Open Research Europe

High-speed atomic force microscopy (HS-AFM) experiments provide unique insights into nanoscale biological processes by directly visualizing the surface topography dynamics of biomolecules, such as proteins, over extended periods of time and generating large imaging data sets. However, the lack of computational methods for automated data analysis has thus far limited the full utilization of these data for the quantitative understanding of single-molecule dynamic. Here, we present the AFM Analysis Arena (A3) within BioAFMviewer as a software platform for automated, high-throughput quantitative analysis of AFM topographic imaging data within a user-friendly interactive graphical interface, implementing a convenient workflow from raw HS-AFM (AFM) imaging to statistical analysis. In several applications to HS-AFM imaging data, we demonstrate methods of automatized molecule detection and dynamic tracking to highlight the available capacities, aiming to transform HS-AFM measurements from observations towards an analytical tool in nanoscale biology.

Population genomic studies have revealed strong regional structure in Plasmodium knowlesi across Southeast Asia. To facilitate routine genomic surveillance, we developed a targeted amplicon sequencing panel that preserves the large- and small-scale population genetic structure. We screened published genomes for short, highly polymorphic targets and a 30-SNP barcode. We validated multiplexed amplification and sequencing using a portable Oxford Nanopore Technologies MinION sequencer on 28 clinical infections from four southern Thai provinces collected between 2018-2023. Twenty-seven loci yielded sequence data and 23 infections with data from at least eight loci were retained for analysis. Across the 23 Thai infections and 104 published Malaysian genomes, 586 diverse SNPs were observed. The amplicon panel and 30-SNP barcode retained the major population structure observed in genome-wide data, including a clear distinction between Bornean and Peninsular Malaysian infections and two clusters in Borneo. Across newly sequenced Thai infections, 25 loci were diverse and showed substantial diversity (mean expected heterozygosity = 0.69). All infections were effectively monoclonal. Pairwise identity-by-descent revealed substantial recent relatedness, with 46 of 253 pairs at or above 0.5. Two Thai genetic groups (n = 12 and n = 11) were observed, mirroring previously observed structure in peninsular Malaysia. Groups were not significantly associated with province or collection year. In conclusion, targeted sequencing can reveal geographically informative population structure across scales and thus support surveillance.

Cannabis sativa L. cultivation in tropical regions or under indoor conditions requires supplemental lighting to maintain plant yields. Night-break (NB) photoperiods offer a cost-effective alternative to conventional day extension, although genotypic responses have not been clearly described. This study evaluated the effects of continuous and pulsed NB treatments on the growth, phenology, physiology, productivity, and cannabinoid accumulation of three Cannabis sativa genotypes representing two chemotypes. Plants were exposed to a commercial 18-h photoperiod (T1), a continuous 1-h NB (T2), or four 15-min NB pulses (T3) with equal total duration. Continuous NB produced responses comparable to the commercial photoperiod. In contrast, pulsed NB accelerated phenological progression by two to three weeks and reduced final plant height by 15-28%, depending on genotype, without impairing photosynthetic performance or decreasing floral dry biomass. Harvest index increased under both NB treatments, indicating enhanced biomass allocation to reproductive structures, while cannabinoid accumulation remained primarily genotype-dependent with only minor treatment-specific modulation. These findings demonstrate that NB efficacy depends not only on the duration of nocturnal light but also on its temporal distribution, highlighting temporal organization of nocturnal light as a key regulator of photoperiodic responses and a promising strategy for optimizing cannabis cultivation.

Menstrual health remains a neglected public health challenge in India, where persistent stigma, incomplete education, inadequate sanitation, and limited access to menstrual health resources can adversely affect health and social participation. Institution-based educational programs represent an underexplored opportunity to improve menstrual health literacy while promoting more inclusive discussions surrounding menstruation. Bridging this gap, an exploratory, prospective educational evaluation was conducted at a hospital in Coimbatore, Tamil Nadu and an engineering college in Wayanad, Kerala. Anonymous pre- and post-course surveys assessed menstrual health knowledge, attitudes, practices, perceived barriers, and perspectives on emerging digital health technologies. Quantitative responses were summarized descriptively, and open-ended responses were reviewed to identify recurring perspectives that contextualized the quantitative findings. The standardized educational program integrated evidence-based menstrual health education with discussions of bioengineering and wearable technologies. Eighty-three participants completed the pre-course survey, 68 attended the educational program, and 20 completed the post-course survey. Because responses could not be linked at the individual level, the survey cohorts were compared descriptively. At baseline, 80 of 82 respondents (97.6%) recognized menstruation as a biological process, yet only 38 of 82 (46.3%) indicated that it was not solely a woman's issue. The mean agreement that menstruation remained taboo was 7.44 of 10 (SD 1.99; n=78), and 51 of 77 respondents (66.2%) reported that they or others avoided activities during menstruation. Painful periods were identified as a barrier by 56 of 67 respondents (83.6%), and 18 of 67 (26.9%) considered institutional or community toilets insufficiently clean and private for menstrual management. Open-ended responses illustrated how pain, fear of leakage, stigma, and inadequate facilities constrained participation in school, physical activity, and social life. These findings document a marked gap between basic biological recognition and the social normalization and practical support of menstruation in two South Indian institutions, and support further evaluation of gender-inclusive, context-responsive menstrual health education using paired assessments, stronger follow-up, comparison groups, and long-term outcomes.

Background: Hand hygiene is a key public health intervention for preventing infectious diseases, including respiratory and gastrointestinal infections. Although the COVID-19 pandemic improved awareness and compliance, sustained adherence remains a challenge, with evidence showing a decline in practice after outbreaks, particularly among young adults in high-contact settings such as universities. Therefore, this study aimed to assess the knowledge, attitudes, and practices of handwashing hygiene among university students at KCMC University, Moshi, Tanzania, in the post-COVID-19 era. Methods: A cross-sectional study was conducted among 380 students at KCMC University. Data were collected using a pretested structured questionnaire, which captured information on socio-demographic characteristics, knowledge, attitudes, and practices regarding handwashing hygiene. Data were analyzed using descriptive and inferential statistics. Associations between variables were assessed using logistic regression and multivariable analysis, and a p-value of <0.05 was considered statistically significant. Results: A total of 380 students participated in the study, with a mean age of 23.5 {+/-} 3.5 years. More than half 209 (55.0%) were aged 18-23 years, while 203 (53.4%) were female. Most participants had access to hand hygiene facilities 338 (88.9%). 338 (88.9%) participants had good knowledge of hand hygiene but attitudes were mostly negative 193 (50.8%), and practices were suboptimal, with 201 (52.9%) showing poor hand hygiene practices. Good hand hygiene knowledge was associated with access to hand hygiene facilities (AOR=2.84, 95% CI: 1.25-6.42, p=0.012) and formal training on hand hygiene (AOR=3.48, 95% CI: 1.24-9.76, p=0.018). Positive attitudes were associated with being in the fourth year or above (AOR=2.12, 95% CI: 1.16-3.89, p=0.015). Good hand hygiene practices were associated with access to hand hygiene facilities (AOR=2.67, 95% CI: 1.18-6.01, p=0.018) and formal training on hand hygiene (AOR=2.98, 95% CI: 1.19-7.48, p=0.020). Conclusion: Although knowledge and awareness of handwashing hygiene were generally high among university students, inconsistencies between knowledge and actual practices were observed in the post-COVID-19 era. Strengthening health education programs and ensuring continuous access to handwashing facilities may improve adherence to proper hand hygiene practices among university students.

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

Background: Valid and feasible measures of preschoolers' 24-hour movement behaviours are needed for population-level research. We developed a Japanese version of the Movement Behaviour Questionnaire-Child (MBQ-C) and evaluated its measurement properties and practicality. Methods: After translation and cross-cultural adaptation following the COSMIN procedure, including cognitive interviews, we evaluated the questionnaire in a cross-sectional validation study of Japanese children aged 3-6 years. Concurrent validity was assessed against hip-worn accelerometry and a 7-day diary; we also examined test-retest reliability, measurement error, agreement between response formats and modes, and practicality (completion time, burden, and clarity). Analyses used Spearman correlations, intraclass correlation coefficients, the SEM, smallest detectable change (SDC), and Wilcoxon signed-rank tests. Results: Of 208 children, 170 provided valid accelerometer and diary data for the concurrent-validity analysis, and 187 completed the MBQ-C for the questionnaire-based analyses. Concurrent validity was weak for active and energetic play (Spearman {rho} = 0.12-0.26), strong for total screen time (0.78-0.85), and moderate for total sleep (0.48-0.58). Test-retest reliability was moderate to good (ICC, 0.66-0.81), and within-child variability was lowest for sleep (CV 3%) and highest for the physical activity items (21-33%). The open- and closed-ended versions showed no significant differences; paper-electronic agreement was good for screen time and sleep but poor for active play. Completion took approximately 6-9 minutes, with low reported burden. Findings were robust in sensitivity analyses. At the group level, SDC for a mean was SDCind/{surd}N: 119.8/{surd}N, 74.0/{surd}N, 112.6/{surd}N, and 92.5/{surd}N min/day for active play, energetic play, total screen time, and total sleep, respectively (N = number of children surveyed). The systematic differences between the two test-retest occasions were +15.7, +6.5, +4.0, and -5.4 min/day, respectively. Conclusions: The Japanese MBQ-C is a practical option for assessing preschoolers' active play, screen time, and sleep at the group level. By quantifying its group-level measurement error, this study provides benchmarks for interpreting group-level differences and change, supporting the use of the MBQ-C in surveillance and evaluation of programmes delivered at scale.

This study developed a streamlined, sensitive and rapid method for co-detection of low concentrations of Salmonella enterica, Shigella flexneri, Listeria monocytogenes, and pathogenic Escherichia coli from soil. First, autoclaved soil from Iowa with serial dilutions of bacteria to confirm individual species recovery rates on selective agar by qPCR under varying primary enrichment times was used. All pathogens were detected by qPCR of colonies recovered from replicate sterile soils spiked with 10^1 or greater bacteria within 5 hours of enrichment. Shiga toxin expressing E. coli (STEC) and L. monocytogenes were consistently co-recovered in autoclaved and natural soil, pathogen spiking concentration, and secondary enrichment time. S.enterica was consistently recovered at autoclaved and natural concentrations, while S. flexneri detection rates fluctuated. Twenty-three soil samples from Kenyan neighborhoods, with prior evidence of contamination, was compared to qPCR detection at each protocol stage: processing, primary enrichment, secondary enrichment, and selective colony isolation. Detection rates substantially increased for S. enterica, STEC and other pathogenic E. coli after enrichment, with optimal detection rates occurring after secondary enrichment. Testing colonies from selective agar resulted in high false negative rates, possibly reflecting competition from native soil microbiota that was absent in spiked sterile soil experiment. Evaluation of the method with other environmental sample types yielded consistent detection patterns. Evidence suggests a protocol including 24-hour primary enrichment, followed by 24-hour secondary enrichment, and qPCR testing of soil DNA at primary and secondary steps is a reproducible and more expedient method for co-detecting common Enterobacteriaceae and L. monocytogenes pathogens in soil.

Background Italy experiences a substantial heat-related mortality burden, but evidence on cause-specific mortality across its heterogeneous climatic settings remains limited. We aimed to quantify cause-specific mortality attributable to summer heat in Italy at national and ecological region levels and to map its distribution across municipalities. Methods We analysed daily deaths classified by underlying cause in eight ICD-10 groups among individuals aged 18-64 years and 65 years or older across 7,864 municipalities during June-August, 2004-2023. Mortality data were linked to area-weighted ERA5-Land daily maximum temperature. Nonlinear temperature-mortality associations were estimated with Bayesian hierarchical Poisson models, fitted separately for each cause, age group, and ecological region, with municipality-level attributable fractions. Findings High temperatures were associated with mortality from a broad range of causes, particularly among individuals aged 65 years or older. Among the study population, heat-attributable fractions were highest for nervous system diseases (14.93%, 95% credible interval 14.22-15.69) and mental and behavioural disorders (14.47%, 13.55-15.39). Circulatory diseases accounted for the largest absolute burden, with 85,524 heat-attributable deaths (82,762-88,271) across the summers of the 20 years considered. Interpretation The mortality burden associated with summer heat in Italy extended well beyond circulatory and respiratory diseases, highlighting the relevance of a broader range of health outcomes. These findings support heat-health strategies that account for cause-specific and geographical differences in heat-related risk, with greater consideration of neurological and mental health outcomes alongside established cardiorespiratory risks. Funding NextGenerationEU (CUP: B53C23002660006); Imperial College Research Fellowship; NIHR Imperial Biomedical Research Center (BRC: NIHR203323).

Background: TB vaccine development is hampered by the natural history of M. tuberculosis: most infected people rarely progress to disease, so trials must be very large and long to accrue sufficient disease endpoints. Current trials enrol IGRA-positive participants (IGRA: Interferon-Gamma Release Assays), but IGRAs remain positive for many years and cannot distinguish recent from remote infection. Tests for recent infection are now in development, and we argue they could make trials substantially more efficient. Here, we investigate how screening participants prior to enrolment with a test for recent M.tb infection could impact the total number of enrollees required for an adequately-powered TB vaccine trial. Methods: Using a statistical model of vaccine trial recruitment, we estimated the number needed to contact, test, and enrol to achieve an 'effective' trial (defined as 50 disease endpoints), under IGRA-gated enrolment (life-long duration of positivity) vs. enrolment with a test for recent infection. We considered five recruitment strategies: non-test-based enrolment for ages 15-49, ages 12-34, and household contacts of recent cases; and test-based enrolment using IGRA, and a test for recent infection with two-year duration of positivity. The model explored trial recruitment in South Africa, Kenya, and India. Results: Compared with an extended-positivity test, a test for recent infection reduced the median number needed to enrol by 4.2- to 6-fold under favourable conditions. It also outperformed non-test-based strategies, including enrolling household contacts. This comes at the cost of substantially more testing, but crude estimates suggest recency-based enrolment would still be cheaper overall provided the new test does not cost more than roughly 5-10 times an IGRA. Conclusions: Diagnostics for recent M.tb infection have the potential to improve vaccine trial efficiency, but more research is now needed to understand test characteristics and to evaluate their use in this setting. Our Viewpoint sets out the test attributes that would need to be established, such as time to positivity, duration of positivity, sensitivity, and specificity, before such tests could be used in vaccine trials.

Background: Amplicon-based next-generation sequencing is a cornerstone of viral genomic surveillance, but it often produces datasets with highly uneven read coverage due to PCR amplification bias. This leads to excessive data generation and substantial computational overhead for downstream bioinformatic analysis. Results: We evaluated the efficacy of two data reduction strategies on 220 SARS-CoV-2 clinical specimens and 12 wastewater samples: random subsampling with SeqKit and read depth normalization with BBNorm. FASTQ files were reduced to target depths from 10X to 10,000X to compare genome coverage, lineage assignment concordance, and computational resource usage. Both strategies markedly reduced total processing time. For clinical isolates, read depth normalization corrected for amplification bias resulting in highly concordant lineage assignments at a target depth of just 200X. Random subsampling preserved inherent bias but produced reliable assignments at depths over 500X. Reducing target depths below 500X in complex wastewater mixtures severely degraded variant deconvolution accuracy and heavily skewed lineage abundance profiles for both methods. Conclusion: Both random subsampling and read depth normalization successfully reduce computational overhead while preserving key clinical sequencing metrics at appropriate reduction settings. The optimal method depends on the analytical goal, ranging from computationally lightweight random subsampling for rapid lineage assignment, to read depth normalization for generating high-quality consensus sequences. However, aggressive data reduction should be avoided in environmental surveillance, where maintaining a high baseline sequencing depth is critical for accurate mixed-variant deconvolution.

Abstract Background Many ecologically important small invertebrates are preserved in ethanol, which compromises DNA integrity and severely limits their potential to be utilized in further genomic research. The lack of soil arthropod representation ultimately results in limited availability of reference genomes and population genomic inference from pooled sequencing (Pool-seq). Results We introduce PoLoCo (Pooled Low-Coverage), a reproducible workflow integrating pooled specimen draft genome assembly from ethanol-preserved specimens with population-level allele-frequency analysis. We assembled a 411 Mb draft genome of Entomobrya nivalis from pooled short reads, achieving 97.7% BUSCO completeness despite notable fragmentation. Comparison with the published reference genome showed partial genomic correspondence while also revealing reference-dependent differences in the retained SNP datasets. The application of PoLoCo to 82 retained pooled populations produced a final distance-thinned allele-frequency dataset suitable for population-scale genomic inference. Case-study execution documented the computational requirements of the major workflow components on standard HPC infrastructure, with the workflow, documentation, and software environments openly available in Zenodo. Conclusions PoLoCo demonstrates that draft genome assemblies and reproducible Pool-seq allele-frequency analyses can be performed with long-preserved sample material from the field. PoLoCo integrates reference construction, sync-based allele-frequency analysis, and benchmarking into a free and open workflow. It establishes a reproducibility benchmark for biodiversity genomics and yields a transferable workflow for ecological and evolutionary initiatives where high-quality DNA or long-read sequencing is not feasible.

Background: Hand hygiene (HH) is one of the most effective measures to prevent healthcare-associated infections (HAIs) and limit the transmission of antimicrobial-resistant pathogens in healthcare facilities (HCFs). Effective HH programs depend not only on the availability of essential resources but also on healthcare workers' (HCWs) knowledge, attitudes, and adherence to recommended HH practices. Despite the critical role of these factors in improving infection prevention and control (IPC), evidence on HH knowledge and practices among HCWs remains limited. In Uganda, such evidence from lower-level district health facilities is particularly scarce. This study assessed HH knowledge and practices, along with associated factors, among HCWs at selected health facilities in the Mukono and Kagadi districts of Uganda. Methods: A cross-sectional study was conducted among 586 HCWs at selected health facilities in the Mukono and Kagadi districts from May to June 2025. Data were collected using a structured questionnaire administered via KoboCollect, covering participants' socio-demographic and professional characteristics, HH knowledge, self-reported HH practices, and facility-related factors. Descriptive statistics summarized the data, and modified Poisson regression with generalized estimating equations (GEE), accounting for clustering of HCWs within facilities, was used to estimate prevalence ratios for factors associated with HH knowledge and practices. Results: Overall, 82.1% of HCWs had adequate HH knowledge, and 43.0% had good HH practice. Nearly all respondents correctly identified the recommended handwashing duration (98.8%), the minimum effective alcohol concentration for alcohol-based hand rub (ABHR) (98.8%), proper HH as the most effective method for preventing HAIs (97.6%), and 89.9% correctly listed all five WHO Moments for HH. HCWs in Mukono were more likely to have adequate HH knowledge than those in Kagadi (aPR = 1.36; 95% CI: 1.15-1.60). HCWs with 6-10 years of experience had higher knowledge of HH than those with less than two years of experience (aPR=1.16, 95% CI: 1.02-1.33). Good HH practice was significantly more common among clinical officers (aPR=1.36, 95% CI: 1.06-1.75) and other cadres (aPR=1.65, 95% CI: 1.14-2.39) than nurses and nursing assistants. Conclusion: HCWs demonstrated high HH knowledge but suboptimal HH practice, suggesting a gap between knowledge and self-reported practice. Interventions should move beyond knowledge dissemination to strengthen compliance through multimodal, cadre-specific strategies, particularly for nurses and nursing assistants.

Chemotaxis assays are widely used to quantify sensory-driven behavioral responses in Caenorhabditis elegans. Sodium azide is commonly used to immobilize animals at assay endpoints, although concerns have been raised that diffusion beyond the endpoint region could affect behavior before a choice is completed. We compared sodium azide-mediated immobilization with post-assay cooling using the repellent 1-octanol and attractant diacetyl. Wild-type animals showed robust avoidance of 30% 1-octanol and attraction to 0.5% diacetyl with either method, across multiple assay durations, and with or without worm food Escherichia coli OP50. These results support post-assay cooling as an alternative endpoint-scoring method.

The COVID-19 pandemic substantially disrupted tuberculosis (TB) prevention, diagnosis, and treatment services worldwide. Despite increased TB notifications in Nepal during the post-pandemic recovery period, the geographic distribution and temporal clustering of notifications remain poorly understood. This study examined the spatial and temporal patterns of TB notifications across Nepal during the COVID-19 recovery period. We conducted a nationwide retrospective ecological analysis of district-level TB notifications across all 77 districts of Nepal from FY 2019/20 to FY 2023/24. Space-time, purely spatial, and purely temporal scan statistics were performed using a discrete Poisson model in SaTScan. Statistical significance was assessed using Monte Carlo simulation with 999 replications. A total of 172,155 TB cases were notified during the study period. TB notification rates increased by 51%, from 92 to 139 per 100,000 population between FY 2019/20 and FY 2023/24. Four statistically significant high-notification space-time clusters were identified (all p < 0.001). The primary cluster included Kathmandu, Lalitpur, Bhaktapur, Makwanpur, Bara, and Parsa districts during FY 2021/22-FY 2023/24 (RR = 1.60). Additional clusters were identified in western Nepal, the eastern Terai, and Rupandehi district. Purely temporal analysis showed a nationwide increase in notifications during FY 2022/23-FY 2023/24 (RR = 1.26, p = 0.001). TB notifications increased substantially during Nepals post-pandemic recovery period, with marked geographic and temporal heterogeneity. Integrating spatial and temporal surveillance may help identify areas requiring geographically targeted TB control strategies.

Protein aggregates are a diverse array of intra- and extracellular structures containing misfolded proteins with limited macromolecular movement. Protein aggregation is induced by aging and cellular stress and is a hallmark of neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. Fluorescent tools to visualize global protein aggregation in whole organisms are, therefore, important for screening genes and compounds that may reduce or alter protein aggregation in connection with improved health outcomes. Fluorescent probes that report low fluidity structures in cells, such as molecular rotor dyes, are gaining popularity and overcome certain limitations of targeted markers such as tagged proteins or antibodies. However, permeability has been a key barrier preventing widespread use of such probes in whole organisms, including in C. elegans. To achieve visualization of non-specific protein aggregates in whole C. elegans nematodes without the need for strain engineering, we have developed a simple soaking protocol. Our protocol overcomes the permeability limitations of the commercially available molecular rotor dye, Proteostat, enabling widespread labeling of protein aggregates in live and post-fixed worms. Here, we present our novel approach and demonstrate quantification of protein aggregates in multiple tissues to detect differences in protein aggregation due to both genotype and treatment effects. We also outline anticipated future applications for this protocol, including its adaptation for other dyes, as well as limitations and special considerations for its use.

Background: Quantifying behavioral drivers of protective action during infectious disease outbreaks remains challenging. App-based experimental epidemic games (epigames) provide a framework for studying these decisions in naturalistic settings, but correspondence between in-game measures and real-life beliefs or behaviors requires evaluation. We examined whether an activity-based opportunity cost influenced voluntary quarantine decisions within an epigame and whether real-life health beliefs transferred into the game and predicted observed choices. Methods: We conducted a two-week randomized controlled trial from May 4 to May 17, 2026, at VinUniversity (VinUni) in Hanoi, Vietnam. Participants using the Epigames app installed in their smartphone were randomized to a low-barrier group, earning 1 point per qualifying contact with other participant when remaining active, or a high-barrier group, earning 2 points per contact. Of 439 enrolled participants, 283 were successfully randomized to one of the two groups, made at least one valid daily quarantine decision, and their data was used in the analysis. Real-life (S1) and in-game (S2) beliefs measured susceptibility, severity, quarantine self-efficacy, and perceived benefits. Belief correspondence was assessed using Holm-adjusted Spearman correlations and prospective cumulative-logit models. Quarantine choices were analyzed using participant-day binomial generalized estimating equations (GEE) and participant-level quasi-binomial models. Results: Participants made 1,823 valid daily decisions and chose quarantine on 458 participant-days (25.1%). Quarantine rates were 30.1% in the low-barrier group and 20.2% in the high-barrier group. The high barrier was associated with 41% lower population-averaged odds of quarantine (GEE odds ratio [OR] 0.59, 95% confidence interval [CI] 0.40-0.86, = 0.0059); the quasibinomial estimate was identical (OR 0.59, 95% CI 0.40-0.86, = 0.0055). Among 251 participants with matched S1-S2 data, all four corresponding beliefs were positively associated (Spearman's = 0.259-0.418; all Holm-adjusted < 0.001). Prospective ordinal models confirmed positive matched-domain associations (ORs 1.39-2.18 per one-point S1 increase). Beliefs showed little robust association with quarantine behavior, and neither S1 nor S2 beliefs jointly modified the randomized effect. Self-efficacy, network degree, and the preregistered gender main effect were also unsupported. Conclusion: A higher activity-based opportunity cost reduced voluntary quarantine within the VinUni epigame, while participants carried relative differences in stated health beliefs from real-life to in-game framing. These beliefs did not reliably predict observed quarantine choices within the game framing. Epigames can support controlled study of incentives and belief correspondence in naturalistic settings such as college campuses, but behavioral validity or real-world generalizability requires further work.

As internet and mobile phone access increases, digital health interventions are increasingly used to support remote and low-resource populations. In many settings, the health impact of digital interventions is constrained by limited or short-lived engagement. We tested if conditional mobile data credit incentives increase digital intervention engagement within a digital intervention trial. This nested cluster randomized controlled trial was conducted with caregivers of children 3-9 months at baseline. Of 72 neighborhood clusters from the treatment arm of the parent trial (evaluating a digital early childhood development intervention), 20 were randomly assigned to treatment and 52 as controls. Eligible participants had a phone provider through which mobile data credits could be most affordably administered. Treated participants received around 250 MB mobile data credits every two weeks, conditional upon using the digital intervention at least once in the same period. Credits were digitally administered via a third-party service. Digital intervention engagement (primary outcome) was monitored continuously until endline data collection approximately 2 years after assignment. Secondary outcomes included caregivers views, behaviors, and early childhood development. We used intent-to-treat analyses to assess all outcomes. This trial was registered as NCT05202106 at clinicaltrials.gov and was completed in 2025. Participants were recruited between September 2021 and March 2023 (N Treatment = 147; N Control = 333). Conditional mobile data credits extended engagement with the digital early childhood development intervention by 6.50 weeks on average (treatment mean = 26.19 weeks, control mean = 19.69; p=0.011). No significant impacts were identified on secondary outcomes. As such, mobile data credits may be a cost-effective way for increasing caregiver engagement with digital health intervention in low resource settings.

This study evaluated the feasibility and preliminary effectiveness of a 12-session group-based nurturing care intervention for adolescent mothers in the Dominican Republic. We conducted a one-group pretest-posttest feasibility study in Boca Chica, Dominican Republic. Participants were adolescent mothers under 20 years of age with a child enrolled in an early childhood development (ECD) center operated by the National Institute for Early Childhood Comprehensive Care. The intervention, based on the World Health Organization's Nurturing Care Framework, addressed ECD, affective care, positive discipline, and maternal well-being. We calculated effect sizes using Cohen's d. Of 14 adolescent mothers who started the program, 11 completed both assessments (78.6% retention). Participants had a mean age of 16.91 years (SD = 1.35), with children averaging 1.15 years (SD = 0.84). Two outcomes showed statistically significant improvements. Parental efficacy (1-4 scale) increased from M = 3.38 to M = 3.84 (d = 0.99, p = .008), with significant gains observed in three subdomains: protective (d = 1.21, p = .003), formative (d = 0.90, p = .014), and bonding competencies (d = 0.81, p = .023). Cognitive stimulation activities (0-1 scale) increased from M = 0.42 to M = 0.65 (d = 0.81, p = .036). Participant satisfaction was high (M = 4.64 on a 5-point scale). ASCENDI demonstrated feasibility and preliminary effectiveness in improving parental efficacy and cognitive stimulation among adolescent mothers. Large effect sizes and high retention and satisfaction support the potential of group-based nurturing care interventions for adolescent mothers in resource-limited settings.

Surface plasmon resonance (SPR) and biolayer interferometry (BLI) are well-established label-free analytical technologies used to determine biomolecular interaction rates (ka, kd) and equilibrium constants (KD). While assays historically rely on multi-cycle kinetics (MCK), this approach requires intermediate surface regeneration, risking sensor degradation and increasing reagent consumption. Single-cycle kinetics (SCK) avoids regeneration by sequentially injecting increasing analyte concentrations. However, implementing SCK workflows on biosensor platforms like the OctetRR8 BLI instrument (Sartorius) remains a challenge due to the lack of built-in or online data analysis tools. To address this, we introduce SCKAnalyzer, a free, user-friendly web application designed for processing and fitting SCK datasets. SCKAnalyzer accepts column-based text files and native raw BLI data (.frd format) from Sartorius instruments, performing data pre-processing and non-linear fitting to a 1:1 interaction model. Additionally, it offers advanced simulation capabilities that allow variable association and dissociation durations across sequential injection steps, a feature absent in existing platforms such as BiacoreTMSimul8. The performance of SCKAnalyzer was experimentally validated using a 3-step SCK method (SCK3) for DNA/RNA duplex formation on a Biacore T200, as well as 4-step (SCK4) and 5-step (SCK5) methods for DNA/DNA duplex formation on an OctetRR8 BLI instrument. Binding parameters (ka, kd, KD) derived from SCKAnalyzer showed excellent agreement with values from established proprietary softwares such as Biacore T200 Evaluation Software v3.2.1 and BIAevaluation v4.1.1. SCKAnalyzer provides a practical alternative to MCK, reducing probe consumption and extending SCK utility across biosensor platforms. SCKAnalyzer is available at sck.iecb.u-bordeaux.fr (source code: github.com/synthaze/sckanalyzer).