The genus Trichilia (Meliaceae) produces bioactive compounds with insecticidal, antioxidant, antifungal and anti-inflammatory properties. Due to the limitations of conventional anti-inflammatory drugs, Trichilia emarginata, native to Brazil's Atlantic Forest, offers a promising source of novel phytochemicals. Leaves were extracted and fractionated using chromatographic methods, and chemical profiling was conducted by UPLC-HRMS. Anti-inflammatory activity was evaluated ex vivo in human whole blood through inhibition of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) release. Fractions CHLO and HAL showed significant dual inhibition compared to the control: PGE2 decreased by 48% (CHLO) and 41% (HAL), while LTB4 decreased by 61% and 48%, respectively (ANOVA, Dunnett's test, p < 0.05). Limonoids, triterpenes and steroid derivatives were identified in CHLO and ACT fractions, whereas phenolic compounds, notably chlorogenic acids, predominated in HAL and the crude extract, highlighting T. emarginata as a promising source of anti-inflammatory metabolites.
Anthranilic diamides (AD) are a modern class of insecticides used as alternatives to pyrethroids and neonicotinoids, particularly against lepidopteran pests. Despite their widespread use and presence in surface waters, little is known regarding their effects on amphibians. The aim of this study was to examine the effects of environmentally-relevant concentrations of AD insecticides chlorantraniliprole (CHLO) and cyantraniliprole (CYAN) on metamorphosis of the toad Rhinella arenarum. Tadpoles were exposed to CHLO or CYAN at concentrations ranging from 5 and 5000 µg/L from stage 27 until metamorphosis completion. Both insecticides produced a non-monotonic acceleration of the time required for individuals to progress through development and a decrease in the proportion of individuals completing metamorphosis, although a delay in metamorphosis was also observed at 5 µg/L of CHLO. Snout-vent length and body weight of metamorphosed toads were not markedly affected by either insecticide. CHLO was more toxic than CYAN, with a lowest observed effect concentration (LOEC) for CHLO on time to metamorphosis defined as 5 µg/L compared to 5000 µg/L for CYAN. The LOEC for reduced metamorphic success defined as 50 µg/L for CHLO compared to 500 µg/L for CYAN. As most effects occurred after stage 39, when metamorphosis depends upon thyroid hormones, it is conceivable that that AD insecticides act as endocrine disruptors. These findings suggest that contamination of surface waters with CHLO and CYAN may disrupt amphibian development in the wild and warrant further research to investigate the possibility of endocrine-disruption by ADs.
Whether and how pathogen-derived antigens themselves drive vascular injury remains an unresolved question in infection biology and cardiovascular research. Using murine model and human pluripotent stem cell-derived vascular organoids (VOs), we demonstrate that Toxoplasma gondii (T. gondii) infection or exposure to its soluble tachyzoite antigens (STAg) compromises vascular integrity, leads to the impairment of endothelium, and heightens inflammation. Mechanistically, STAg upregulates the scavenger receptor CD36 on endothelial cells (ECs), reprogramming them toward a scavenger endothelial cell (SEC)-like phenotype. This shift enhances CD36‑dependent antigen uptake and subsequently perturbs the macroautophagy/autophagy-lysosomal pathway (ALP), culminating in cellular homeostasis disruption. Interestingly, prophylactic folic acid (FA) ameliorates T. gondii STAg-induced vascular injury. FA acts by suppressing CD36 expression, thereby limiting STAg uptake and attenuating ALP hyperactivation, together preserving vascular integrity at the molecular, structural, and metabolic levels. To our knowledge, this study provides the first evidence that pathogen-derived proteins can reprogram ECs into an SEC-like phenotype in a CD36‑dependent manner, with subsequent dysfunction of ALP. Our findings not only identify pathogen-derived proteins as novel mediators of vascular injury but also establish the CD36-ALP axis as a promising therapeutic target. Furthermore, we propose FA as a potent vascular-protective agent.Abbreviation: ALP: autophagy-lysosomal pathway; Chlo: chloroquine; ECs: endothelial cells; ECM: extracellular matrix; ER: endoplasmic reticulum; FA: folic acid; RAPA: rapamycin; SMCs: smooth muscle cells; STAg: soluble tachyzoite antigens; T. gondii: Toxoplasma gondii; TLRs: toll-like receptors; VOs: vascular organoids.
Cytomegalovirus encephalitis (CME) is one of the serious opportunistic complications in human immunodeficiency virus (HIV)-infected patients, characterized by rapid onset, poor prognosis, and high mortality. Until now, there has been limited comprehensive research on the clinical and prognostic characteristics of HIV/CME patients reported in China. We conducted a retrospective study of 43 patients diagnosed with CME among individuals infected with HIV from 2015 to 2023 at Guangzhou Eighth People's Hospital. Among them, 27 patients had a favorable prognosis, while 16 patients had an unfavorable prognosis (which includes death, treatment ineffectiveness, or aggravated condition), as determined by clinical diagnosis. The clinical symptoms and laboratory examination data for the two groups were analyzed. Multivariate analysis and a nomogram were developed using statistical variables. HIV/CME patients with an unfavorable prognosis exhibited more consciousness disorders and nuchal rigidity than those with a favorable prognosis. There were no significant differences between the two groups in terms of clinical characteristics, such as hemiparalysis, meningeal irritation, cerebrospinal fluid (CSF) pressure, and other relevant factors. In the CSF, the chlo-ride (Cl) level was significantly higher in HIV/CME patients with a favorable prognosis, whereas the Cytomegalovirus (CMV)-DNA levels showed the opposite trend. A multivariate analysis of fever, nuchal rigidity, consciousness disorder, and CSF CMV-DNA can be used to predict prognosis in HIV/CME patients, with a C-index of 0.83 (95% CI: 0.64-1.00). The log CSF CMV-DNA copies/mL emerged as an independent risk factor for prognosis. High CSF CMV-DNA (≥300,000 copies/mL) indicated an unfavorable prognosis. This study is the first to propose a prognostic threshold for CSF CMV-DNA (≥300,000 copies/mL) and develop a nomogram that integrates clinical and laboratory features for risk prediction in HIV/CME patients in China. The findings highlight that CSF CMV-DNA ≥ 300,000 copies/mL, along with neurological symptoms such as consciousness disorder and nuchal rigidity, are significant predictors of unfavorable prognosis in HIV/ CME patients. The constructed nomogram offers a clinically useful tool for early risk stratification, which may aid in timely therapeutic decision-making. HIV/CME patients with an unfavorable prognosis showed more obvious signs and symptoms of central nervous system infection, lower CSF Cl levels, and higher CSF CMV-DNA compared to patients with a favorable prognosis. Recognizing these indicators early and administering timely antiviral therapy before the disease progresses to CME are of great value in improving the survival rate of patients. Our findings extend beyond existing international studies by providing novel prognostic evidence in a Chinese cohort and offering a practical predictive tool for clinical risk stratification.
In the agricultural industry, non-thermal plasma has fascinated the scientific community due to its potential applications in the pre-cultivation, cultivation and postharvest processes of crops. Plasma-processed seeds exhibit high germination percentage, fast germination, and enhanced plant growth, antioxidant activity, nutritional composition and yield of several crops. A limited field work was conducted, applying plasma technology from germination to harvest of tomatoes. This experiment was designed to study the consequences of tomato seed treatment using reduced pressure magnetic field-mediated glow discharge (MFGD) plasma for the durations of 3, 6, 9, and 12 min, where 9 min MFGD plasma treatment achieved the best germination percentage with an increase in germination by ∼25.64% compared with the control. Furthermore, plasma-processed water (PPW) was applied to plants grown from 9 min-treated seeds for 4 times at 10 days intervals during the vegetative stage. Experimental results show that in comparison with the control, plant length, stem diameter, and photosynthetic pigments (chlo-a, b and carotene) were enhanced in plants subjected to MFGD seed treatment and PPW spray. In addition, the content of antioxidant enzymes, secondary metabolites, soluble sugar, protein and mineral contents was enhanced. The increments of soluble sugar and protein in the tomato fruits were 41.58% and 146.67%, respectively, with respect to the control. In contrast to the control, the yield was enhanced by ∼30.84%. It can be concluded from this experiment that the plasma-processed seeds and PPW foliar spray collectively contribute significantly to boosting tomato yield and nutrients.
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Background. Despite technological innovations and improvements in stents and devices, sex-related discrepancies are still reported in the outcomes after ST-segment elevation myocardial infarction (STEMI), depending on biological and sex-specific pathophysiological differences, which have not been completely understood. The aim of the present study was to provide real-world data on the prognostic role of sex among patients with STEMI, enclosed into a recent up-to-date international registry. Methods. The ISACS-STEMI COVID-19 is a large-scale retrospective registry, including STEMI patients treated with mechanical reperfusion between 1 March and 30 June, 2019 and 2020. Patients, treated in 109 centers across Europe, Latin America, Southeast Asia, and North Africa, were grouped according to sex. Primary endpoint: In-hospital mortality; secondary endpoints: Time delay, 30-day mortality, and postprocedural Thrombolysis In Myocardial Infarction (TIMI) 3 flow. Results. We included 16,083 patients, 24.3% females (54.3% hospitalized in 2019, 45.7% in 2020). Women with STEMI were older, more often diabetic and hypertensive (p < 0.001), with a higher prevalence of hypercholesterolemia (p = 0.02), longer ischemia time (p = 0.01), ambulance referral (p = 0.03) and cardiogenic shock at presentation (p = 0.05), but less frequently smokers, with a previous cardiovascular event (p < 0.001) or anterior STEMI (p = 0.03) as compared to males. Preprocedural TIMI 0 flow, multivessel disease, need for thrombectomy (p < 0.001 and p = 0.001, respectively), use of Glycoprotein IIbIIIa inhibitors or cangrelor, radial access and implantation of drug-eluting stents (p < 0.001, p < 0.001 and p = 0.001, respectively) were also more common in men. Impaired postprocedural epicardial reperfusion (TIMI flow 0-2) was observed more frequently in females as compared to males (10% vs. 7.2%; adjusted OR [95% CI] = 1.30 [1.13-1.49], p = 0.01). In-hospital mortality was 5.8%, significantly higher among women (8.3% vs. 5%, p < 0.001, adjusted HR [95% CI] = 1.26 [1.06-1.5], p = 0.01). Similar data were observed for 30-day mortality (10.3% vs. 6.2%, p < 0.001, adjusted HR [95% CI] = 1.22 [1.06-1.38], p = 0.007). Conclusions. Among STEMI patients being treated with the most updated standard of care for primary percutaneous coronary intervention, female sex is still associated with higher complexity and impaired prognosis, displaying suboptimal epicardial reperfusion and increased in-hospital and 30-day mortality.
Alcohol use disorder and alcohol withdrawal syndrome impose substantial clinical and economic burdens, with repeated hospitalizations being common. We aimed to systematically review readmission rates following inpatient detoxification, assess variation across study designs and hospital settings, and identify key risk and protective factors. We performed a literature search in Embase and Pubmed on 10/04/2026 focusing on studies assessing in hospital alcohol detoxification. Exclusion criteria included studies on substance use other than alcohol and outpatient or residential treatment. Main outcome was rehospitalization, and meta-analysis was performed to estimate pooled readmission proportions. Secondary outcomes were risk factors and protective factors influencing the rate of rehospitalization. Twenty-five studies were included. The pooled proportion of readmissions following alcohol detoxification was estimated at 17% (95% CI: 14%-21%; 13 studies, n = 287,896) within 1 month, increasing to 44% (95% CI: 36%-52%; 8 studies, n = 2,877) at 1 year. Substantial between-study heterogeneity was observed. Subgroup analyses found no significant differences by hospital setting or time period. Findings for study aim and study design were mixed and based on limited data A small number of studies suggested associations with housing stability, employment, and treatment engagement. This meta-analysis suggests that approximately one in six patients are readmitted within 1 month and nearly half within 1 year after inpatient alcohol detoxification. However, readmission rates varied considerably across settings and populations. Future research should evaluate targeted interventions to reduce readmissions among high-risk patient groups.
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Despite the large use of renin-angiotensin system inhibitors (RASI) in STSegment Elevation Myocardial Infarction (STEMI) patients, few data have been reported on the prognostic impact of chronic RASI at admission in patients suffering from STEMI, especially during the COVID-19 pandemic. Therefore, the current study investigated the prognostic impact of chronic RASI at admission in patients suffering from STEMI, including both SARS-CoV-2 positive and negative individuals, enrolled before and during the COVID-19 pandemic. We included STEMI patients who received primary percutaneous coronary intervention (PPCI) and were enrolled in the ISACS-STEMI COVID-19 registry. In the present sub-analysis, patients were allocated according to chronic RASI therapy at admission. The primary study endpoint was the occurrence of in-hospital mortality. Secondary endpoints were postprocedural TIMI 3 flow and mortality at 1 month. The overall population was 15,693 patients, including 6,213 patients pretreated with RASI. Several differences in baseline characteristics were observed between the two groups. No difference was observed in the prevalence of SARS-CoV-2 infection. After correction for all baseline confounders, including procedural features, chronic pretreatment with RASI was independently associated with improved postprocedural TIMI 3 flow (OR [95% CI] = 1.14 [1.03-1.35], p = 0.042), lower in-hospital mortality (adjusted OR [95% CI] = 0.64 [0.54-0.75], p < 0.001), and lower 30-day mortality (adjusted OR [95% CI] = 0.62 [0.53-0.73], p < 0.001). This is the largest study investigating the prognostic impact and benefits of chronic RASI pre-treatment in STEMI patients undergoing PPCI, including those treated during the COVID19 pandemic. We found that chronic RASI treatment at hospital admission was associated with significant improvement in reperfusion and reduction in mortality, without any negative effect in SARS-CoV-2 positive patients. Results should be interpreted considering the retrospective, nonrandomized nature of the study.
Few studies in humans have revealed differences in the inflammatory responses between biological sexes when encountering serious infections. Our study aimed to investigate how those differences were presented among sexes and age groups from 30 days before (D-30) through 30 days after (D30) a bloodstream infection (BSI). We did a retrospective population-based cohort study, including patients aged > 15 years with their first-time BSI between 2007 and 2016. Based on aggregated data, we computed daily mean levels of C-reactive protein (CRP) and neutrophils in the D-30/D30 period, separately for females and males within the age groups 15-49 and ≥ 50 years. For each age group, we used adjusted multilevel mixed effects linear regression analyses to detect differences in daily mean levels between females and males. A total of 24,074 patients had 268,648 specimens with CRP and 138,482 with neutrophils. CRP and neutrophils peak values were significantly higher in females, reaching their highest values among the ≥ 50 years group. For all age groups, peak values occurred for CRP at D1 and for neutrophils at D0. Neutrophil values were more equal between the sexes, although higher levels were found in the ≥ 50 year age group among females after D-4. Females and males with BSI exhibited different trajectories and different peak values close to the BSI episode, in particular in females in the ≥ 50-year age group. Severe infections, such as BSI, need further investigation regarding sex differences, stratified into age groups for expected female menopause.
Despite advances in the treatment of cutaneous melanoma, there is still a high percentage of patients who fail to respond or develop resistance to treatment. Establishing robust in vitro melanoma models will enable mechanism-based drug screening while reducing animal testing. In this work, a three-dimensional (3D) melanoma skin model (3DMSM) was developed on a porous scaffold. The culture of three melanoma cell lines (SKMEL-1, A375, and G361) in co-culture with human fibroblasts, melanocytes, and keratinocytes allowed the formation of the dermis, and stratified epidermis. Tumors were established in this model using two methodologies: adding previously formed melanoma cell aggregates (CA) or seeding melanoma cells directly into the dermis (CD). In this model, melanoma cells remain in their original microenvironment and, after proliferation, invade the basal layer. The model recapitulates correct melanocyte localization, epidermal disruption, extracellular matrix (ECM) remodeling, including collagen deposition, and epithelial-to-mesenchymal transition (EMT). Additionally, the cytokine profiles studied indicate that the model could mirror the inflammatory and immune-evasive traits of melanoma. Overall, 3DMSM provides a useful tool for understanding the mechanisms of melanoma progression and invasion, and for developing personalized medicine strategies through the implementation of a patient-derived model.
BACKGROUND: Aromatase inhibitors (AI) are used to treat hormone-receptor-positive breast cancer, but have several side effects that often lead to premature discontinuation/switch. Physical activity (PA) can attenuate side effects and improve quality of life. However, most cancer survivors fail to meet PA guidelines. Theory-based PA interventions are effective in breast cancer survivors, but scarce. PAC-WOMAN tested the effects of two PA promotion interventions (supervised exercise vs brief counseling) on primary outcomes – PA, sedentary behavior, quality of life –, and secondary outcomes – body composition, fitness, physical function, sleep, body image, depression, psychological well-being, pain, and motivational variables – in women on AI. METHODS: This pragmatic randomized controlled trial included 110 women with stage I-III hormone-receptor-positive breast cancer on AI (56.1 ± 7.6 yr; 28.1 ± 5.9 kg/m2; 23.4 ± 20.1 months on AI), randomized to: 1) brief PA counseling (PAC); 2) structured exercise (ExG); 3) waiting-list control. Primary and secondary outcomes were evaluated with validated instruments at the end of the intervention period. Repeated measures’ Anovas, adjusted for age, AI duration, body mass index and chronic diseases, were conducted. Bonferroni corrections were applied (significance level at p < 0.0083). RESULTS: Significant group-by-time interactions were observed for moderate-vigorous PA (MVPA), exercise motivations and affect, muscle strength and leg endurance (p ≤ 0.008). Objectively measured PA showed no significant effects in either intervention group, except for a near-significant MVPA increase of 107 min/week [95% CI: 4; 209] over time in ExG. Larger improvements were observed in all self-reported PA indicators in ExG, while PAC revealed gains only in light PA and active choices. ExG improved global health status, physical functioning and physiological indicators (e.g., fitness, body composition) over time; PAC enhanced future perspectives, body image functioning, breast symptoms, and other psychological outcomes, with changes in depressive symptoms (-1.4 [-2.4; -0.3]) and life satisfaction (0.8 [0.2; 1.5]) approaching significance. CONCLUSIONS: PAC-WOMAN showed that ExG and PAC yielded distinct benefits for breast cancer survivors on AI. ExG improved total PA, fitness, physical function, physical quality of life, and alleviated pain symptoms. PAC primarily enhanced light PA and psychological outcomes such as body image and life satisfaction. Together, these findings highlight the potential scalability of PAC alongside the robust physical benefits of ExG. TRIAL REGISTRATION: April 2023 – NCT05860621; https://doi.org/10.17605/OSF.IO/ZAQ9N ; UMIN000050945.
Non-structural protein 1 (NS1) of influenza A virus is a multifunctional virulence factor and represents a promising anti-influenza target, considering its conserved and druggable structure. As antiviral target, NS1-RNA-binding domain (RBD) remains unexplored, despite its critical role in replication. In this study, we applied a "Map-and-Mutate" strategy to identify and functionally validate highly conserved and druggable regions within the NS1-RBD. Using large-scale sequence alignments and structural characterization, we integrated conservation and druggability analyses to predict conserved druggable pockets and top-ranked hot spots, mutate the five most promising residues (L15, W16, R19, R35, and L43) and study their impact on viral fitness. In vitro, the mutations W16 and R35 caused most significant reduction in viral fitness; however, L15 and R19 also impaired replication. Combined mutations involving W16 and either L15 or L43 exerted a cumulative effect, reducing viral replication, hemagglutination titers and neuraminidase activity. This study demonstrates that most residues identified and investigated using the "Map-and-Mutate" strategy negatively impact viral fitness, underscoring the approach's value in pinpointing novel antiviral targets. Together with our prior research, this study reinforces the importance of NS1 as a promising antiviral target, providing a rationale for designing and developing therapies with a higher resilience to viral resistance.
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Umbilical cord blood (CB)-derived natural killer (NK(CB)) cells are a promising immunotherapeutic modality due to their cytotoxicity, allogeneic compatibility, and reduced immunogenicity relative to peripheral blood NK cells (NK(PB)). Although several commercial culture media have been optimized for NK(PB) expansion, their suitability for NK(CB) cells remains insufficiently characterized. This study compares the expansion efficiency, phenotypic shifts, functional activation, and cost-effectiveness of NK(CB) cells expanded ex vivo under feeder-free static culture conditions with IL-2-supplemented culture media. CB-derived CD56⁺ cells isolated by magnetic-activated cell sorting were cultured in six candidate culture media, after which CTS™ NK-Xpander™ (NKX) and NK MACS® (NKM) with 1% (NKM1) or 2% (NKM2) supplement were selected for further evaluation. NKX achieved the highest average fold expansion (18.8 ± 7.0), followed by NKM2 (16.8 ± 7.3) and NKM1 (14.3 ± 3.5). All conditions increased cytotoxicity and CD107a degranulation, with NKX consistently yielding stronger functional responses. Phenotypic analysis revealed a shift toward a CD56brightCD16⁻ profile, particularly in NKM cultures, accompanied by cytokine-associated CD16 downregulation. Cost analysis identified NKX and NKM1 as the most cost-efficient conditions, whereas NKM2 doubled manufacturing costs due to higher supplement requirements. In conclusion, NKX and NKM media effectively support feeder-free ex vivo expansion of NK(CB) cells with high viability and robust functional activation. Phenotypic changes, donor-dependent variability, and CD16 downregulation should be considered when designing clinically translatable manufacturing strategies. These findings are essential to enhance scalability, reduce manufacturing costs, and advance NK(CB) cells as an accessible and effective immunotherapy, facilitating their integration into clinical-grade manufacturing and scalable bioprocessing pipelines.
Several scores have been developed to facilitate risk stratification and early discharge following primary angioplasty, particularly the Zwolle Risk Score (ZRS). However, validation in large-sized studies is still lacking. Therefore, the aim of the current study was to validate the use of the ZRS in a contemporary global population, including patients who were treated during the SARS-CoV-2 pandemic and enrolled in a large intercontinental observational study. The ISACS-STEMI COVID-19 is a large-scale retrospective multicenter registry involving primary PCI centers from Europe, Latin America, South-East Asia, and NorthAfrica, including patients treated from March 1st until June 30th, in 2019 and 2020]. ZRS was calculated for each patient. The patients were additionally categorized according to the following values of the ZRS [≤3; 4-6; 7-9; ≥10]. Our study outcomes were in-hospital and 30-day mortality. The discriminatory capacity of the ZRS was assessed by the area under the ROC curve [c statistic] as an index of model performance. Our population is represented by 16084 STEMI patients undergoing mechanical reperfusion enrolled in 109 centers. The score showed a very good performance in the predicting mortality both in-hospital [AUC=0.83 [0.82-0.85], p<0.0001] and at 30- day follow-up [AUC=0.82 [0.81-0.84, p<0.0001]. The results were confirmed when the ZRS was separately applied to patients treated in 2019 and 2020, with good stability across time. ZRS was able to identify a large cohort [n=10672, 66.3%] of low-risk patients [score ≤3] with a very low mortality rate at 2 days [1%] and between 3 and 10 days [0.7%], with a very good negative predictive value for in-hospital [98.3%] and 30-day mortality [97.7%], with similar results in 2019 and 2020. This study is the first to demonstrate the good prognostic performance of the ZRS in a large-scale contemporary global multicenter validation set. Similar results were obtained both in the pre-pandemic and the COVID-19 era. ZRS ≤3 identified a very low-risk population that could be discharged early, even during the COVID-19 pandemic, with expected advantages in the availability of hospital beds and nursing staff, costs of medical care, and in-hospital risk of contagion.
Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary approach in immunotherapy that has shown remarkable success in the treatment of blood cancers. Many preclinical studies are currently underway worldwide to extend the CAR-T-cell therapy benefits to a broad spectrum of cancers, using rodent models. Alternative in vivo platforms are essential for overcoming the drawbacks associated with rodent models, including immunodeficiency in humanized models, ethical concerns, extended time requirements, and cost. In this work, we used the chicken chorioallantoic membrane (CAM) assay to evaluate the in vivo efficacy of cluster-of-differentiation 19 (CD19)-targeting CAR-T cells expressing a second-generation CAR construct against human lymphoma derived from the Raji cell line. Our results confirm the efficacy of selected CAR-T cells on tumor growth, metastasis, and angiogenesis. Further, the chicken embryo has an intrinsic active immune system. Therefore, the dialog between CAR-T cells and endogenous immune cells, as well as their participation in the tumor challenge, has also been studied. In conclusion, our study demonstrates that the chicken CAM assay provides a relevant in vivo, 3Rs (Replacement, Reduction and Refinement)-compliant new approach methodology (NAM), which is well-suited for the current needs of preclinical research on CAR-T-cell therapy.
As digital learning becomes more common in higher education, learners in online and blended learning environments may encounter challenges such as insufficient engagement, distraction, and course non-completion. Therefore, promoting sustained participation and long-term learning persistence has become an important issue in educational technology and instructional design. In recent years, gamification has attracted increasing attention because of its potential to support learner engagement, feedback, progress visualization, and social interaction. However, its longer-term motivational mechanisms in educational contexts remain insufficiently clarified. This study examined the relationships among gamification design elements, intrinsic motivation, and learning persistence in a 16-week blended digital game-based learning course. A one-group post-intervention survey design was adopted. A total of 337 students from a university in Taichung, Taiwan participated in the study, and data were collected through paper-based self-report questionnaires at the end of the course. Exploratory factor analysis and reliability analysis indicated acceptable construct validity and internal consistency of the measurement scales. Structural equation modeling showed that gamification design elements were significantly and positively associated with intrinsic motivation, and intrinsic motivation was significantly and positively associated with learning persistence. Gamification design elements were also directly associated with learning persistence. The mediation analysis further indicated that intrinsic motivation partially mediated the relationship between gamification design elements and learning persistence. The model fit indices suggested acceptable model fit, and the standardized path coefficients indicated meaningful but moderate positive associations. Overall, the findings provide preliminary empirical support for the proposed model and suggest that need-supportive gamified instructional design may be related to higher intrinsic motivation and stronger learning persistence. The study contributes to gamified learning research by interpreting gamification design as a need-supportive instructional structure rather than merely as a reward-based strategy.
Late HIV diagnosis is associated with a higher impact on treatment outcomes and a potential for prolonged transmissibility of HIV-1 infection. The consensus definition for late HIV diagnosis is problematic. It was updated in 2022; however, this definition relies on information that might not be clinically available. This study aimed to assess late HIV diagnosis using alternative parameters, in addition to the definition of clusters of differentiation (CD4) cell count, namely, sequence ambiguity rate and estimated time of infection inferred through phylogenetic analysis. Clinical, socio-demographic, and genotypic information from 3668 antiretroviral therapy-naïve individuals living with HIV was retrieved from the REGA database. Individuals were classified according to three approaches: (i) CD4 cell count, (ii) sequence ambiguity rate, and (iii) phylogenetic reconstruction using TreeTime to estimate the time of most recent common ancestor (MRCA) as a proxy for time of infection. Based on CD4 cell count, 53.8% of individuals had a late diagnosis and 46.2% had a non-late diagnosis. Based on sequence ambiguity rate, 57.8% had a chronic and 42.2% had a recent infection, and 86.4% had an estimated time of infection of more than 3 years, whereas 13.6% had less than 3 years. A total of 114 individuals were classified as diagnosed late by CD4 criteria and showed evidence of recent infection based on low ambiguity rates and MRCA estimates under 3 years. These individuals had significantly lower viral loads than those with true late diagnoses (median 61,358 vs 134,730 copies/ml; P <0.001). Overall, 41% of individuals were consistently classified across all three methods. The definition of late diagnosis remains a major challenge. Alternative and complementary methods, such as the use of viral loads, combined with some more clinical information, may improve the lack of baseline data.