Background Oligoasthenoteratospermia (OAT) represents a major cause of male infertility. The molecular mechanisms underlying sperm morphological defects, particularly their potential interplay with environmental exposure-associated molecular alterations, remain poorly understood and warrant exploratory investigation. Methods Semen and plasma samples were collected from normal controls (NC, n = 6), mild morphological defect (MMD, n = 6), and severe morphological defect (SMD, n = 6) subjects in this exploratory pilot study designed to detect large-effect signals. Integrated proteometabolomic profiling was performed using 4D proteomics and LC-MS. Bioinformatic analysis focused on screening differential expression, functional enrichment, exploring potential interactions between exposure-associated metabolites and proteins, and identifying coherent patterns to potentially differentiate groups. Results Comparative analysis identified biphasic dysregulation patterns in key cytoskeletal proteins. The prominent hub ACTN4 exhibited depletion in MMD sperm while displaying pathological spatial dislocation with 83% showing midpiece signal loss and fragmented tail fluorescence, accompanied by extracellular vesicle release in SMD sperm. Quantitative immunofluorescence validation confirmed these divergent phenotypes, that is a 56% systemic reduction in MMD compared to controls (p < 0.0001), with SMD sperm also showing 41% absolute depletion versus NC (p < 0.0001) despite proteomic upregulation, which indicates functional loss through vesicular shedding. Plasma metabolomics revealed distinct profiles between severity groups, quantifying 357 differentially abundant metabolites (including prioritized selenometabolite-bile acid interactions) between SMD and MMD. Exploratory correlation analysis prioritized inverse associations including arsenocholine (an established environmental arsenic exposure biomarker) and ACTN4 (rho = -0.867, p = 0.0025), glycodeoxycholic acid versus PPP1CC (rho = -0.917, p < 0.001), and Se-adenosylselenomethionine-PGK1 (rho = -0.867, p = 0.0025), reflecting ultrastructural damage patterns. Conclusion This severity-stratified analysis demonstrates that sperm morphological defects in OAT associate with compartment-specific ACTN4 depletion patterns, that is quantitative loss systemically impairing head-tail coupling in MMD versus spatial dislocation with pathological vesiculation causing functional nullification in SMD. The exploratory findings suggest potential associations with multisystem exposure effects, particularly indicated by the arsenocholine-ACTN4 correlation, though causal relationships cannot be established from this observational study. In addition, exposure-protein associations involving PPP1CC and PGK1 also support the hypothesis of environmental perturbation affecting sperm integrity, though these observations require validation in independent cohorts.

Epimedium (Berberidaceae) is a fundamental herb in traditional Chinese medicine, historically prescribed for Kidney deficiency syndromes manifesting as reproductive decline and age-related debility. Its classic function of tonifying the Kidney and invigorating the Yang is supported by a complex, multi-component pharmacological profile. It is a cornerstone of traditional Chinese medicine and the 'monarch' drug in classical formulas like Er-xian Tang. As the most abundant and systematically studied active monomer in Epimedium extracts, ICA combines the antioxidant backbone of flavonoids with the biological membrane affinity conferred by its isoprene side chain. This dual nature enables it to exhibit potent biological activity and signaling regulation capabilities across diverse tissues. In recent years, ICA's regulatory role in male reproductive physiology has garnered increasing attention. It can significantly improve male reproductive function by regulating redox homeostasis, hormonal biosynthesis, and the balance between apoptosis and proliferation. It may also be used in combination with pharmacological agents or biotechnological approaches to enhance therapeutic efficacy further. Therefore, this review systematically examines the targets and mechanisms through which ICA improves male reproductive function and evaluates its value as monotherapy or as a component of combination therapies, providing a theoretical framework and translational research direction for managing male reproductive disorders and enhancing assisted reproductive technologies outcomes.

Integrated in-incubator microscopy systems and all-in-one time-lapse culture incubators are often expensive and proprietary, which limits accessibility and customization. Open-source alternatives exist but often involve tradeoffs in imaging performance, illumination control, or adaptability. Here, we describe a do-it-yourself (DIY), low-cost, three-dimensional (3D)-printed time-lapse imaging platform designed for use inside standard cell culture incubators. The device employs a Raspberry Pi-based controller with stepper motor actuation and a programmable light-emitting diode (LED) backlight that enables both bright-field and oblique illumination modes. The system supports automated multi-well imaging, scheduled time-lapse acquisition, and real-time preview through open-source Python software. Performance validation demonstrated reliable long-term imaging of cultured cells and early embryos, with stable operation under incubator conditions. Optical resolution testing using a United States Air Force (USAF) 1951 target confirmed a minimum resolvable feature of approximately 1.55 µm. Motion evaluation showed low drift and high repeatability, with positioning accuracy closely matching commanded displacements. The system is compatible with multiple plate formats and can be adapted for specialized culture setups. Overall, this platform provides a cost-effective, reproducible, and customizable solution for long-term live-cell and embryo imaging in laboratory settings.

Age-related impaired wound healing presents distinct challenges compared to conventional wounds, primarily due to dysregulated phase kinetics of wound repair and chronic cellular senescence, which collectively disrupt the dynamic and orderly progression of tissue regeneration. Effective intervention requires a stage-specific and temporally programmed therapeutic strategy. To address this, we first performed transcriptome sequencing (RNA-seq) to elucidate the differential healing trajectory between young and aged wounds. Herein, we construct a photo-responsive, Fisetin-functionalized DNA nanocage (TAF) for spatiotemporally precise therapy of aged wounds. TAF is composed of a tetrahedral DNA nanocage loaded with a miR-29-targeting antisense oligonucleotide (ASO) covalently linked through a photocleavable linker and noncovalently encapsulating the senolytic agent Fisetin. The TAF platform enables a programmed therapeutic strategy: (a) the intrinsic antioxidant capacity of DNA scavenges excessive reactive oxygen species (ROS) during the early inflammatory phase; (b) photo-triggered ASO release in the proliferative phase promotes collagen synthesis; and (c) sustained Fisetin release eliminates senescent cells, fostering a pro-regenerative microenvironment. In vivo studies in aged mice reveal that TAF-mediated therapy significantly accelerates early wound closure and enhances high-quality tissue regeneration. In summary, TAF establishes an efficient nanomedicine platform that improves aged tissue repair through stage-specific, temporally programmed regulation.

The circadian regulation of autophagy in airway epithelial inflammation remains poorly understood, particularly in the context of asthma. This study investigated how nuclear receptor subfamily 1 group D member 1 (NR1D1)-mediated downregulation of myosin-like BCL2-interacting protein (BECLIN1) influences autophagy in airway epithelial cells to alleviate inflammation. An ovalbumin-induced asthma mouse model and a Beas-2b cell line with type 2 inflammation were established. Rhythmic fluctuations in circadian clock genes (Circadian locomotor output cycles Kaput [Clock], Brain and muscle Arnt-like protein 1 [Bmal1], and Nr1d1) and autophagy-related genes (Autophagy Related 5 [Atg5], Beclin1, and microtubule-associated proteins 1A/1B light chain 3B [Lc3]) were monitored. In vitro, NR1D1 expression was modulated using SR9009 or siRNA, and effects on BECLIN1 and LC3 were assessed. BECLIN1 expression was altered via plasmid transfection to evaluate effects on NR1D1. NR1D1 bonded with and repressed the BECLIN1 promoter. In asthmatic mice and Beas-2b cells, NR1D1 expression was reduced, whereas ATG5, BECLIN1, and LC3 were elevated. CLOCK, BMAL1, NR1D1, ATG5, BECLIN1, and LC3 levels in the lung tissue showed cyclic fluctuations, with NR1D1 and BECLIN1 negatively correlated. SR9009 reduced BECLIN1 and LC3 levels, while NR1D1 knockdown increased them in vitro. BECLIN1 overexpression elevated NR1D1, whereas its inhibition reduced NR1D1. Circadian and autophagy-related genes showed pronounced periodic oscillation in asthmatic mice, coinciding with airway inflammation. NR1D1 negatively regulates BECLIN1 to suppress autophagy and mitigate airway inflammation in asthma, indicating a possible therapeutic target.

Cutaneous malignant melanoma is a highly aggressive skin cancer with poor prognosis. Although microRNA (miRNA)-based gene therapy shows promise, its clinical translation is limited by rapid degradation in serum and the barrier function of the stratum corneum. Herein, we engineer a programmable, targeted, and non-invasive nanoplatform, termed MARS (MUC1-Aptamer-functionalized, tetrahedral framework nucleic acid-based miRNA delivery system for gene Silencing). The therapeutic miR-30a-5p is encapsulated within a self-assembled DNA tetrahedron, an architecture that confers exceptional structural stability. The framework is specifically functionalized with an MUC1 aptamer to act as a navigator for active tumor targeting while simultaneously carrying the gene-silencing payload. In vitro assays demonstrate that MARS successfully delivers miR-30a-5p into A375 cells, effectively silencing the transcription factor E2F7 and subsequently triggering the intrinsic apoptotic pathway. Importantly, in vivo studies using a xenograft mouse model reveal that topically applied MARS can penetrate the skin barrier and accumulate in tumor tissues, achieving a tumor growth inhibition rate of 66.86%, comparable to intravenous anti-PD-L1 immunotherapy but with superior safety, compliance, and cost-effectiveness. This study presents MARS as a robust strategy that integrates stability, programmable dual-functionality (targeting and gene silencing), and non-invasive transdermal capabilities, providing a promising strategy for localized melanoma treatment.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy, exhibiting pronounced invasive characteristics and a dismal prognosis. Conventional therapeutic modalities, including radiotherapy, laser therapy, and surgery, are frequently invasive and can lead to complications, underscoring the need for the development of efficacious, safe, and noninvasive therapeutic approaches. This study investigated a tetrahedral framework nucleic acid (tFNA)-based bioswitchable microRNA (miRNA) delivery system, designated BiRDS, engineered for the inhibition of UM through the use of miRNA suppressors via noninvasive eyedrops. The BiRDS construct exhibited a tetrahedral structure, which was small in size, easily synthesizable, stable, and biosafe, and was able to efficiently carry miR-30a-5p into UM cells. Functionally, BiRDS was observed to inhibit the proliferation, migration, and invasion of UM cells while promoting apoptosis through the miR-30a-5p/E2F7 axis. It is noteworthy that BiRDS nanoeyedrops were able to penetrate the complex ocular barrier structure and reach the fundus, thereby inhibiting the growth of UM in a xenograft model. As a patient-friendly, eyedrop-based miRNA delivery system, BiRDS not only inhibited UM without enucleation of the eyeball but was also expected to improve patient compliance and quality of life while providing a safer alternative for ocular drug administration. This work substantiates BiRDS nanoeyedrops as a potential paradigm shift in the local treatment of early UM, facilitating its application in treating other ocular diseases via miRNA therapies.

BACKGROUND:Air pollution exposure (both individual and joint) is associated with dementia, but its relation to early-onset dementia (EOD) and late-onset dementia (LOD) remains inconclusive. Meanwhile, the modification by genetic predisposition and mediation by accelerated biological aging are also unclear. METHODS:A cohort of 285 774 dementia-free participants from the UK Biobank was analyzed. Exposure levels of four major air pollutants (PM2.5, PM10, NO2, and NOx), two air pollution scores (APS1 and APS2) were obtained, and their associations with all-cause dementia (ACD), EOD and LOD were assessed via Cox models. Genetic predisposition to dementia was evaluated and the mediation role of PhenoAge-Accel was investigated under the counterfactual framework. RESULTS:During a median follow-up of 13.4 years, 3 898 participants developed ACD, including 231 with EOD and 3 650 with LOD. Per IQR increase of PM2.5, PM10, NO2, NOx, APS1 and APS2 was associated with 6.5% (95%CIs) (2.3%-10.9%), 6.8% (2.2%-11.5%), 4.6% (0%-9.5%), 5.3% (0.7%-10.0%), 6.8% (2.7%-11.1%), or 6.7% (2.2%-11.4%) higher risk of incident ACD, exhibiting a stronger effect on EOD than LOD. Participants with the highest polygenic risk score and air pollution scores possessed the greatest risk of ACD, EOD, and LOD. PhenoAge-Accel moderately mediated the influence of air pollution exposure on ACD risk, especially among low genetic risk participants, with slightly lower mediation effects for EOD than LOD. Similar results were found when adopting KDMAge-Accel. CONCLUSIONS:Long-term joint exposure to air pollutants exhibited stronger associations with EOD than LOD, and accelerated biological aging serves as a partial mediator in this adverse connection.

BACKGROUND:Respiratory syncytial virus (RSV) is the most common pathogen of acute lower respiratory tract infection in children under 5 years old. Here, we aims to investigate the role of the central clock gene BMAL1 in RSV infection. METHODS:The rhythmic oscillation of clock genes, pro-inflammatory factors, viral loads and p-p38/p38 in the lung of mice were observed. BMAL1 knockdown mice, Beas-2B cells and jet-lagged mice were infected with RSV. The virus copy numbers, expression of inflammatory factors and clock genes, and pathological inflammatory infiltration of the lung were measured. Transcriptomic analysis was performed to find the possible pathway. RESULTS:The expression of clock genes in the lungs of mice exhibited circadian rhythmicity. The rhythmic oscillation amplitude and trend of clock genes, inflammatory factors, DUSP1, and p-p38/p38 in the lung of RSV infected mice were altered. BMAL1-knockdown and jet-lagged mice showed exacerbated RSV infection and RSV-induced inflammation in the lung. Analysis of transcriptomes showed that BMAL1 regulated RSV-induced inflammation through the p38 MAPK axis. Regulation of RSV infection by BMAL1 was also confirmed in Beas-2B cells. CONCLUSIONS:RSV infection affects the expression of clock genes in Beas-2B cells and lungs of mice, and alters the rhythmic oscillation patterns of pro-inflammatory factors, DUSP1 and p-p38/p38 in the lungs. BMAL1 disruption is identified as a potential pathological factor of RSV-associated inflammation. p38 MAPK pathway is involved in the regulation of RSV infection and RSV-induced inflammation by BMAL1.

This letter aims to provide valuable insights into broader evidence triangulation (i.e., a well-designed primary association analysis followed by elaborate approaches to control residual confounding effects from various design and modeling perspectives) for clarifying the association between air pollutants and health outcomes. It also highlights the importance of selecting appropriate instrumental variable for instrument-based causal modeling, emphasizing that all causal questions can be effectively addressed within the Mendelian randomization framework.

PURPOSE:Some studies have shown that circadian rhythms are associated with the development and progression of hepatocellular carcinoma (HCC). In this study, we aimed to elucidate the characterization, prognostic significance and targeting value of circadian rhythm gene CSNK1E in HCC. METHODS:In this study, relevant datasets were downloaded from TCGA and GEO databases and analyzed for differences respectively. The key modules of the prognosis-related gene set were identified using WGCNA, and the intersection of the key module genes with 48 circadian rhythm genes was taken and analyzed in single-cell data. The identification of key circadian rhythm genes in HCC aimed to analyze the expression, prognostic significance, and clinically relevant features of CSNK1E in cancer. The expression characteristics of CSNK1E were further examined by immunohistochemistry, western blotting (WB), and Real-time quantitative PCR (qRT-PCR). The effects of CSNK1E on the phenotypes of HCC cells were evaluated using Cell Counting Kit-8 (CCK8), flow cytometry, Transwell assay and Wound healing assay. Furthermore, transcriptomic analysis identified HIPPO signaling pathway as a potential pathway involving CSNK1E. The functional role of CSNK1E in HIPPO signaling was subsequently validated through western blotting (WB) and quantitative real-time PCR (qRT-PCR) assays. RESULTS:We constructed a prognostic model of key circadian rhythm-related genes (CSNK1E, CSNK1D, CSNK2A1, and CSNK2B) to predict the prognosis and survival of HCC patients. The high-risk group had a worse prognosis compared to the low-risk group, which was confirmed by ROC curves and survival curves. CSNK1E expression levels were significantly associated with clinicopathological features and identified as a robust and independent prognostic biomarker in HCC. Higher CSNK1E expression levels were associated with poorer overall survival in various clinical subgroups. The cell experiment showed that CSNK1E knockdown suppressed cell proliferation, migration, and invasion while promoting apoptosis; its overexpression produced opposite effects. Moreover, CSNK1E may mediate HCC cell proliferation through Hippo signaling pathway. CONCLUSIONS:The finding that the circadian gene CSNK1E contributes to HCC progression through activation of HIPPO signaling pathway suggests that it may be a promising therapeutic target for HCC.

Due to ethnic heterogeneity in genetic architecture, genetic risk score (GRS) constructed within the European population generally possesses poor portability in underrepresented non-European populations, but substantial genetic similarity exists across diverse ancestral groups. We here explore the prediction performance of early exposures and GRS on body mass index (BMI) through leveraging genetic similarity knowledge acquired from Europeans into non-Europeans. We present a linear mixed prediction model for BMI in three distinct UK Biobank cohorts under the transfer learning framework, where we consider Asians (n = 7487) and Africans (n = 7533) as target samples and Europeans (n = 280,575) as informative auxiliary samples. Besides environmental and behavior exposures, we incorporate multiple BMI-related variants, by which the GRS is constructed via transfer machine learning techniques informed by genetic similarity shared across target and auxiliary samples. The use of GRS gained more predictive odds for BMI than the model with traditional risk factors alone in the Asian and African cohorts, leading to an approximately 3.6

Circadian rhythm disruption (CRD) is a critical contributor to sleep, metabolic, and neuropsychiatric disorders. Ultrasound (US) stimulation represents a non-invasive neuromodulatory modality, yet its impact on CRD remains unexplored. This study is the first to propose and systematically assess the effects of transcranial low-intensity focused ultrasound (LIFU) stimulation targeting the suprachiasmatic nucleus (SCN) in a chronic jet lag mouse model. Circadian and anxiety-related behaviors were evaluated through spontaneous locomotor activity monitoring and open field testing. Plasma melatonin and cortisol levels were quantified via ELISA, and neuronal activity in the SCN, dorsomedial hypothalamus (DMH), and subparaventricular zone (SPZ) was assessed using c-Fos immunofluorescence. Results revealed that US stimulation markedly reduced the phase difference in the US-SCN group to −2.6 h, in contrast to −7.15 h in the CJL group, indicating enhanced circadian alignment with the normal control group in the US-SCN. In the US-SCN group, melatonin and cortisol levels significantly decreased from 214.70 $~\pm ~$ 3.54 pg/mL and 217.21 $~\pm ~$ 5.62 pg/mL to 172.45 $~\pm ~$ 10.16 pg/mL (p = 0.001) and 187.35 $~\pm ~$ 2.57 pg/mL (p = 0.002), respectively. Additionally, anxiety-like behaviors were alleviated in the US-SCN group, as evidenced by a 61.40% increase in the time spent exploring the center zone. Neuronal activity in the SCN, DMH, and SPZ was significantly enhanced. Histological analysis confirmed no tissue damage following LIFU stimulation, demonstrating its safety. LIFU stimulation of the SCN effectively alleviates CRD. This non-invasive approach offers a promising therapeutic strategy for CRD and advancing neuromodulation-based chronotherapy.

Physiological time series classification is a crucial topic with broad and vital applications in biomedical research. Deep learning can automatically capture key features through training and is an effective physiological time series classification method. However, due to the high cost of collecting and labeling biomedical data, obtaining sufficient training samples in many practical application scenarios is often challenging. Insufficient training samples may lead to severe overfitting problems in deep learning models and reduce their generalization ability. To address this limitation, we propose a new physiological time series classification model for small sample sizes, called WEFormer. Specifically, the model introduces a time series foundation model with frozen weights as a generalized feature extractor, which can enhance the expressive power of the whole model and reduce the extra work of extracting features. Additionally, the model employs a wavelet to decompose the input time series into different frequency sub-bands and, in subsequent training, highlights the critical sub-bands while suppressing extraneous noise. Extensive experiments on the human emotion corpora (WESAD) and the cognitive load dataset (MOCAS) demonstrate that the proposed model outperforms previous methods and achieves significant performance gains with small sample sizes. We also conducted an ablation study to show the benefits of each component in WEFormer.

Abstract Background To investigate the association between cigarette smoking, smoking cessation and the trajectory of cardiometabolic multimorbidity (CMM), and further to examine the association of age at smoking initiation and smoking cessation with CMM. Methods This study included 298,984 UK Biobank participants without cardiometabolic diseases (CMDs) (including type 2 diabetes, coronary heart diseases, stroke, and hypertension) at baseline. Smoking status was categorized into former, current, and never smokers, with age at smoking initiation and smoking cessation as a proxy for current and former smokers. The multi-state model was performed to evaluate the association between cigarette smoking, smoking cessation and CMM. Results During a median follow-up of 13.2 years, 59,193 participants developed first cardiometabolic disease (FCMD), 14,090 further developed CMM, and 16,487 died. Compared to former smokers, current smokers had higher risk at all transitions, with hazard ratio (95% confidence interval) = 1.59 (1.55 ∼ 1.63) vs. 1.18 (1.16 ∼ 1.21) (P = 1.48 × 10− 118) from health to FCMD, 1.40 (1.33 ∼ 1.47) vs. 1.09 (1.05 ∼ 1.14) (P = 1.50 × 10− 18) from FCMD to CMM, and 2.87 (2.72 ∼ 3.03) vs. 1.38 (1.32 ∼ 1.45) (P < 0.001) from health, 2.16 (1.98 ∼ 2.35) vs. 1.25 (1.16 ∼ 1.34) (P = 1.18 × 10− 46) from FCMD, 2.02 (1.79 ∼ 2.28) vs. 1.22 (1.09 ∼ 1.35) (P = 3.93 × 10− 17) from CMM to death; whereas quitting smoking reduced the risk attributed to cigarette smoking by approximately 76.5% across all transitions. Reduced risks of smoking cessation were also identified when age at quitting smoking was used as a proxy for former smokers. Conclusions Cigarette smoking was associated with a higher risk of CMM across all transitions; however, smoking cessation, especially before the age of 35, was associated with a significant decrease in CMM risk attributed to cigarette smoking.

AIMS:The relationship between the long-term joint exposure to ambient air pollution and incidence of myocardial infarction (MI) and modification by genetic susceptibility remain inconclusive. METHODS AND RESULTS:We analysed 329 189 UK Biobank participants without MI at baseline. Exposure concentrations to particulate matter (PM2.5 and PM10), nitrogen dioxide (NO2), and nitrogen oxides (NOx) were obtained. Air pollution score assessing the joint exposure was calculated, and its association with MI was evaluated via Cox model under the P value aggregation framework. Genetic susceptibility to MI was evaluated by incorporating polygenic risk score (PRS) into models. Risk prediction models were also established. During a median follow-up of 13.4 years, 9993 participants developed MI. Per interquartile range increase of PM2.5, PM10, NO2, and NOx resulted in 74% [95% confidence intervals (CIs) 69%-79%], 67% (63%-72%), 46% (42%-49%), and 38% (35%-41%) higher risk of MI. Compared with the lowest quartile (Q1) of air pollution score, the multivariable adjusted hazard ratio (HR) (95%CIs) of Q4 (the highest cumulative air pollution) was 3.50 (3.29-3.72) for MI. Participants with the highest PRS and air pollution score possessed the highest risk of incident MI (HR = 4.88, 95%CIs 4.35-5.47). Integrating PRS, air pollution exposure, and traditional factors substantially improved risk prediction of MI. CONCLUSION:Long-term joint exposure to air pollutants including PM2.5, PM10, NO2, and NOx is substantially associated with increased risk of MI. Genetic susceptibility to MI strengthens such adverse joint association. Air pollutions together with genetic and traditional factors enhance the accuracy of MI risk prediction.

Trans-ethnic genome-wide association studies have revealed that many loci identified in European populations can be reproducible in non-European populations, indicating widespread trans-ethnic genetic similarity. However, how to leverage such shared information more efficiently in association analysis is less investigated for traits in underrepresented populations. We here propose a statistical framework, trans-ethnic genetic risk score informed gene-based association mixed model (GAMM), by hierarchically modeling single-nucleotide polymorphism effects in the target population as a function of effects of the same trait in well-studied populations. GAMM powerfully integrates genetic similarity across distinct ancestral groups to enhance power in understudied populations, as confirmed by extensive simulations. We illustrate the usefulness of GAMM via the application to 13 blood cell traits (i.e. basophil count, eosinophil count, hematocrit, hemoglobin concentration, lymphocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, monocyte count, neutrophil count, platelet count, red blood cell count and total white blood cell count) in Africans of the UK Biobank (n = 3204) while utilizing genetic overlap shared in Europeans (n = 746 667) and East Asians (n = 162 255). We discovered multiple new associated genes, which had otherwise been missed by existing methods, and revealed that the trans-ethnic information indirectly contributed much to the phenotypic variance. Overall, GAMM represents a flexible and powerful statistical framework of association analysis for complex traits in underrepresented populations by integrating trans-ethnic genetic similarity across well-studied populations, and helps attenuate health inequities in current genetics research for people of minority populations.

Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1- /- ), hepatocyte-specific Bmal1 knockout mice (L-Bmal1- /- ), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1- /- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.