, increasing or decreasing patterns, and trajectories characterized by early or late mortality. Importantly, trajectories remained independent, indicating that changes in one PA component were not linked to changes in others. Conclusion: Older men and women follow distinct and independent long term PA trajectories across components, underscoring that PA behaviour cannot be described by a single dimension. Significance/Implications: The observed independence and heterogeneity of trajectories suggest that muscle strength, mechanical strain, and turning actions capture meaningful and distinct aspects of PA that are not reflected by traditional measures alone. Future PA-strategies could incorporate these dimensions and acknowledge sex-specific patterns to better reflect natural movement. The independence of components suggests that future interventions should target multiple dimensions, as changes in one component may not translate to others. Such an approach may support more tailored and sustainable PA interventions in later life.

Background/Objective: The Network Pain Rehabilitation Limburg (NPRL) was established to provide integrated, biopsychosocial-based rehabilitation care for patients with chronic musculoskeletal pain, emphasizing the delivery of appropriate care by the right person at the right place and cost. This study examines the perceived interprofessional collaboration practice (ICP) and work satisfaction among primary care healthcare professionals engaged in NPRL. Patients and Methods: A mixed-methods approach involved seven general practitioners (GPs), twenty-four therapists (physiotherapists and occupational therapists), and five mental health practice nurses in eleven semi-structured focus groups and one interview conducted from 2017 to 2020. The Interprofessional Collaboration Attainment Survey quantitatively measured healthcare professionals' ICP abilities before and after NPRL participation. Qualitative analysis, structured around existing ICP frameworks and the Quadruple Aim, was based on interview data. Results: Findings revealed stable ICP and work satisfaction, with discussions focusing on transitioning to a biopsychosocial perspective on chronic pain and its implications, along with concerns about GP burden and insurer reimbursement issues. Significant enhancements were noted in communication and team functioning (p < 0.05). Conclusions: Overall, healthcare professionals reported positive experiences with NPRL's integrated approach, showcasing dedication to providing rehabilitation care for chronic musculoskeletal pain in primary care. Recommendations for improving ICP included advocating for a broader societal biopsychosocial view of chronic pain, introducing case managers in primary care to support GPs, and exploring alternative reimbursement models with insurers. However, significant transformations to impact work satisfaction and ICP may necessitate more time and consideration.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@417649org.highwire.dtl.DTLVardef@1bcd186org.highwire.dtl.DTLVardef@15fecb3org.highwire.dtl.DTLVardef@acdf8d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

IntroductionIndividuals with COPD can be classified according to their levels of physical activity (PA) and physical capacity (PC). The relationship between nutrition and body composition within these classifications remains unclear. ObjectivesTo compare the body composition and food intake of people with COPD and verify the associations. MethodsCross-sectional exploratory analysis study in which body composition and food intake were assessed in individuals with COPD. Classification was based on six-minute walk test (PC) and accelerometry(PA): Quadrant "can do, dont do" (I-preserved PC, low PA); quadrant "can do, do do" (II-preserved PC, preserved PA). Results72 individuals with COPD, 39 in quadrant I and 33 in quadrant II, with mean ages of (69 {+/-} 6) (67 {+/-} 7), respectively. Group I had a higher proportion of males, whereas group II had a higher proportion of females. A positive trend in skeletal muscle mass (p=0.011) (B= 2.883) and a negative trend in basal metabolic rate (p=0.010) (B=-0.092) for group I. ConclusionBrazilians with COPD classified in quadrants I and II showed similar results in terms of body composition and food intake. A positive trend in skeletal muscle mass was observed for the group I. These findings align with the pathophysiological model of COPD, in which the preservation of muscle mass and adequate protein intake support functional capacity and the maintenance of higher physical activity levels.

BackgroundThe COVID-19 pandemic significantly disrupted healthcare services globally, particularly in low-resource settings. This study explores the impact of the pandemic on physiotherapy services in Nepal. MethodsA cross-sectional study was conducted. Qualitative data were collected through semi-structured interviews with 12 physiotherapists, while quantitative data were gathered from an onsite survey of 29 health facilities at six different districts of Province III of Nepal. Inductive thematic analysis approach was used to analyze the qualitative data, and descriptive statistics were used for the closed ended questions. ResultsThe findings were categorized into sub-themes under two major themes: i) Pandemic effect on physiotherapy services and patient care and ii) Adaptation, innovation and collaboration. The study revealed a significant disruption in physiotherapy services with a notable decline in patient flow and service availability. Most patients, especially those with disabilities and post-operative needs, experienced worsening conditions due to limited access to care. There was an increased recognition of the role of physiotherapy in acute respiratory care and post-COVID-19 recovery. Tele-rehabilitation was explored as an alternative care method but faced challenges in implementation. More than half (62.07%) of the centers reported uninterrupted physiotherapy services, whereas almost one third (31.03%) experienced service suspension. Most centers (89.7%) had personal protective equipment available, and majority (86.2%) of the physiotherapists worked in multidisciplinary team: fever clinics, triage, emergency care, respiratory physical therapy, and nursing and administrative support were among the expanded roles. Several centers (37.9%) used virtual care with telephone consultation serving as the primary modality. Virtual service was mostly absent in centers where in-person services persisted. ConclusionThe COVID-19 pandemic significantly impacted physiotherapy services in Nepal, leading to service disruptions and compromised patient care. It highlighted the need to further incorporate physiotherapy into the healthcare system and enhance rehabilitation services to improve continued patient care.

BACKGROUND: Cranioplasty is an essential procedure to restore cranial integrity, protect neural structures, and improve cosmetic outcomes. However, commercially available implants are often costly, limiting their accessibility in public healthcare systems. Three dimensional (3D) printing offers a low cost alternative for producing patient-specific solutions. METHODS: A retrospective case series of eight patients undergoing cranioplasty using customized polymethylmethacrylate (PMMA) implants fabricated with 3D printed molds was conducted. Computed tomography (CT) scans were used for segmentation and digital modeling. Patient specific molds were designed and printed preoperatively. Variables analyzed included design time, printing time, intraoperative workflow, and clinical outcomes. RESULTS: Design time ranged from approximately 1 hour for small defects to 3 hours for larger defects. Printing time ranged from 2 3 hours for smaller defects and up to 8 10 hours for larger reconstructions. Satisfactory aesthetic outcomes were achieved in 7 of 8 patients (87.5%). No major implant related complications were observed. CONCLUSION: Low cost 3D printing for PMMA cranioplasty is a feasible, accessible, and effective technique for cranial reconstruction, particularly in resource limited settings. Keywords: Cranioplasty; 3D printing; Cranial defect reconstruction; Low cost surgery; Patient specific implants; Polymethylmethacrylate; Skull reconstruction

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

Background Trials in occupational populations, such as surgeons, face feasibility challenges due to high workload, restricted availability, and clinical heterogeneity, which may compromise recruitment, adherence, and retention. Objective To prespecify the feasibility framework and progression criteria for an internal pilot phase embedded within a pragmatic randomized controlled trial (RCT) comparing Mechanical Diagnosis and Therapy with generalized exercise in surgeons with chronic spinal pain. Design Protocol for a prespecified internal pilot phase embedded within a pragmatic, two-arm, parallel-group RCT. Methods The internal pilot will include the first four months of recruitment and aims to randomize at least 12 participants. Feasibility will be assessed across predefined domains, i.e., recruitment, eligibility, consent, intervention uptake, adherence, retention, data completeness, and treatment fidelity. Each domain is operationally defined and linked to prespecified progression criteria to ensure interpretability and decision-making utility. Criteria will be interpreted collectively to guide trial continuation. A minimal qualitative process evaluation will be embedded. Ethics and dissemination The host trial has received ethical approval (N-20240046) and is registered at ClinicalTrials.gov (NCT07293130). The findings from the internal pilot will be reported in a separate feasibility manuscript.

Reliable interpretation of electrocardiograms (ECGs) requires precise identification of P, QRS, and T (PQRST) wave boundaries. However, it remains challenging due to noise, signal quality variability, and inherent morphological diversity particularly in recordings from children. This study systematically compares the performance of leading deep neural networks (DNN) and heuristic-based delineation algorithms on ambulatory single-lead ECG signals focusing on temporal accuracy. Experiments were conducted using the publicly available LUDB dataset and a private validation dataset comprising 21,759 annotated single-lead wave segments from 611 children recorded using KardiaMobile ECG sensor. DNN were first trained on the LUDB dataset and subsequently tested on the validation dataset. The delineation performance was assessed using Sensitivity (Se) and positive-predictive-value (P+) metrics. The best-performing heuristic based and DNN models reached Se and P+ of (98.9% vs 97.9%) for P, (99.8% vs 99.2%) for QRS, and (98.7% vs 95.9%) for T wave fiducials, respectively. The lowest standard-deviation (in ms) of wave onset/offset delineation was achieved by attention based 1DU-Net model; {+/-}16.6/{+/-}16.3 for P-wave, {+/-}14.0/{+/-}16.3 for QRS, and {+/-}26.3/{+/-}18.8 for T-wave, respectively. The findings indicate that optimized heuristic models can perform comparably to complex DNN, highlighting their efficiency and suitability for real-time ECG delineation in digital health monitoring applications.