Developmental and temporal characteristics of clonal sperm mosaicism.

Throughout development and aging, human cells accumulate mutations resulting in genomic mosaicism and genetic diversity at the cellular level. Mosaic mutations present in the gonads can affect both the individual and the offspring and subsequent generations. Here, we explore patterns and temporal stability of clonal mosaic mutations in male gonads by sequencing ejaculated sperm. Through 300× whole-genome sequencing of blood and sperm from healthy men, we find each ejaculate carries on average 33.3 ± 12.1 (mean ± SD) clonal mosaic variants, nearly all of which are detected in serial sampling, with the majority absent from sampled somal tissues. Their temporal stability and mutational signature suggest origins during embryonic development from a largely immutable stem cell niche. Clonal mosaicism likely contributes a transmissible, predicted pathogenic exonic variant for 1 in 15 men, representing a life-long threat of transmission for these individuals and a significant burden on human population health.

Landscape-scale benefits of protected areas for tropical biodiversity.

The United Nations recently agreed to major expansions of global protected areas (PAs) to slow biodiversity declines1. However, although reserves often reduce habitat loss, their efficacy at preserving animal diversity and their influence on biodiversity in surrounding unprotected areas remain unclear2-5. Unregulated hunting can empty PAs of large animals6, illegal tree felling can degrade habitat quality7, and parks can simply displace disturbances such as logging and hunting to unprotected areas of the landscape8 (a phenomenon called leakage). Alternatively, well-functioning PAs could enhance animal diversity within reserves as well as in nearby unprotected sites9 (an effect called spillover). Here we test whether PAs across mega-diverse Southeast Asia contribute to vertebrate conservation inside and outside their boundaries. Reserves increased all facets of bird diversity. Large reserves were also associated with substantially enhanced mammal diversity in the adjacent unprotected landscape. Rather than PAs generating leakage that deteriorated ecological conditions elsewhere, our results are consistent with PAs inducing spillover that benefits biodiversity in surrounding areas. These findings support the United Nations goal of achieving 30% PA coverage by 2030 by demonstrating that PAs are associated with higher vertebrate diversity both inside their boundaries and in the broader landscape.

Somatic mosaicism reveals clonal distributions of neocortical development.

The structure of the human neocortex underlies species-specific traits and reflects intricate developmental programs. Here we sought to reconstruct processes that occur during early development by sampling adult human tissues. We analysed neocortical clones in a post-mortem human brain through a comprehensive assessment of brain somatic mosaicism, acting as neutral lineage recorders1,2. We combined the sampling of 25 distinct anatomic locations with deep whole-genome sequencing in a neurotypical deceased individual and confirmed results with 5 samples collected from each of three additional donors. We identified 259 bona fide mosaic variants from the index case, then deconvolved distinct geographical, cell-type and clade organizations across the brain and other organs. We found that clones derived after the accumulation of 90-200 progenitors in the cerebral cortex tended to respect the midline axis, well before the anterior-posterior or ventral-dorsal axes, representing a secondary hierarchy following the overall patterning of forebrain and hindbrain domains. Clones across neocortically derived cells were consistent with a dual origin from both dorsal and ventral cellular populations, similar to rodents, whereas the microglia lineage appeared distinct from other resident brain cells. Our data provide a comprehensive analysis of brain somatic mosaicism across the neocortex and demonstrate cellular origins and progenitor distribution patterns within the human brain.

FLAN: feature-wise latent additive neural models for biological applications.

MOTIVATION: Interpretability has become a necessary feature for machine learning models deployed in critical scenarios, e.g. legal system, healthcare. In these situations, algorithmic decisions may have (potentially negative) long-lasting effects on the end-user affected by the decision. While deep learning models achieve impressive results, they often function as a black-box. Inspired by linear models, we propose a novel class of structurally constrained deep neural networks, which we call FLAN (Feature-wise Latent Additive Networks). Crucially, FLANs process each input feature separately, computing for each of them a representation in a common latent space. These feature-wise latent representations are then simply summed, and the aggregated representation is used for the prediction. These feature-wise representations allow a user to estimate the effect of each individual feature independently from the others, similarly to the way linear models are interpreted. RESULTS: We demonstrate FLAN on a series of benchmark datasets in different biological domains. Our experiments show that FLAN achieves good performances even in complex datasets (e.g. TCR-epitope binding prediction), despite the structural constraint we imposed. On the other hand, this constraint enables us to interpret FLAN by deciphering its decision process, as well as obtaining biological insights (e.g. by identifying the marker genes of different cell populations). In supplementary experiments, we show similar performances also on non-biological datasets. CODE AND DATA AVAILABILITY: Code and example data are available at https://github.com/phineasng/flan_bio.

Recombination resolves the cost of horizontal gene transfer in experimental populations of Helicobacter pylori.

Horizontal gene transfer (HGT) is important for microbial evolution, yet we know little about the fitness effects and dynamics of horizontally transferred genetic variants. In this study, we evolve laboratory populations of Helicobacter pylori, which take up DNA from their environment by natural transformation, and measure the fitness effects of thousands of transferred genetic variants. We find that natural transformation increases the rate of adaptation but comes at the cost of significant genetic load. We show that this cost is circumvented by recombination, which increases the efficiency of selection by decoupling deleterious and beneficial genetic variants. Our results show that adaptation with HGT, pervasive in natural microbial populations, is shaped by a combination of selection, recombination, and genetic drift not accounted for in existing models of evolution.

Peroxisomal ß-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer.

BACKGROUND: Peroxisomes are central metabolic organelles that have key roles in fatty acid homoeostasis. As prostate cancer (PCa) is particularly reliant on fatty acid metabolism, we explored the contribution of peroxisomal ß-oxidation (perFAO) to PCa viability and therapy response. METHODS: Bioinformatic analysis was performed on clinical transcriptomic datasets to identify the perFAO enzyme, 2,4-dienoyl CoA reductase 2 (DECR2) as a target gene of interest. Impact of DECR2 and perFAO inhibition via thioridazine was examined in vitro, in vivo, and in clinical prostate tumours cultured ex vivo. Transcriptomic and lipidomic profiling was used to determine the functional consequences of DECR2 inhibition in PCa. RESULTS: DECR2 is upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa (CRPC). Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and therapy-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. DECR2 influences cell cycle progression and lipid metabolism to support tumour cell proliferation. Further, co-targeting of perFAO and standard-of-care androgen receptor inhibition enhanced suppression of PCa cell proliferation. CONCLUSION: Our findings support a focus on perFAO, specifically DECR2, as a promising therapeutic target for CRPC and as a novel strategy to overcome lethal treatment resistance.

Implementing living-donor pediatric liver transplantation in Southern Vietnam: 15-year results and perspectives.

BACKGROUND: ND2 in Ho Chi Minh City is currently the only public center that performs PLT in Southern Vietnam. In 2005, the first PLT was successfully performed, with support from Belgian experts. This study reviews the implementation of PLT at our center and evaluates the results and challenges. METHODS: Implementation of PLT at ND2 required medico-surgical team building and extensive improvement of hospital facilities. Records of 13 transplant recipients from 2005 to 2020 were studied retrospectively. Short- and long-term complications, as well as the survival rates, were reported. RESULTS: The mean follow-up time was 8.3 ± 5.7 years. Surgical complications included one case of hepatic artery thrombosis that was successfully repaired, one case of colon perforation resulting in death from sepsis, and two cases of bile leak that were drained surgically. PTLD was observed in five patients, of whom three died. There were no cases of retransplantation. The 1-year, 5-year, and 10-year patient survival rates were 84.6%, 69.2%, and 69.2%, respectively. There were no cases of complication or death among the donors. CONCLUSION: Living-donor PLT was developed at ND2 for providing a life-saving treatment to children with end-stage liver disease. Early surgical complication rate was low, and the patient survival rate was satisfactory at 1 year. Long-term survival decreased considerably due to PTLD. Future challenges include surgical autonomy and improvement of long-term medical follow-up with a particular emphasis on prevention and management of Epstein-Barr virus-related disease.

Clinical characteristics of COVID-19 patients treated in emergency COVID-19 hospitals in Vietnam: Experience from Phutho province, Vietnam.

Background: This study aimed to evaluate the clinical characteristics, patient's management approaches, and outcomes of the COVID-19 patients in Phu Tho Province, Vietnam. Methods: A retrospective, multicenter study of 2166 COVID-19 patients in 13 hospitals in Phutho Province, Vietnam. The subjects were divided into 3 groups based on vaccination status: unvaccinated group, 1st dose of vaccine group, 2nd dose of vaccine group. The clinical characteristics, management approaches, and outcomes were collected and compared between the 3 groups. Results: The hospitalization rate of the 3 groups decreased from the unvaccinated group, the 1st dose of vaccinated group, to the 2nd dose of vaccinated group, 42.61%; 30,24% and 27,15% respectively. The 19-40 years old group had the highest hospitalization rate (38,1%) together with the group that had not accepted the full COVID 19 vaccination dose (57,64%). The 2nd dose of vaccinated group had the lowest percentages of high temperature, cough, dyspnea, chest pain and sore throat. The unvaccinated group had the highest heart rate, respiratory rate and SpO2 compared to the two other groups. The percentage needing Immunomodulation and Anticoagulant Therapy was highest (6.8% and 1.4 % respectively) in the unvaccinated group. The percentage receiving Antiviral Therapy was highest (42,5%) in those who had received the 2nd dose of vaccine. Conclusions: COVID-19 vaccination improved the symptoms of the patients and should be accepted in all ages.

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish.

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.

The mediation model of social status on the link between parental attachment, aggressive behavior, and psychological well-being: Evidence from two studies in Vietnamese adolescents and young adults.

Little is known about how insecure attachment affects aggressive behavior and well-being among Vietnamese youth. Using structural equation modeling, we investigate the mediating role of subjective social status (SSS) on the paths from insecure attachment to overt aggressive behavior (OAB) and psychological well-being (PW) in a sample of 1753 Vietnamese adolescents (Mage = 16.136, SD = 0.784) and test whether the results will be replicated in another sample of 601 Vietnamese young adults (Mage = 19.93, SD = 1.35). Participants complete a survey comprising demographic information, attachment styles, SSS, OAB, and PW questionnaires. Our main findings include: (a) anxious attachment positively related to OAB in both samples, (b) anxious attachment was only negatively associated with adolescents' PW, (c) avoidant attachment was positively correlated to OAB in adolescents but negatively correlated in young adults, and (d) avoidant attachment was negatively related to PW in both samples. In addition, (e) in adolescents, the mediation role of SSS was significant in all paths, but (f) SSS only mediated the link from avoidant attachment to PW of young adults. The present study suggests that aggressive behavior might not be associated with social status or attachment in the same way in adolescents compared to young adult Vietnamese samples.

Formation of templated inclusions in a forebrain α-synuclein mouse model is independent of LRRK2.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

Cost of Acute and Sequelae Care for Japanese Encephalitis Patients, Bangladesh, 2011-2021.

Japanese encephalitis (JE) is associated with an immense social and economic burden. Published cost-of-illness data come primarily from decades-old studies. To determine the cost of care for patients with acute JE and initial and long-term sequelae from the societal perspective, we recruited patients with laboratory-confirmed JE from the past 10 years of JE surveillance in Bangladesh and categorized them as acute care, initial sequalae, and long-term sequelae patients. Among 157 patients, we categorized 55 as acute, 65 as initial sequelae (53 as both categories), and 90 as long-term sequelae. The average (median) societal cost of an acute JE episode was US $929 ($909), of initial sequelae US $75 ($33), and of long-term sequelae US $47 ($14). Most families perceived the effect of JE on their well-being to be extreme and had sustained debt for JE expenses. Our data about the high cost of JE can be used by decision makers in Bangladesh.

DECODE: a computational pipeline to discover T cell receptor binding rules.

MOTIVATION: Understanding the mechanisms underlying T cell receptor (TCR) binding is of fundamental importance to understanding adaptive immune responses. A better understanding of the biochemical rules governing TCR binding can be used, e.g. to guide the design of more powerful and safer T cell-based therapies. Advances in repertoire sequencing technologies have made available millions of TCR sequences. Data abundance has, in turn, fueled the development of many computational models to predict the binding properties of TCRs from their sequences. Unfortunately, while many of these works have made great strides toward predicting TCR specificity using machine learning, the black-box nature of these models has resulted in a limited understanding of the rules that govern the binding of a TCR and an epitope. RESULTS: We present an easy-to-use and customizable computational pipeline, DECODE, to extract the binding rules from any black-box model designed to predict the TCR-epitope binding. DECODE offers a range of analytical and visualization tools to guide the user in the extraction of such rules. We demonstrate our pipeline on a recently published TCR-binding prediction model, TITAN, and show how to use the provided metrics to assess the quality of the computed rules. In conclusion, DECODE can lead to a better understanding of the sequence motifs that underlie TCR binding. Our pipeline can facilitate the investigation of current immunotherapeutic challenges, such as cross-reactive events due to off-target TCR binding. AVAILABILITY AND IMPLEMENTATION: Code is available publicly at https://github.com/phineasng/DECODE. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein Phosphatase 2A Activation Via ApoER2 in Trophoblasts Drives Preeclampsia in a Mouse Model of the Antiphospholipid Syndrome.

[Figure: see text].

Estimating the economic burden of respiratory syncytial virus infections in infants in Vietnam: a cohort study.

BACKGROUND: Little information is available on the costs of respiratory syncytial virus (RSV) in Vietnam or other low- and middle-income countries. Our study estimated the costs of LRTIs associated with RSV infection among children in southern Vietnam. METHODS: We conducted a prospective cohort study evaluating household and societal costs associated with LRTIs stratified by RSV status and severity among children under 2 years old who sought care at a major pediatric referral hospital in southern Vietnam. Enrollment periods were September 2019-December 2019, October 2020-June 2021 and October 2021-December 2021. RSV status was confirmed by a validated RT-PCR assay. RSV rapid detection antigen (RDA) test performance was also evaluated. Data on resource utilization, direct medical and non-medical costs, and indirect costs were collected from billing records and supplemented by patient-level questionnaires. All costs are reported in 2022 US dollars. RESULTS: 536 children were enrolled in the study, with a median age of 7 months (interquartile range [IQR] 3-12). This included 210 (39.2%) children from the outpatient department, 318 children (59.3%) from the inpatient respiratory department (RD), and 8 children (1.5%) from the intensive care unit (ICU). Nearly 20% (105/536) were RSV positive: 3.9 percent (21/536) from the outpatient department, 15.7% (84/536) from the RD, and none from the ICU. The median total cost associated with LRTI per patient was US$52 (IQR 32-86) for outpatients and US$184 (IQR 109-287) for RD inpatients. For RSV-associated LRTIs, the median total cost per infection episode per patient was US$52 (IQR 32-85) for outpatients and US$165 (IQR 95-249) for RD inpatients. Total out-of-pocket costs of one non-ICU admission of RSV-associated LRTI ranged from 32%-70% of the monthly minimum wage per person (US$160) in Ho Chi Minh City. The sensitivity and the specificity of RSV RDA test were 88.2% (95% CI 63.6-98.5%) and 100% (95% CI 93.3-100%), respectively. CONCLUSION: These are the first data reporting the substantial economic burden of RSV-associated illness in young children in Vietnam. This study informs policymakers in planning health care resources and highlights the urgency of RSV disease prevention.

Event-Related Potentials of Single-Sided Deaf Cochlear Implant Users: Using a Semantic Oddball Paradigm in Noise.

INTRODUCTION: In individuals with single-sided deafness (SSD), who are characterised by profound hearing loss in one ear and normal hearing in the contralateral ear, binaural input is no longer present. A cochlear implant (CI) can restore functional hearing in the profoundly deaf ear, with previous literature demonstrating improvements in speech-in-noise intelligibility with the CI. However, we currently have limited understanding of the neural processes involved (e.g., how the brain integrates the electrical signal produced by the CI with the acoustic signal produced by the normal hearing ear) and how modulation of these processes with a CI contributes to improved speech-in-noise intelligibility. Using a semantic oddball paradigm presented in the presence of background noise, this study aims to investigate how the provision of CI impacts speech-in-noise perception of SSD-CI users. METHOD: Task performance (reaction time, reaction time variability, target accuracy, subjective listening effort) and high density electroencephalography from twelve SSD-CI participants were recorded, while they completed a semantic acoustic oddball task. Reaction time was defined as the time taken for a participant to press the response button after stimulus onset. All participants completed the oddball task in three different free-field conditions with the speech and noise coming from different speakers. The three tasks were: (1) CI-On in background noise, (2) CI-Off in background noise, and (3) CI-On without background noise (Control). Task performance and electroencephalography data (N2N4 and P3b) were recorded for each condition. Speech in noise and sound localisation ability were also measured. RESULTS: Reaction time was significantly different between all tasks with CI-On (M [SE] = 809 [39.9] ms) having faster RTs than CI-Off (M [SE] = 845 [39.9] ms) and Control (M [SE] = 785 [39.9] ms) being the fastest condition. The Control condition exhibited significantly shorter N2N4 and P3b area latency compared to the other two conditions. However, despite these differences noticed in RTs and area latency, we observed similar results between all three conditions for N2N4 and P3b difference area. CONCLUSION: The inconsistency between the behavioural and neural results suggests that EEG may not be a reliable measure of cognitive effort. This rationale is further supported by different explanations used in past studies to explain N2N4 and P3b effects. Future studies should look to alternative measures of auditory processing (e.g., pupillometry) to gain a deeper understanding of the underlying auditory processes that facilitate speech-in-noise intelligibility.

A Study of Event-Related Potentials During Monaural and Bilateral Hearing in Single-Sided Deaf Cochlear Implant Users.

OBJECTIVES: Single-sided deafness (SSD) is characterized by a profoundly deaf ear and normal hearing in the contralateral ear. A cochlear implant (CI) is the only method to restore functional hearing in a profoundly deaf ear. In a previous study, we identified that the cortical processing of a CI signal differs from the normal-hearing ear (NHE) when directly compared using an auditory oddball paradigm consisting of pure tones. However, exactly how the brain integrates the electrical and acoustic signal is not well investigated. This study aims to understand how the provision of the CI in combination with the NHE may improve SSD CI users' ability to discriminate and evaluate auditory stimuli. DESIGN: Electroencephalography from 10 SSD-CI participants (4 participated in the previous pure-tone study) were recorded during a semantic acoustic oddball task, where they were required to discriminate between odd and even numbers. Stimuli were presented in four hearing conditions: directly through the CI, directly to the NHE, or in free field with the CI switched on and off. We examined task-performance (response time and accuracy) and measured N1, P2, N2N4, and P3b event-related brain potentials (ERPs) linked to the detection, discrimination, and evaluation of task relevant stimuli. Sound localization and speech in noise comprehension was also examined. RESULTS: In direct presentation, task performance was superior during NHE compared with CI (shorter and less varied reaction times [~720 versus ~842 msec], higher target accuracy [~93 versus ~70%]) and early neural responses (N1 and P2) were enhanced for NHE suggesting greater signal saliency. However, the size of N2N4 and P3b target-standard effects did not differ significantly between NHE and CI. In free field, target accuracy was similarly high with the CI (FF-On) and without the CI (FF-Off) (~95%), with some evidence of CI interference during FF-On (more variable and slightly but significantly delayed reaction times [~737 versus ~709 msec]). Early neural responses and late effects were also greater during FF-On. Performance on sound localization and speech in noise comprehension (S CI N NHE configuration only) was significantly greater during FF-On. CONCLUSIONS: Both behavioral and neural responses in the semantic oddball task were sensitive to CI in both direct and free-field presentations. Direct conditions revealed that participants could perform the task with the CI alone, although performance was suboptimal and early neural responses were reduced when compared with the NHE. For free-field, the addition of the CI was associated with enhanced early and late neural responses, but this did not result in improved task performance. Enhanced neural responses show that the additional input from the CI is modulating relevant perceptual and cognitive processes, but the benefit of binaural hearing on behavior may not be realized in simple oddball tasks which can be adequately performed with the NHE. Future studies interested in binaural hearing should examine performance under noisy conditions and/or use spatial cues to allow headroom for the measurement of binaural benefit.

Impacts of COVID-19 crisis and some related factors on the mental health of 37150 Vietnamese students: a cross-sectional online study.

BACKGROUND: University students are vulnerable to changes due to COVID-19 pandemic. Although warning has been made about the impact of this crisis on students' mental health, there are barely any sufficient study. This work investigated how the pandemic affected the mental health of students at the Vietnam National University of Ho Chi Minh City (VNU-HCMC) and efficiency of available mental health supportive methods. METHODS: An online survey was conducted among students at Vietnam National University of Ho Chi Minh City (VNU-HCMC) from October 18, 2021, to October 25, 2021. Microsoft Excel 16.51 (Microsoft, USA) and R language, Epi packages 2.44 and 4.1.1 (rdrr.io) were used for data analysis. RESULTS: Thirty-seven thousand one hundred fifty students participated in the survey, including 48.4% female and 51.6% male. Online learning pressure was mainly recorded (65.1%). Many students suffered from sleeping disorders (56.2%). Some reported being victims of abuse (5.9%). Female students expressed a significantly higher level of distress than males, particularly the feeling of ambiguity about the purpose of life (p-value < 0.0001, OR: 0.94, 95% CI: [0.95-0.98]). Third-year students suffered higher stress levels than others, especially in online learning (68.8%, p-value < 0.05). Mental health statuses among students of different lockdown status regions were not significantly different. Therefore, lockdown status did not affect the stress levels of students which suggested that poor mental health outcomes seemed to root in the suspension of everyday university life rather than the prohibition of going out. CONCLUSIONS: During COVID-19, students experienced lots of stress and mental problems. These findings underscore the importance of academic and innovative activities, bringing attention to the needs of interactive study and extra-curricular activities.