The integration of high-dimensional and multi-modal biomarker data remains a central challenge in precision medicine, hindered by noise, weak individual signals, and heterogeneous data structures. We propose AdaMixNet, an adaptive mixed-effects deep learning framework that unifies nonlinear fixed-effects modeling with kernel-based random-effects estimation to robustly and accurately predict complex disease outcomes. By leveraging feature screening to distinguish sparse, high-impact biomarkers from dense, low-signal features, AdaMixNet captures both strong and subtle biological effects across diverse data modalities. Through comprehensive simulations and applications to two large-scale real cohorts, i.e., METABRIC (breast cancer) and ADNI (Alzheimer’s disease), AdaMixNet shows robust overall performance, often outperforming strong machine-learning and statistical baselines in simulation and achieving competitive or best results for several real-data outcomes while maintaining good performance across sample sizes from 1000 to 20,000. AdaMixNet offers a generalizable and interpretable framework for integrating high-dimensional and multi-modal omics profiles with low-dimensional clinical data, accelerating the translation of molecular insights into clinical applications. Modern medicine collects large amounts of biological data, such as gene-expression profiles, genetic variants, and medical test results. However, combining these different types of data to better predict disease remains difficult. The data are often noisy, and important signals can be weak or hidden among many measurements. In this study, we developed a method called AdaMixNet. It is a computer-based tool that learns from both strong and subtle biological signals while also using standard clinical information. This helps improve the accuracy of predicting disease outcomes. We tested AdaMixNet using simulated data and two large real-world studies of breast cancer and Alzheimer’s disease. Our method reduced prediction errors compared to existing approaches. This work may help researchers and doctors better use complex biological data to support more personalized healthcare decisions in the future. Dai et al. develop AdaMixNet, an adaptive mixed-effects deep learning framework that integrates high-dimensional omics data with clinical variables for disease outcome prediction. Across simulations and the METABRIC and ADNI cohorts, AdaMixNet reduces prediction error by up to 25% and outperforms existing approaches.

Testosterone production by testicular Leydig cells (LCs) in male mammals is energetically demanding and prone to mitochondrial damage. Despite these challenges, LCs exhibit remarkable longevity and minimal turnover, suggesting the existence of specialized mechanisms that maintain LC mitochondrial homeostasis under such constrains. Here we identify a mitochondrial transfer network between LCs and different testicular macrophage (tMac) subpopulations. Leydig cells release extracellular vesicles containing defective mitochondria, which are eliminated by CD206hi tMacs in a TREM2-dependent process. Deletion of Trem2 in tMacs disrupts this transfer, leading to impaired testosterone synthesis. Conversely, LCs acquire extracellular vesicles containing functional mitochondria from MHCIIhi tMacs through ITGβ1-VCAM1 interactions. Loss of Vcam1 in LCs hinders this mitochondrial transfer, thereby compromising testosterone production. Together, our findings reveal an unrecognized mitochondrial transfer network between LCs and tMacs that safeguards LC homeostasis and testosterone production, offering valuable insights into intercellular communication mechanisms that maintain tissue homeostasis.

Background:Psychosocial stress is a key risk factor for coronary heart disease (CHD), particularly in postmenopausal women who face both a high stress burden and elevated cardiovascular risk. DNA methylation (DNAm) - a critical epigenetic modification bridging environment and health - remains understudied as a contributor to stress- related CHD. Methods:We conducted an epigenome-wide association study (EWAS) of stress in the Women's Health Initiative (WHI), an ancestrally diverse cohort of postmenopausal women (n=3,857). At screening visit, participants completed a questionnaire assessing stressful life events and provided whole blood for DNAm. Incident CHD was then longitudinally ascertained (follow-up mean/SD: 16.7/8.4 years), and DNAm signatures were evaluated as CHD predictors using Cox regression. Predictive models were independently validated in the Jackson Heart Study (JHS; n=3,053) and Multi-Ethnic Study of Atherosclerosis (MESA; n=870). The bulk-level DNAm associations were computationally deconvolved at the cell-type-specific level using tensor composition analysis (TCA). Results:The EWAS in WHI identified 841 stress-related DNAm sites (99 hypermethylated, 742 hypomethylated with stress) after FDR correction, with 13 significant after Bonferroni correction, including sites located on immune and CHD- related genes (e.g., TNF , ALDH2 ). Methylation risk scores (MRSs) integrating the 841 FDR-significant sites (MRS 841 ) and 13 Bonferroni-significant sites (MRS 13 ) predicted incident CHD (HR=1.33-1.37; p≤0.0008) and mediated 16.5-17.7% of the association between stress and CHD. In JHS and MESA, MRS 13 independently predicted CHD (HR=1.34; p=0.036), whereas MRS 841 was suggestively associated with CHD (HR=1.27; p=0.087). TCA indicated that the greatest number of stress-related sites predictive of CHD was specifically in monocytes (133 total), with directions consistent with bulk-level associations (9 hypermethylated, 124 hypomethylated with stress). Conclusion:Our study supports methylation risk scores as novel biomarkers of stress- related CHD and uncovers epigenetic regulation in monocytes as a potential underlying mechanism. These findings highlight biological pathways linking stress and disease and may promote personalized interventions in high-risk populations.

Postpartum depression (PPD) is linked to neuroimmune dysregulation. Brexanolone, an intravenous formulation of the neurosteroid allopregnanolone and the first FDA-approved treatment for PPD, produces rapid and sustained antidepressant effects. However, its long-term mechanisms of action remain unclear. This study evaluated brexanolone’s prolonged impact on two groups of biomarkers in whole blood: inflammatory mediators and growth/differentiation/neurotrophic factors. Whole blood was also maintained in culture (4 h) and subjected to lipopolysaccharide (LPS) stimulation of the TLR4 inflammatory pathway. Ten individuals with moderate-to-severe PPD received brexanolone and were assessed before, and at 6 h, ~7, and ~30 days post-infusion. BDNF significantly increased and remained elevated through 30 days, representing a sustained neurotrophic response. In contrast, inflammatory mediators CCL11, IL-6, TNF-α, and IL-18 showed rapid reductions by 6 h. TNF-α suppression lasted up to 7 days, while CCL11 and IL-6 remained suppressed through 30 days. These changes were associated with reductions in Hamilton Depression Rating Scale (HAM-D) scores over time. LPS-stimulated whole blood cultures revealed suppression of TLR4-induced CCL11, IL-1β, IL-6, IL-8, IL-18, TNF-α, HMGB1, and MIP-1β at 6 h. IL-8, IL-18, and TNF-α remained suppressed through 7 days, while IL-1β and CCL11 remained suppressed through 30 days, aligning with sustained HAM-D score improvements. Biomarker × time interactions suggested dynamic regulation of inflammatory and neurotrophic pathways. Given the small sample size, these findings should be interpreted as a pilot study, but they indicate that brexanolone promotes both rapid and sustained anti-inflammatory and neurotrophic effects supporting lasting symptom remission in PPD.

BACKGROUND:Alcohol use disorder (AUD) lacks objective clinical tests; current screening (AUDIT) relies on self-report and can miss risk. Building on our recent whole-blood analysis, where immune dysregulation, particularly IL-1β, predicted AUD risk, we tested whether Toll-like receptor (TLR) stimulation would further unmask risk-related immune signatures. METHODS:Whole blood from 28 young adults (Low-risk: AUDIT <6; High-risk: AUDIT ≥6) was stimulated in culture with lipopolysaccharide (LPS, TLR4) or imiquimod (IMQ, TLR7). Fourteen immune mediators were quantified using Luminex multiplex assays. Group and stimulus effects were tested with aligned rank transform (ART) factorial models; principal component analysis (PCA) summarized the multivariate structure. Predictive associations with AUDIT were assessed via linear regression and Random Forest analyses. RESULTS:IL-1β, IL-3, IL-6, IL-7, IL-8, IL-18, CCL11, MCP-1, and MIP-1β were elevated in the high-risk group following stimulation. LPS evoked stronger responses than IMQ for IL-1β, IL-6, IL-8, and MIP-1β, whereas MCP-1 was higher with IMQ. PCA distinguished high- from low-risk groups, driven by CCL11, MCP-1, IL-7, IL-3, IL-6, IL-18, MIP-1β, and IL-1β. LPS-evoked IL-1β, IL-3, and CCL11 predicted AUDIT scores (adjusted R2 = 0.22-0.37). IMQ-evoked CCL11, IL-18, MIP-1β, IL-1β, and IL-6 were also significant predictors (adjusted R2 = 0.16-0.29). After outlier filtering, LPS associations persist, and IMQ-evoked CCL11 and IL-18 remain. Random Forests predicted AUDIT with R2 = 0.33 (LPS) and R2 = 0.27 (IMQ), with top features IL-3, IL-18, IL-1β, CCL11 (LPS) and IL-6, IL-18, CCL11, IL-1β (IMQ). CONCLUSIONS:LPS and IMQ stimulations unmasked immune response patterns that separated high- from low-risk individuals, with exaggerated pro-inflammatory responses in the high-risk group. IL-1β, IL-3, IL-18, and CCL11 repeatedly predicted AUDIT scores, with IL-1β and IL-3 LPS-dominant and CCL11 and IL-18 LPS/IMQ stimulus-shared. Stimulation-evoked mediator profiling may complement self-report screening and improve risk stratification in drinkers. Further studies are needed to address the exploratory nature of the results.

Many neuropsychiatric conditions share overlapping features underpinned by shared genetics. In this study, we examined the co-aggregation of sub-threshold variation in neuropsychiatric phenotypes in biological parents of autistic children to provide insights into the intergenerational transmission of genetic liability for autism. Autistic, neuropsychiatric (i.e., anxiety, depression, ADHD), and cognitive traits were characterized in biological parents of children enrolled in a longitudinal developmental study: 189 families of autistic children and 100 families with no autistic children were included. Families were further characterized as having only one (simplex) or more than one (multiplex) autistic child. Maternal and paternal traits were compared across groups using analysis of variance. Between-parent and within-parent correlations across trait domains examined patterns of familial trait aggregation and assessed shared versus unique contributions to the inheritance of autism. Logistic regression assessed the predictive utility of parental traits for simplex vs. multiplex group membership. Mean levels of paternal autistic traits (F(2, 224) = 5.67, FDR-adjusted p-value (q) = 0.013) and maternal anxious (F(2, 262) = 11.14, q < 0.001) and depressive (F(2, 262) = 7.08, q = 0.005) traits differed between groups. Post-hoc tests revealed elevated autistic traits in multiplex fathers (q = 0.009) and elevated anxious (q < 0.001) and depressive (q = 0.004) traits in multiplex mothers compared to parents of non-autistic children; simplex parents did not differ from either of the other groups. Parental traits jointly accounted for 7.9

Testicular aging commonly leads to testosterone deficiency and impaired spermatogenesis, yet the underlying mechanisms remain elusive. Here, we show that Leydig cells are particularly vulnerable to aging processes in testis. Single-cell RNA sequencing identifies the expression of Hmgcs2, the gene encoding rate-limiting enzyme of ketogenesis, decreases significantly in Leydig cells from aged mice. Additionally, the concentrations of ketone bodies β-hydroxybutyric acid and acetoacetic acid in young testes are substantially higher than that in serum, but significantly diminish in aged testes. Silencing of Hmgcs2 in young Leydig cells drives cell senescence and accelerated testicular aging. Mechanistically, β-hydroxybutyric acid upregulates the expression of Foxo3a by facilitating histone acetylation, thereby mitigating Leydig cells senescence and promoting testosterone production. Consistently, enhanced ketogenesis by genetic manipulation or oral β-hydroxybutyric acid supplementation alleviates Leydig cells senescence and ameliorates testicular aging in aged mice. These findings highlight defective ketogenesis as a pivotal factor in testicular aging, suggesting potential therapeutic avenues for addressing age-related testicular dysfunction.

OBJECTIVE:Postpartum psychosis (PPP) is a rare but severe postpartum psychiatric emergency that occurs during a critical time in mother-infant attachment. This study investigated the efficacy, safety, and tolerability of brexanolone, a neuroactive steroid and synthetic form of allopregnanolone, for adult females with PPP. METHODS:In this open-label, proof-of-concept study, ten patients with PPP received brexanolone as a single, continuous, 60-h intravenous infusion titrated to a target dose of 90 μg/kg/h over a period of 2.5 days. Primary outcomes were change in total score on the Young Mania Rating Scale (YMRS) and Positive and Negative Syndrome Scale (PANSS); Secondary outcomes included change in Hamilton Depression Rating Scale (HAMD) from baseline to Day 7. Adverse events were monitored. Patients were followed through Day 90. RESULTS:All ten patients completed the treatment protocol without dose reductions and without intolerable adverse side effects. Change in mean PANSS total score from baseline to Day 7 was statistically significant (p = 0.00006; 95 % CI [19.60-38.40]), as were YMRS scores (p = 0.01; 95 % CI [2.69-16.01]) and HAM-D scores (p = 0.0003; 95 % CI [5.22-11.98]). CONCLUSIONS:This study suggests the potential for neuroactive steroids as rapid-acting, adjunctive treatment for PPP and provides support for future research to assess the role of neuroactive steroids and synthetic forms of allopregnanolone in the treatment of PPP.

Crohn’s disease (CD) is a chronic non-specific inflammatory bowel disease with an increasing incidence worldwide. Patients with CD are facing elevated risk for mental disorders (MD) than healthy people, and chronic psychological stress is considered to trigger deterioration and relapse of CD. The microbiome-gut-brain axis (MGBA) is recognized as a crucial factor in unraveling this connection. Whereas, so far, few studies have revealed the relationship of the microbiota communities and tryptophan catabolites of the terminal ileum mucosa on gut-brain communication. A total of 52 patients with CD, along with 11 patients with colorectal cancers recruited as controls, were enrolled in this study. The participants completed Patient Health Questionnaire-9 and Generalized Anxiety Disorder-7 Questionnaire. The terminal ileal mucosa was collected during surgery. We profiled the microbiota composition of 37 patients and quantified the tryptophan catabolites of 28 patients utilizing 16 S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry, respectively. In addition, bioinformatics methods were used to elucidate the interrelationships between psychological states, microbial communities, and tryptophan catabolites. CD patients with MD showed a significant reduction in microbial diversity within the ileal mucosa. Regarding microbial composition, Prevotella was relatively enriched in CD patients with MD, along with lower relative abundances of Akkermansia and Faecalibacterium. Furthermore, significant disparities in the levels of Picolinic acid (PA), Kynurenic acid (KYNA), Nicotinic acid (N-Acid), and Indole-3-carbaldehyde (ICAld) were detected within the ileal mucosa of CD patients comorbid with MD. A pronounced correlation was observed between PA levels and anxiety scale scores. The heightened abundance of Prevotella may be closely associated with altered levels of PA, N-Acid, and KYNA. Alterations in the microbial composition of the terminal ileum may interact with changes in tryptophan metabolism and are associated with MD in patients with CD undergoing surgery.

Alcohol use disorder (AUD) is associated with chronic inflammation and immune dysregulation, yet no validated immune-based markers exist to support assessment or monitoring. This study identifies interleukin-1 beta (IL-1β) in whole blood as a promising candidate biomarker of AUD risk, based on Alcohol Use Disorders Identification Test (AUDIT) scores. Twenty-eight non-treatment-seeking adults, with AUDIT scores between 2 and 22, provided whole blood samples. We aimed to identify biomarkers that signal immune changes associated with early AUDIT score risk, where interventions may be most effective. Luminex multiplex immunoassays quantified 14 immune-related mediators in combined cell lysates and supernatants. IL-1β, IL-18, IL-7 and CCL11 were significantly elevated in individuals with higher AUDIT scores. IL-1β showed the largest effect size (Cohen's d) and was the most consistent predictor of both AUDIT and AUDIT-Consumption (AUDIT-C) scores across random forest and linear regression analyses. Moderated multiple regression (MMR) confirmed that IL-1β predicted both scores independent of other immune mediators. Receiver operating characteristic (ROC) analyses demonstrated discriminative potential, with IL-1β achieving an AUC of 0.81 (good discrimination) for AUDIT ≥ 6 (true positive rate [TPR] = 0.71; false positive rate [FPR] = 0.14) and an AUC of 0.94 (excellent discrimination) for AUDIT-C thresholds (TPR = 0.80; FPR = 0.00). Principal component analysis (PCA) revealed greater immune variability in the high-risk group, particularly among proinflammatory mediators, suggesting immune dysregulation. This study demonstrates the utility of integrating whole blood immune profiling with high-sensitivity multiplex immunoassays, and applying both traditional statistical methods and machine learning to explore potential biomarkers for AUD risk. IL-1β is a statistically robust and clinically relevant candidate biomarker of AUD risk assessed by AUDIT scores. These findings require replication in larger, independent samples to determine their translational potential in addiction medicine.

Declining rates of male fertility pose a significant clinical challenge, while the mechanisms underlying testicular interstitial function remain incompletely understood. Here, we conducted a comprehensive analysis of the single-cell transcriptomic landscape of the murine testicular interstitium across the postnatal lifespan. The investigation unveiled a previously unrecognized population of Cd34+/Sox4+ mesenchymal cells nestled within the interstitium, hinting at their potential as Leydig cell (LC) progenitors. With the aging process of Cd34+/Sox4+ mesenchymal cells, we observed a decline in glutathione levels within the testicular interstitium. Remarkably, these Cd34+/Sox4+ mesenchymal cells exhibited clonogenic self-renewal capacity and a robust propensity to differentiate into LCs. Intriguingly, when transplanted into LC-disrupted or failure models, Cd34+/Sox4+ cells efficiently colonized the testicular interstitium, resulting in a notable increase in testosterone production. Exploring the epigenetic landscape, we identified critical transcription factors, most notably Sox4, governing the stem cell fate of Cd34+/Sox4+ mesenchymal cells. Overall, this comprehensive lifespan-resolved single-cell atlas of testicular interstitial cells provides fundamental insights into LC progenitor biology and regenerative capacity during aging.

Nestin, a well-characterized intermediate filament protein expressed in stem cells, is increasingly recognized for its non-canonical roles in diverse subcellular compartments. Here, a novel mitochondrial localization of Nestin in human mesenchymal stem cells (hMSCs) is identified, where it functions as a critical protector against mitochondrial dysfunction and cellular senescence. It is demonstrated that Nestin is imported into the mitochondrial intermembrane space via its N-terminal mitochondrial targeting sequence through Translocase of the Outer Mitochondrial Membrane 20 (TOM20)-dependent machinery. Within mitochondria, Nestin directly interacts with Mic60 to maintain cristae architecture and sustain oxidative phosphorylation. Genetic ablation of mitochondrial Nestin triggers cristae disorganization, respiratory deficiency, and premature senescence in hMSCs. Strikingly, targeted restoration of the Mic60-binding Tail3 domain of Nestin is sufficient to rescue cristae morphology, mitochondrial function, and senescence phenotypes. These findings establish a non-filamentous role for Nestin in mitochondrial quality control and propose a new therapeutic strategy for age-related disorders through modulation of mitochondrial Nestin-Mic60 interactions.

Leydig cells (LCs), which originate from mesenchymal-like progenitors, are the primary testosterone-producing cells in the testis. However, the roles of postnatal LC progenitors in pubertal development, homeostasis, and injury response remain poorly understood. Here, we demonstrate that neonatal testicular Gli1+ cells give rise to nearly all LCs during pubertal development. Conditional deletion of nuclear receptor subfamily 5, group A, member 1 (Nr5a1) in Gli1+ progenitors significantly impairs testicular development by inhibiting pubertal LC formation. Prepubertal exposure to cyclophosphamide disrupts LC formation, further impairing the development of the reproductive system. During adulthood, testicular Gli1+ cells contribute to the slow turnover of LCs, whereas conditional deletion of Nr5a1 has little impact on testicular homeostasis. Following hemicastration, Gli1+ cells rapidly differentiate into LCs in response to unilateral LC depletion, demonstrating that adult Gli1+ cells act as a functional reserve. These findings highlight the critical role of postnatal Gli1+ cells as progenitors of LCs for testicular pubertal development, adult homeostasis, and regeneration.

Emerging evidence suggests that genetic variants and environmental toxicants may synergistically contribute to DSD. To test this hypothesis, we employed LhcgrW495X/+ (luteinizing hormone/chorionic gonadotropin receptor) male mice subjected to prenatal Di-(2-ethylhexyl) phthalate (DEHP) exposure, a model designed to investigate steroidogenic gene expression in gene-environment interactions. Pregnant wild-type (WT) dams (mated with LhcgrW495X/+ heterozygote (HET) received varying levels of DEHP: no exposure, low-dose (100 mg/kg/d) DEHP, and high-dose (1000 mg/kg/d) DEHP during gestation, which led to prenatal exposure in male offspring. Male offspring were divided into HET (LhcgrW495X/+) and WT groups based on genotype in three levels of DEHP exposure. The study assessed phenotypic characteristics (DSD, testosterone levels, and semen quality) and examined the expression of steroidogenic genes (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd3b2). LhcgrW495X/+ male offspring without DEHP exposure exhibited normal phenotypes and steroidogenic gene profiles. Low-dose DEHP had no detectable effects on WT offspring, but synergistically induced DSD in LhcgrW495X/+ male offspring by interfering with steroidogenic gene expression (Lhcgr, Hsd17b3, Hsd3b2). High-dose DEHP caused DSD in both genotypes, but the severity of DSD and interference with steroidogenic gene expression were more pronounced in LhcgrW495X/+ male offspring. This study verifies that Genetic variants (LhcgrW495X/+) and environmental toxicants (DEHP) synergistically induce DSD, thereby elucidating the pathogenesis of DSD. Interfering with steroidogenic gene expression may be an important synergistical mechanism. This finding highlights the clinical imperative to minimize prenatal exposure to endocrine disruptors, particularly in pregnancies with variants of DSD. The pathogenesis of disorders/differences of sex development (DSD) remains incompletely understood. While genetic variants and environmental toxicants are hypothesized to synergistically contribute to DSD, no animal model has been established to validate this interaction, and the mechanistic basis remains elusive. Here, we propose that such synergy may impair steroidogenic gene networks, thereby disrupting testosterone synthesis and inducing DSD. To test this, we employed LhcgrW495X/+ (luteinizing hormone/chorionic gonadotropin receptor) male mice exposed prenatally to di-(2-ethylhexyl) phthalate (DEHP), a model recapitulating human gene-environment interplay. Pregnant dams (mated with LhcgrW495X/+ males) received no, low-dose(100 mg/kg/d), or high-dose(1000 mg/kg/d) DEHP during gestation, which led to prenatal exposure in male offspring. Male offspring were divided into HET (LhcgrW495X/+) and WT groups by genotype (LhcgrW495X/+ or WT) in three levels of DEHP exposure. This design enabled simultaneous evaluation of genetic (Lhcgr) and environmental (DEHP) effects on male offspring. Notably, male offspring carrying the LhcgrW495X/+ variant or exposed to low-dose DEHP alone exhibited normal phenotypes. However, prenatal exposure to low-dose DEHP selectively induced DSD in LhcgrW495X/+ male offspring via interfering with steroidogenic gene expression (Lhcgr, Hsd17b3, Hsd3b2). High-dose DEHP caused DSD in both genotypes, but with significantly greater severity in LhcgrW495X/+ offspring. In conclusion, our findings suggest that genetic variants (LhcgrW495X/+) and environmental toxicants (DEHP) synergistically disrupt steroidogenesis, leading to DSD. Clinically, our findings underscore the need for rigorous avoidance of prenatal endocrine disruptors, particularly in pregnancies with variants of DSD.

Background: Testicular aging is associated with diminished fertility and certain age-related ailments, and effective therapeutic interventions remain elusive. Here, we probed the therapeutic efficacy of exosomes derived from human umbilical cord mesenchymal stem cells (hUMSC-Exos) in counteracting testicular aging. Methods: We employed a model of 22-month-old mice and administered intratesticular injections of hUMSC-Exos. Comprehensive analyses encompassing immunohistological, transcriptomic, and physiological assessments were conducted to evaluate the effects on testicular aging. Concurrently, we monitored alterations in macrophage polarization and the oxidative stress landscape within the testes. Finally, we performed bioinformatic analysis for miRNAs in hUMSC-Exos. Results: Our data reveal that hUMSC-Exos administration leads to a marked reduction in aging-associated markers and cellular apoptosis while promoting cellular proliferation in aged testis. Importantly, hUMSC-Exos facilitated the restoration of spermatogenesis and elevated testosterone synthesis in aged mice. Furthermore, hUMSC-Exos could attenuate inflammation by driving the phenotypic shift of macrophages from M1 to M2 and suppress oxidative stress by reduced ROS production. Mechanistically, these efficacies against testicular aging may be mediated by hUMSC-Exos miRNAs. Conclusions: Our findings suggest that hUMSC-Exos therapy presents a viable strategy to ameliorate testicular aging, underscoring its potential therapeutic significance in managing testicular aging.

STUDY QUESTION:What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER:The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY:Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION:Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS:scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE:The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA:The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION:Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS:These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest.

Adaptive metabolic responses and innate metabolites hold promising therapeutic potential for stroke, while targeted interventions require a thorough understanding of underlying mechanisms. Adiposity is a noted modifiable metabolic risk factor for stroke, and recent research suggests that it benefits neurological rehabilitation. During the early phase of experimental stroke, the lipidomic results showed that fat depots underwent pronounced lipolysis and released fatty acids (FAs) that feed into consequent hepatic FA oxidation and ketogenesis. Systemic supplementation with the predominant ketone beta-hydroxybutyrate (BHB) is found to exert discernible effects on preserving blood-brain barrier (BBB) integrity and facilitating neuroinflammation resolution. Meanwhile, blocking FAO-ketogenesis processes by administration of CPT1α antagonist or shRNA targeting HMGCS2 exacerbated endothelial damage and aggravated stroke severity, whereas BHB supplementation blunted these injuries. Mechanistically, it is unveiled that BHB infusion is taken up by monocarboxylic acid transporter 1 (MCT1) specifically expressed in cerebral endothelium and upregulated the expression of tight junction protein ZO-1 by enhancing local β-hydroxybutyrylation of H3K9 at the promoter of TJP1 gene. Conclusively, an adaptive metabolic mechanism is elucidated by which acute lipolysis stimulates FAO-ketogenesis processes to restore BBB integrity after stroke. Ketogenesis functions as an early metabolic responder to restrain stroke progression, providing novel prospectives for clinical translation.