To compare the differential activation characteristics of Toll-like receptor 5 (TLR5) agonist KFD and Toll-like receptor 7 (TLR7) agonist R837 on nasal epithelial cells (NECs) and bone marrow-derived dendritic cells (BMDCs), and to determine whether their combination produces synergistic enhancement as nasal mucosal adjuvants.

Mouse NECs and BMDCs were stimulated with 200 ng/mL KFD or R837 alone or in combination (n=3). At 3 h after stimulation, transcriptome sequencing was performed to analyze gene expression profiles, with key gene expression validated by qPCR. IL-6 levels in the supernatants were measured by ELISA at 12 h post-stimulation. Female C57BL/6 mice (6 to 8 weeks old, weighing 18 to 20 g) were randomly divided into negative control (normal saline), KFD alone (2 μg) group, R837 alone (2 μg) group, and KFD+R837 combination (2 μg KFD+2 μg R837) groups (n=5). At 18 h after intranasal administration, nasal lavage fluid (NALF) and bronchoalveolar lavage fluid (BALF) were collected to detect IL-6 levels, and the expression of costimulatory molecules CD80/CD86 on dendritic cells (DCs) in cervical lymph nodes (CLNs) was analyzed by flow cytometry. Using ovalbumin (OVA) as a model antigen for intranasal immunization, mice were randomly divided into normal saline (negative control), OVA alone (25 μg), OVA+KFD (25 μg OVA+2 μg KFD), OVA+R837 (25 μg OVA+2 μg R837), and OVA+combination adjuvant (25 μg OVA+1 μg KFD+1 μg R837) groups (n=5). Mice were immunized intranasally at weeks 0, 4, and 8. Serum and saliva samples were collected at 2 weeks after the final immunization, and OVA-specific IgG/IgA antibody levels were detected by ELISA.

Transcriptomic analysis revealed that KFD primarily activated NECs, with differentially expressed genes enriched in innate immune pathways such as TNF and NF-κB. In contrast, R837 potently activated BMDCs, with differentially expressed genes involved in TNF and IL-17 signaling pathways. In vitro BMDC experiments confirmed that R837 upregulated Tnf and Cxcl10 mRNA levels more strongly than KFD (P<0.01). The combination synergistically enhanced the expression of Tnf, Cxcl10, and Cd40 (P<0.001) and IL-6 secretion (KFD+R837 combination vs single agent, P<0.000 1). After intranasal administration, both KFD and R837 significantly upregulated CD80/CD86 expression on CLN DCs, with the combination group showing the most significant CD80 upregulation (KFD+R837 combination vs single agent, P<0.05). KFD alone significantly elevated IL-6 levels in NALF, which was not further enhanced by the combination. In the OVA immunization model, the combination adjuvant group exhibited significantly higher levels of serum OVA-specific IgG and salivary OVA-specific IgA than the OVA group (P<0.000 1), which were superior to either single adjuvant (serum IgG: OVA+ combined adjuvants vs OVA+R837, P<0.05; salivary IgA: OVA+ combined adjuvants vs OVA+KFD, OVA+ combined adjuvants vs OVA+R837, both P<0.05).

TLR5 and TLR7 agonists differentially activate NECs and DCs. Their combination synergistically activates DCs and significantly enhances both systemic and local antigen-specific immune responses to mucosal vaccines.

Intestinal organoids serve as important tools for drug screening, but the extent to which they recapitulate in vivo tissue microenvironments and drug responses remains inadequately evaluated. This study aims to verify systematic differences between intestinal organoids and in vivo intestinal crypts in fundamental biological characteristics and responses to nobiletin (NOB) through parallel comparison.

A standard mouse small intestinal organoid culture system was established, with synchronous isolation of homologous mouse small intestinal crypt tissues. Whole-transcriptome sequencing was employed to analyze gene expression profiles, flow cytometry was used to analyze cell subpopulation proportions, and Western blotting was performed to detect key protein expression levels. In vivo crypts and ex vivo organoids were treated with different concentrations of NOB: in vivo administration via gavage at 200 mg/kg for 12 h; organoids cultured for 48 h followed by 100 μmol/L NOB treatment for 12 h. GSEA, GO, and KEGG enrichment analyses and morphological observations were conducted on crypt cells from both groups to comprehensively evaluate the regulatory effects of NOB on cell cycle, metabolic flux, and signaling pathways.

Intestinal organoids effectively recapitulated core biological characteristics of in vivo crypts, including active proliferation of intestinal stem cells, multi-lineage differentiation potential, and CD24-marked cell clustering patterns. However, organoids exhibited specific metabolic reprogramming and immune-silent phenotypes, with differential glycosylation modification of the stem cell marker OLFM4. NOB treatment further amplified model-dependent differences: in in vivo crypts, NOB primarily downregulated the genes related to immune defense and oxidative stress, without significant effects on crypt morphology or OLFM4⁺ cell numbers; in organoids, NOB significantly suppressed core anabolic pathways including DNA replication, cell cycle, and amino acid and nucleotide synthesis, downregulated the expression of stem cell markers, and markedly reduced organoid budding number (P<0.001).

Intestinal organoids and in vivo tissues share commonalities while exhibiting differences at both baseline states and drug response levels. Organoids can recapitulate intestinal epithelial self-renewal processes but differ from in vivo tissues in metabolic programming and immune response. NOB mainly modulates immune microenvironment-related pathways in the in vivo context, whereas it directly targets epithelial cell-autonomous proliferation and metabolic programs in organoids.

Myelodysplastic syndromes (MDS) are associated with a high risk of progression to leukemia, highlighting the critical need to explore novel therapeutic agents. Based on network pharmacology prediction and molecular docking, we hypothesized that luteolin (LUT) might induce cellular senescence and apoptosis in MDS cells by targeting the Akt signaling pathway. This study aims to verify its targets and molecular mechanisms through experimental studies.

By integrating multiple databases, such as PharmMapper and GeneCards, common targets of LUT and MDS were screened. A protein-protein interaction (PPI) network was constructed using the STRING database and visualized with Cytoscape software to identify key targets. The DAVID database was employed for the functional enrichment analysis of Gene Ontology (GO) and the pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) for the common targets. Molecular docking between LUT and the key targets was performed using AutoDock software, and the docking efficacy was evaluated by analyzing the binding energy. Subsequently, molecular dynamics simulations were carried out using Gromacs to observe the ligand-receptor interactions and conformational changes. MUTZ-1 and MOLM-13 cells were used as experimental models. After treating the cells with different concentrations of LUT for 24 h （MUTZ-1：0、15、30、60 μmol/L，MOLM-13：0、4、8、16 μmol/L）, cell viability was measured by CCK-8 assay, DNA synthesis capacity was assessed by EdU assay, colony formation was examined by soft agar assay, morphological changes were observed by transmission electron microscopy (TEM), intracellular reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential, and apoptosis rates were detected by flow cytometry, senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) staining, mRNA expression levels of senescence-associated secretory phenotype (SASP) factors were determined by RT-qPCR, and protein expression levels were examined by Western blotting.

A total of 568 LUT targets and 2 348 MDS-related targets were screened, yielding 228 intersecting targets. The intersecting targets were imported into the DAVID database. GO functional enrichment analysis demonstrated that biological processes were enriched in the responses to oxidative stress and cellular senescence; cellular components were enriched in protein kinase complex and secretory granule lumen; and molecular functions were enriched in protein kinase activity and cytokine receptor binding. KEGG pathway enrichment analysis revealed predominant enrichment in key pathways including the Akt signaling pathway, cellular senescence, and apoptosis. The PPI network constructed using the STRING database and Cytoscape software identified the top 10 key targets, as IL6, NFKB1, STAT3, TNF, JUN, BCL2, ALB, CASP3, Akt1 and IL1B. Molecular docking results indicated that the binding energy between LUT and Akt1 was -6.25 kcal/mol, suggesting a strong interaction. The molecular dynamics simulation results demonstrated stable binding between LUT and the Akt1 target. In vitro experimental results showed that LUT inhibited the proliferation of both MUTZ-1 and MOLM-13 cells in a dose-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of LUT acting on MUTZ-1 cells for 24 h was 29.960 μmol/L, and that on MOLM-13 cells for 24 h was 8.654 μmol/L. Treatment of MUTZ-1 and MOLM-13 cells with various concentrations of LUT for 24 h significantly inhibited DNA synthesis and colony formation (P<0.01). The treatment of LUT at different concentrations for 24 h resulted in significant increases in intracellular ROS and mitochondrial ROS levels in a dose-dependent manner (P<0.05), decrease in mitochondrial membrane potential in a dose-dependent manner (P<0.05), and elevation of apoptosis rates in a dose-dependent manner (P<0.05). TEM revealed obvious apoptotic morphological features in MUTZ-1 and MOLM-13 cells after 24 h of LUT treatment. The protein levels of Cleaved PARP and Cleaved caspase-3, and the pro-apoptotic protein BAX were significantly upregulated, and that of the anti-apoptotic protein BCL-2 was significantly downregulated. SA-β-Gal staining showed that the proportion of senescent cells was significantly increased in MUTZ-1 and MOLM-13 cells after LUT treatment for 24 h, along with enhanced expression of related SASP factors (P<0.001), and significant upregulation of senescence-associated proteins p53, p21 and p16. After LUT treatment for 24 h, the protein expression of p-Akt (Ser473) and p-MDM2 (Ser166) was significantly downregulated, while the total Akt protein expression showed no significant change.

LUT may induce mitochondria-dependent apoptosis and senescence in MDS cells by targeting the Akt signaling pathway, thereby inhibiting the proliferation of MDS cells.

Posterior communicating artery aneurysm(PCoAA) exhibit complex anatomical relationships, and microsurgical clipping of large or posteromedially projecting aneurysms remains technically challenging with considerable postoperative morbidity. Therefore, selecting optimal surgical approach has become a key clinical concern. This study compares the efficacy, safety, and overall performance of the Dolenc approach versus the conventional pterional approach for clipping large or posteromedially projecting PCoA aneurysms.

A prospective nonrandomized controlled study design was adopted. A total of 79 patients with large or posteromedially projecting PCoA aneurysms consecutively treated in Department of Neurosurgery, Liangping Hospital of the First Affiliated Hospital of Chongqing Medical University between September 2019 and September 2025 were enrolled, and alternatively assigned to the Dolenc approach group (n=41) or conventional pterional approach group (n=38) based on admission sequence. The primary endpoint was the favorable outcome rate at 3 months postoperatively, assessed using Glasgow Outcome Scale (GOS), with GOS score of 4 to 5 defined as a favorable outcome and score of 1 to 3 as an unfavorable outcome. Secondary endpoints included operative time, postoperative length of hospital stay, distribution of GOS scores, and aneurysm neck residual on digital subtraction angiography (DSA)/CT angiography (CTA) at 3 months postoperatively (defined as contrast filling of the aneurysm neck). Firth’s bias-reduced penalized likelihood logistic regression analysis was used to evaluate the association between surgical approach and unfavorable outcome (with age stratification as covariate), reporting odds ratio (OR), 95% confidence interval (CI), and P value. In addition, a standardized three-domain (technical-safety-efficiency) scoring system and a composite score were constructed for descriptive comparison, along with cost-effectiveness and subgroup analyses.

For the primary endpoint, the favorable outcome rate was 97.6% (40/41) in the Dolenc group and 78.9% (30/38) in the conventional group, with an absolute difference of 18.6% (95%CI: 4.8% to 32.4%, P=0.009). Postoperative length of hospital stay was shorter in the Dolenc group (13.46±2.43 vs 16.05±2.46 d), with a mean difference of -2.59 d (95%CI: -3.69 to -1.49, P<0.001). Operative time was comparable between groups (3.98±0.60 vs 3.97±0.62 h), with a mean difference of 0.01 h (95%CI: -0.26 to -0.28, P=0.944). Regarding functional outcomes, the Dolenc group had a greater proportion of patients achieving a GOS score of 5 than the conventional group (61.0% vs 18.4%), with an absolute difference of 42.6% (95%CI: 23.2% to 61.9%, P<0.001), and the distribution of GOS scores in the Dolenc group was shifted toward better outcomes, with lower proportions of patients having GOS scores of 4 and 3 than the conventional pterional group (χ²=14.96, P<0.001). For imaging follow-up, the neck remnant rate was lower in the Dolenc group (2.4% vs 13.2%), with an absolute difference of -10.8% (95%CI: -22.5% to -1.0%, P=0.072). Multivariable analysis showed that the conventional pterional approach was associated with unfavorable outcome (OR=10.20, 95%CI: 1.20 to 87.04, P=0.034). The model was significant overall (χ²=7.56, df=2, P=0.02), with good calibration (Hosmer-Lemeshow test P=0.82), and moderate discrimination (AUC=0.75, 95%CI: 0.61 to 0.86). Both the three-domain scores and the composite score were higher in the Dolenc group (composite score: 0.873 vs 0.739). Subgroup analyses suggested the Dolenc approach demonstrated greater advantage in high-risk/complex subgroups, with difference of 0.256 for Hunt-Hess grade Ⅲ-Ⅳ (95%CI: 0.056 to 0.475, P=0.025) and 0.157 for large aneurysms (95%CI: 0.016 to 0.305, P=0.049); however, these findings should be interpreted with caution given the limited sample sizes in some subgroups.

In this cohort, for microsurgical clipping of large or posteromedially projecting PCoA aneurysms, the Dolenc approach is associated with a higher rate of 3-month favorable postoperative outcomes and a shorter length of hospital stay, without an apparent increase in operative time.

“One-stop” interventional therapy for non-valvular atrial fibrillation (NVAF) traditionally relies on transesophageal echocardiography (TEE) guidance, which carries limitations including anesthesia requirements and esophageal injury risks. Whether digital subtraction angiography (DSA) combined with transthoracic echocardiography (TTE) guidance can balance therapeutic efficacy with procedural simplification remains unclear. This study aims to compare the safety and efficacy of “one-stop” procedure for NVAF under different imaging guidance modalities.

Based on predefined inclusion and exclusion criteria, this retrospective cohort study was conducted on consecutive NVAF patients undergoing combined “one-stop” interventional therapy in Department of Cardiology of our hospital between August 1, 2014 and July 31, 2024. According to intraoperative guidance methods, they were divided into the DSA combined with TTE group (DSA group, n=77) and the DSA combined with TEE group (TEE group, n=209). Relevant clinical data of the patients were collected from the hospital big data intelligent platform medical record system, including gender, age, comorbidities, and results of TTE and TEE examinations. Postoperative follow-up included atrial fibrillation recurrence, stroke, bleeding, device-related thrombosis (DRT), cardiac tamponade, hospitalization due to heart failure, and cardiac death. The safety and efficacy of the “one-stop” procedure were compared between the 2 groups.

There were 286 patients enrolled in this study, including 170 males and 116 females, with 77 in the DSA group and 209 in the TEE group. All patients completed at least 6 months of follow-up, with a mean duration of 36.49±14.26 months. No statistically significant differences were observed in baseline characteristics between the 2 groups (P>0.05). The DSA group demonstrated shorter mean procedural time (202.4±39.3 vs 214.1±33.4 min, P=0.013). The incidence of DRT was higher in the DSA group than in the TEE group (3.9% vs 1.4%), though the difference was not statistically significant (P=0.411). Atrial fibrillation recurrence occurred in 10 patients (13.0%) in the DSA group and 19 patients (9.1%) in the TEE group, with no significant intergroup difference. Regarding clinical endpoints, no significant differences were observed between the 2 groups in terms of stroke, major bleeding, cardiac tamponade, heart failure rehospitalization, or cardiac death. Kaplan-Meier survival curve analysis revealed no statistical differences between the 2 groups in composite endpoints, such as major adverse cardiovascular events (MACE), and in the efficacy composite endpoint, including atrial fibrillation recurrence, heart failure rehospitalization, stroke, or transient ischemic attack.

For NVAF patients undergoing one-stop procedures, DSA guidance can shorten procedural time and achieve comparable safety and efficacy to TEE guidance, with the exception of a trend toward increased DRT incidence.

Infertility represents a major global reproductive health challenge. Metabolite levels in both partners are highly correlated and may serve as potential confounders for each other. However, how the overall preconception plasma metabolic profiles of both partners jointly influence natural conception outcomes remains unclear. Based on the Chongqing Preconception Reproductive Health and Birth Outcomes Cohort (PREBIC), this study aims to explore the association between preconception plasma metabolite levels in couples of reproductive age and conception outcomes.

The Chongqing PREBIC was launched in January 2019 at the Chongqing Health Center for Women and Children, recruiting couples of reproductive age undergoing preconception health examinations to establish baseline cohorts and collect blood samples. Follow-up was then performed every 3 months for clinical pregnancy outcomes following 24 months of unprotected intercourse. The study ultimately included 1 292 couples of reproductive age for analysis. According to natural conception status, participants were assigned into a pregnant group (n=967) and a non-pregnant group (n=325). Baseline plasma samples were subjected to standardized metabolomic testing using the Bruker IVDR NMR platform, yielding 167 metabolic parameters. Elastic network regression was employed to screen key metabolites, and logistic regression models were constructed to evaluate the association between metabolites and 2-year pregnancy outcomes, and confounding factors such as age, body mass index (BMI), smoking and alcohol consumption were adjusted.

Among the 1 292 couples of reproductive age, the 2-year pregnancy rate was 74.85%. Eighty-two metabolites showed positive correlations between partners, with a mean correlation coefficient of 0.15. After controlling for inter-variable correlations using elastic network model, 14 male metabolites and 20 female metabolites were found to be associated with conception outcomes. Logistic regression revealed gender-specific associations between plasma metabolite levels in both partners and conception outcomes: in males, triglycerides in low-density lipoprotein were positively associated with conception outcomes (Q4 vs Q1: RR=1.12, 95%CI: 1.03 to 1.19, P adjust=0.047). In females, low-density lipoprotein, apolipoprotein A in high-density lipoprotein, apolipoprotein B in low-density lipoprotein, creatinine, and leucine were negatively associated with conception outcomes. Acetic acid in in both partners was positively correlated with conception outcomes (Q4 vs Q1: male RR=1.13, 95%CI: 1.04 to 1.21, P adjust=0.047; female RR=1.18, 95%CI: 1.05 to 1.28, P adjust=0.007).

Preconception metabolite levels in couples of reproductive age may act on future reproductive potential in a gender-specific manner, with common key metabolites identified. Countermeasures　Based on these findings, it is recommended that the health policy-making departments accelerate the transition of preconception care services from focusing solely on women toward a dual-partner co-management model. Incorporate the pre-pregnancy metabolism of both males and females of reproductive age into routine preconception eugenics health services, with priority monitoring of metabolic indicators, including lipid homeostasis and energy metabolism; develop precision intervention measures according to metabolic differences between genders; improve the urban-rural integrated preconception health management system covering both partners; and enhance reproductive health at the public health source level, providing policy basis and technical support for optimizing birth population quality.

China has achieved remarkable progress in tuberculosis (TB) control, with the incidence rates declining continuously; however, the disease burden among females remained substantial with pronounced regional heterogeneity. The differential impacts of climatic factors on TB onset and their driving mechanisms remains poorly understood, particularly lacking zone-specific evidence based on climatic gradients. This study aims to analyze the heterogeneous effects of climatic factors and air pollutants on female TB incidence across 6 climatic zones and identify key driving pathways, thereby providing evidence-based support for developing gender-sensitive, climate-adapted precision prevention and control strategies.

A retrospective descriptive study design was adopted. Based on China’s climate zoning standards and typical climate characteristics, 6 representative regions were selected, corresponding to the 6 major climate zones: Cold-Dry Zone, Hot-Humid Zone, Warm-Humid Zone, Temperate Continental Zone, Alpine Zone, and Temperate Monsoon Zone. Sample representativeness was evaluated across 3 dimensions: geographic coverage (encompassing northern, southern, eastern, western, and central China), climate consistency (consistent with the core climate characteristics of the corresponding regions), and population epidemiological representativeness. A total of 9 570 female patients with newly diagnosed TB in 6 regions from 2017 to 2024, along with official meteorological and pollutant monitoring data during the same period, were collected. Two categories of measurement indicators were established for cases and meteorological pollution variables. After preprocessing such as imputation of missing values and construction of lagged 1 to 3 period variables, a panel data set was formed. With female TB case counts as the dependent variable, stepwise regression analysis was applied, and model performance was evaluated using R², VIF, and F/t tests.

The overall demographic and clinical characteristics of female TB patients exhibited highly significant heterogeneity across the 6 climatic zones (P<0.001). The TB incidence in the females increased with advancing age, showing a fluctuating downward trend across all regions with significant inter-zone differences. The alpine zone demonstrated high incidence rates, initially declining then rising, decreasing from 155/100 000 in 2017 to 36/100 000 in 2022, subsequently increasing to 91/100 000 in 2024. Regression models for all climatic zones passed multicollinearity tests (VIF<10) and demonstrated extremely significant statistical significance (P<0.001): R²=0.452 for the Cold-Dry Zone, R²=0.589 for the Hot-Humid Zone, R²=0.610 for the Warm-Humid Zone, R²=0.626 for the Temperate Continental Zone, R²=0.641 for the Alpine Zone, and R²=0.580 for the Temperate Monsoon Zone. The combined model yielded R²=0.584 (F=71.548, P<0.001). Lag effects of 2 to 3 periods were present across all regions. Core driving factors were zone-specific, with lag 1 period PM10 identified as the core positive driver of female TB onset.

Climatic conditions and air pollutants exert significantly heterogeneous effects on female TB incidence across China’s 6 climatic zones. The 2 to 3 period lag effects essentially align with the incubation period of Mycobacterium tuberculosis infection. Regional differences in the incidence are closely associated with climatic characteristics, population behavioral patterns, and pollutant exposure profiles. Countermeasures Building upon the existing technical guidance system for TB prevention and control, it is recommended to incorporate early warning rules tailored to local climatic conditions and pollutant lag-based screening window standards. Screening resources and health education efforts at the primary care level should be reallocated based on core pollutant drivers in each region. Data interfaces among meteorological, environmental, and TB surveillance systems should be integrated to establish linked assessment mechanisms, enabling zone-specific, time-specific precision early warning and targeted interventions.

The pattern of socioeconomic status (SES) inequality across the natural history continuum of cervical cancer (from precancerous lesions to invasive cancer) remains unclear. This study aims to systematically investigate the associations between different dimensions of SES and risk at various stages of cervical lesions, providing scientific evidence for identifying high-risk populations and formulating targeted prevention and control strategies.

A case-control design was adopted. A total of 508 female patients who visited Department of Gynecology at the First Affiliated Hospital of Army Medical University between October 2024 and September 2025 were enrolled. Based on pathological diagnosis combined with international classification of diseases， 10th revision（ICD-10) coding, cervical intraepithelial neoplasia (CIN), cervical carcinoma in situ, and invasive cervical cancer were designated as 3 case groups, with the women free of cervical lesions serving as the control group. Four SES indicators, that is, education level, household income, occupational income, and area-level economic development, were collected via questionnaires. Given that the outcome, variable was ordinal and the proportionul odds assumption was violated three independent binary logistic regression models were constructed to analyze the associations between each SES indicator and different lesion stages, estimating ORs and 95%CIs, with adjustment for age, body mass index (BMI), and human papillomavirus (HPV) vaccination status as confounders. Multicollinearity was assessed using variance inflation factor (VIF), and sensitivity analysis was performed using multinomial logistic regression.

A total of 508 patients were enrolled, including 74 (14.6%) in the lesion-free group, 213 (41.9%) in the CIN group, 110 (21.7%) in the carcinoma in situ group, and 111 (21.8%) in the invasive cervical cancer group. Multivariate logistic regression analysis revealed that high school education and above was associated with reduced risks of CIN (OR=0.475, 95%CI: 0.258 to 0.877, P=0.017) and invasive cervical cancer (OR=0.361, 95%CI: 0.175 to 0.746, P=0.006), but showed no statistically significant association with carcinoma in situ. occupational income >5 000 yuan was associated with a reduced risk of invasive cervical cancer (OR=0.332, 95%CI: 0.137 to 0.806, P=0.015). Women from non-affluent areas exhibited a 92% increased risk of invasive cervical cancer compared with those from affluent areas (OR=1.920, 95%CI: 1.005 to 3.668, P=0.048). No significant associations were observed between household income and cervical lesions at any stage. All VIF values for variables in Model 3 were <1.12, indicating absence of multicollinearity. Sensitivity analysis using multinomial logistic regression with the lesion-free group as the common reference showed that the relative risk ratios (RRRs) of SES indicators across lesion stages were consistent in direction with the independent binary models, suggesting robust findings.

The impact of socioeconomic disparities on cervical lesion risk among Chinese women exhibits dimensional heterogeneity. High school education and above is independently associated with reduced risks of CIN and invasive cervical carcinoma, whereas residence economic level and occupational income primarily influence the risk of invasive cervical cancer. Countermeasures Integration of screening into grassroots family planning systems, implementation of data-driven bottleneck analysis and micro-interventions, and strengthening of community mobilization and family engagement are recommended to systematically eliminate accessibility barriers faced by populations with low education level and in non-affluent areas regarding HPV vaccination, screening, and treatment, ensuring equitable progress toward cervical cancer elimination goals for disadvantaged populations.

The progression of chronic kidney disease (CKD) is closely associated with the disruption of renal spatial immune microenvironment. This study aimed to develop an integrated experimental and analytical system for dissecting the immune microenvironment of CKD using imaging mass cytometry (IMC), thereby enabling panoramic analysis of single-cell spatial immune microenvironment in CKD renal tissues.

① Antigenic markers targeting renal structural units, stromal cells, and immune cells were screened to construct a mass cytometry imaging antibody panel; all metal-conjugated antibodies were validated for specificity by immunohistochemistry. ②Two normal kidney biopsy specimens adjacent to ureteral carcinoma, 2 diabetic nephropathy (DN), and 2 IgA nephropathy (IgAN) renal biopsy specimens were collected, stained with the aforementioned antibodies, and subjected to image data acquisition using the Hyperion imaging system to obtain multiplexed spatial data. ③ An integrated Ilastik and CellProfiler workflow was established for single-cell segmentation pipeline tailored for renal tissues, and cell identity annotation was achieved using a combination of manual gating and Phenograph unsupervised clustering. ④ Spatial proximity relationships between cells were defined, and permutation testing was employed to simulate random distribution models to examine spatial relationships between tissue cells and immune cells. ⑤ k-nearest neighbors (kNN) algorithm was applied for spatial clustering, and cellular neighborhoods (CNs) were used to delineate the spatial functional units within renal tissue, followed by analysis of the immunological characteristics and relative abundance of these units.

①A 37-color antibody panel was constructed and optimized, with all antibodies demonstrating high specificity and low background noise; this panel enabled accurate identification of renal structural cells, stromal cells, and infiltrating immune cells. ② High-dimensional IMC imaging data were successfully obtained from normal and CKD renal tissues using the Hyperion imaging system. ③ With the aid of the single-cell segmentation pipeline, a high-resolution single-cell atlas of human kidney was systematically mapped, comprising 21 structural cell types and 17 immune cell subtypes. Immune cell populations such as CD15⁺ macrophages, GzmK⁺ CD4⁺ effector T cells, and 3 distinct double-positive renal tubular cells were identified, revealing differential infiltration of immune cells in DN and IgAN tissues. ④ A single-cell spatial interaction analysis framework was established, demonstrating a disease-specific spatial interaction network of immune cells in CKD tissues, and identifying myofibroblasts as a critical spatial hub connecting tissue injury and immune cell infiltration.⑤ Spatial neighborhood analysis delineated 9 functional units of renal tissue, including glomerular, proximal tubular, ascending limb of the loop of Henle, vascular and fibrotic, and immune cell-enriched regions. In diseased tissues, the proportion of normal functional regions was decreased, whereas the proportions of fibrotic and immune-enriched regions were increased significantly. Moreover, immune cell-enriched zones maintained close colocalization with various functional regions, while vascular and fibrotic regions were predominantly adjacent to collecting duct and ascending limb of the loop of Henle.

An IMC methodology tailored for the spatial analysis of renal tissue is successfully developed. This platform enables high-resolution, panoramic visualization of spatial distribution of immune cells in chronic renal tissues, dissecting spatial interactions between immune and structural cells and characteristics of local immune microenvironment. This technology holds promise to advance the development of spatially informed precision diagnostics and targeted therapeutic strategies for renal diseases.

To investigate the molecular mechanisms by which branched-chain amino acids (BCAAs) catabolic disorder induces glomerular podocyte injury, and to elucidate its role and mechanism in promoting the pathogenesis of diabetic nephropathy (DN) in a high-fat diet (HFD)-induced mouse model of obesity.

(1) Cellular Experiments: ① Primary glomerular podocytes were isolated from 6- to 8-week-old male C57BL/6J mice (weight 20 to 25 g) and cultured, and then were randomly divided into 3 groups (n=4): low-glucose (LG, 5.5 mmol/L) group, high-glucose (HG, 25 mmol/L) group, and HG+3 mmol/L BCAA group. Western blotting was used to detect the expression of BCAA metabolism-related molecules (PPM1K, BCKD, and BCKD pSer293), podocyte functional markers (Nephrin, Synaptopodin, and WT1), and the apoptotic molecule Cleaved Caspase-3. Immunohistochemistry staining and Western blotting were performed to detect the expression levels of mTOR signaling pathway molecules (p70s6K pThr389, p70s6K, ATF4 pSer166, and ATF4) and apoptotic pathway molecules (Chop and Trib3). ② ATF4-knockout fibroblast cells were randomly divided into control (n=4) and BCAA (n=4) groups. Western blotting was used to detect the expression levels of apoptosis pathway molecules. ③ Primary glomerular podocytes isolated from 6- to 8-week-old C57BL/6J mice were randomly divided into 3 groups (n=4): HG (25 mmol/L) group, HG + 3 mmol/L BCAA group, and HG + 3 mmol/L BCAA + rapamycin (HG + BCAA + Rap) group. Western blotting was performed to assess the expression levels of mTOR signaling pathway and apoptotic pathway molecules. (2) Animal Experiments: An HFD-induced mouse model of obesity was established. Male C57BL/6J mice aged 6 to 8 weeks were randomly divided into an HFD group, an HFD/Paired group (food intake matched to the HFD/BCAA group), an HFD/BCAA group (4% BCAAs supplemented in drinking water), and an HFD/BCAA + Rap intervention group. Periodic acid-Schiff (PAS) staining, Oil Red O staining, and detection of renal triglyceride content were performed to assess renal pathological morphology and lipid deposition. Biochemical assays were applied to detect the urinary microalbumin and creatinine levels, as well as to calculate the urinary albumin-to-creatinine ratio. Glomerular podocyte functional markers (Nephrin and Synaptopodin) and fibrosis-related molecules (Collagen IV) were detected by immunofluorescence assay and RT-PCR. The expression levels of renal lipid metabolism-related molecules (FABP4, CD36, PGC1α, and PPARα), as well as the mTOR signaling pathway and apoptotic pathway molecules were detected by immunohistochemistry and Western blotting.

(1) Cellular Experiments: ① High-glucose treatment decreased PPM1K expression and increased BCKD pSer293 expression in the podocytes (P<0.000 1). BCAAs downregulated the expression of Nephrin (P<0.000 1) and Synaptopodin (P<0.01) and upregulated Cleaved Caspase-3 expression (P<0.000 1) in the podocytes under high glucose condition. BCAAs significantly upregulated the protein levels of p70s6K pThr389, p70s6K, ATF4 pSer166, ATF4, Chop, and Trib3 in the podocytes (P<0.000 1). ② Knockout of ATF4 significantly blocked BCAA-induced Chop and Trib3 expression activated by mTOR (P<0.000 1). ③ The mTOR inhibitor Rap significantly suppressed the expression of ATF4 pSer166, ATF4, Chop, and Trib3 (P<0.000 1). (2) Animal Experiments: ① Exogenous BCAA supplementation increased the urinary albumin-to-creatinine ratio (P<0.000 1), aggravated the damage to glomerular filtration barrier, downregulated the expression of WT1, Nephrin, and Synaptopodin (P<0.000 1), and upregulated glomerular p70s6K pThr389 (P<0.01) and Chop (P<0.000 1) expression. Additionally, BCAA supplementation promoted renal tubular cast formation and renal lipid accumulation (P<0.000 1), and upregulated the expression of FABP4 and CD36 in renal tubules of HFD-fed mice. ② The mTOR inhibitor Rap blocked the mTOR-ATF4-Chop/Trib3 pathway, downregulated the expression of ATF4 pSer166, ATF4, Chop, and Trib3 (P<0.000 1), and ameliorated renal lipid deposition (P<0.000 1), renal tubular cast formation, and proteinuria (P<0.000 1).

BCAA metabolism disorder induces glomerular podocyte dysfunction and apoptosis by activating the mTOR-ATF4-Chop/Trib3 signaling pathway, leading to renal lipid deposition, renal tubular cast formation, and progression of renal fibrosis, thereby promoting the development and progression of DN.