Objective·To investigate the spatial epigenetic characteristics of spontaneous colon tumors in Apcmin/+mice.Methods·A spatial assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq)technology platform was established using an eight-month-old male Apcmin/+mouse model with spontaneous colon tumors.One tumor from a mouse was harvested and embedded in OCT compound for serial cryosectioning;one tissue section was stained with hematoxylin-eosin(H-E)to observe its histological characteristics,while an adjacent section was processed using spatial ATAC-seq technology to generate spatially resolved DNA libraries,followed by sequencing to obtain spatial chromatin accessibility data.Another tumor from the same mouse was digested into a single-cell suspension,in which viable single cells were sorted by flow cytometry and processed for single-cell RNA sequencing.The results were integrated with spatial chromatin accessibility data to jointly analyze the epigenetic characteristics of the colon tumor microenvironment.Results·A stable spatial ATAC-seq platform was successfully established,dividing the tumor into malignant,non-malignant,and malignant-non-malignant boundary regions.Transcription factors enriched in malignant regions included NK2 homeobox 5(NKX2-5)and transcription factor 3(TCF3).Analysis of transcription factor enrichment in the 3 regions revealed two distinct expression trends:one showing a gradual decrease from malignant to boundary to non-malignant regions,and the other exhibiting high expression in malignant and boundary regions but low expression in non-malignant regions.Gene analysis across regions revealed significant upregulation of hypoxia response,transforming growth factor(TGF),and Kirsten rat sarcoma viral oncogene homolog(KRAS)signaling pathways in malignant regions,with cell cycle-related functions markedly enhanced.Analysis of cell-cell interactions in the tumor microenvironment revealed significant differences in interaction strength:strong interactions within non-malignant regions,moderate interactions between boundary and non-malignant regions,and weak interactions between malignant and boundary regions as well as between malignant and non-malignant regions.Conclusion·Colon tumors in Apcmin/+mice exhibit high spatial heterogeneity;malignant regions were enriched with transcription factors including TCF3,and cell interactions between malignant regions and boundary/non-malignant regions were relatively weak.

Mental disorders pose a significant challenge to global public health,profoundly affecting the quality of life of a vast number of individuals and imposing a heavy health burden on society.Nonetheless,there remains a substantial gap between the current societal capacity to provide prevention,diagnosis,and treatment for mental disorders and the existing demand for such services.In recent years,the development and application of artificial intelligence(AI)technologies have provided unprecedented opportunities to enhance mental healthcare services.As one of the fastest-growing fields of AI,generative AI has played a pivotal role in analyzing diverse forms of data,including medical image processing,protein structure prediction,clinical document generation,auxiliary diagnostic discrimination,and clinical decision support.These advancements have significantly strengthened capabilities in clinical diagnosis,data reconstruction,and adjunctive therapeutic interventions.This review highlights the potential applications of generative AI in advancing fundamental psychiatric research,identifying early risk factors for mental disorders,and assisting clinicians in diagnosis and treatment.Additionally,it addresses the challenges and limitations currently facing the application of generative AI to mental healthcare,including biases,privacy breaches,and insufficient interpretability.Finally,the review summarizes strategies to enhance AI's capacity to deliver mental health services,aiming to leverage new technologies to reduce the global burden of mental disorders and improve the quality of life of affected individuals.

Objective·To elevate the therapeutic effect of artesunate(ART)on NOD/Ltj mice(non-obese diabetic mice,the spontaneous models of Sj?gren syndrome),and explore its potential impact on the distribution of B lymphocyte subsets.Methods·ICR mice were used as the blank control group,and NOD/Ltj mice were randomly divided into disease and ART groups.NOD/Ltj mice and ICR mice were treated with ART(10 mg/kg)or its vehicle(0.5%CMC)by oral gavage every other day for 4 weeks.Body weight,salivary flow rate,submandibular gland index,and spleen index were measured.Cytokines in plasma,including interleukin-6(IL-6),interferon-γ(IFN-γ),and B-cell activating factor(BAFF)in serum,were detected by cytometric bead array(CBA).Hematoxylin-Eosin(H-E)staining of submandibular glands was used to observe the infiltration of lymphocyte.Flow cytometry was applied to analyze the distribution of B lymphocyte subsets in the spleen.The mRNA expression of Prdm1,Il-6r,Il-6,and Stat3 in spleen B lymphocytes was detected by RT-qPCR.The effect of ART on B cells was further detected by CCK-8 and Annexin V-FITC/PI staining by flow cytometry.Results·Compared to the disease group,ART significantly improved the symptoms of Sj?gren syndrome in NOD/Ltj mice.ART treatment also resulted in a reduction in the levels of BAFF,IL-6,and IFN-γ in the plasma(all P<0.05).Moreover,lymphocyte infiltration around the glandular ducts in the submandibular glands was greatly improved in the ART group compared with the disease group.Flow cytometry analysis revealed that the proportion of Na?ve B cells in the ART group was significantly increased compared with the disease group,along with a significant reduction in the proportions of double-negative B cells,switched memory B cells,and plasmablasts(all P<0.05).The relative mRNA expression levels of Prdm1,Il-6r,and Stat3 in the ART group were significantly lower than those in the disease group(all P<0.05).The CCK8 assay results showed that after 6 h of treatment,with the extension of the culture time,cell proliferation in the ART group was significantly inhibited;after 24 h of treatment,the number of apoptotic cells in the ART group was significantly higher than that in the control group(P<0.001).Conclusion·ART demonstrates therapeutic effects in NOD/Ltj mice,potentially through modulating the distribution of peripheral B lymphocyte subsets.It can inhibit the expression of Prdm1,thereby regulating the differentiation of B lymphocytes into plasma cells and plasmablasts.

Objective·To investigate the expression level of O-GlcNAc transferase(OGT)in non-small cell lung cancer(NSCLC)and its impact on lung cancer proliferation,as well as to explore the underlying mechanisms.Methods·The expression of OGT in NSCLC tumors and adjacent normal tissues was detected by immunohistochemistry(IHC).The dataset(GSE31210)from the GEO database was analyzed to assess the correlation between OGT expression and NSCLC patient prognosis.siRNA transfection was performed to knock down OGT expression in H460 and H1299 cells,followed by total RNA extraction and transcriptome sequencing.Pathway enrichment analysis was conducted on differentially downregulated genes in the knockdown group compared with the control group,and Western blotting was used to validate the enrichment results.The effects of OGT knockdown on cell proliferation and colony formation in H460 and H1299 cells were evaluated using the cell counting kit-8(CCK-8)assay and colony formation assay,respectively.The impact of overexpressing downstream genes was also examined.Stable OGT-knockdown cell lines were generated using shRNA and subcutaneously inoculated into nude mice to monitor tumor growth.Results·IHC revealed that OGT expression was significantly upregulated in NSCLC tumor tissues compared to adjacent normal tissues.Patients with high OGT expression exhibited shorter survival times and poorer prognoses than those with low expression.Transcriptome sequencing demonstrated that genes downregulated after OGT knockdown were primarily enriched in the mitogen-activated protein kinase(MAPK)signaling pathway.Western blotting showed that total extracellular regulated protein kinase 1/2(ERK1/2)levels remained unchanged in H460 and H1299 cells after OGT knockdown,while phosphorylated ERK1/2(p-ERK1/2)and its downstream proto-oncogene JUNB protein were markedly reduced.Suppression of OGT expression attenuated the proliferation rate and colony formation capacity of H460 and H1299 cells,whereas JUNB overexpression rescued the proliferation defects induced by OGT knockdown.Notably,H460 cells with stable OGT knockdown formed significantly smaller tumors in nude mice.Conclusion·OGT is highly expressed in NSCLC and correlates with poor prognosis.Knockdown of OGT inhibits NSCLC cell proliferation and clonogenicity in vitro,and tumor growth in vivo.Mechanistically,OGT appears to promote NSCLC progression by activating the ERK/JUNB signaling axis.

Objective·To determine hyaluronan(HA)expression in the endocrine-resistant microenvironment of estrogen receptor-positive(ER+)breast cancer and elucidate its impact on the acquired resistance.Methods·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells.An immunofluorescence(IF)assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells.Using an established adaptive endocrine-resistant breast cancer mouse model,HA expression in resistant breast cancer tissues was assessed by immunohistochemistry(IHC)assay.Single-cell RNA sequencing(scRNA-seq)and RNA sequencing(RNA-seq)were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells.Flow cytometry(FCM)was utilized to measure the proportion of CD44+CD24-cells in MCF7/FulR.The correlation between HA synthesis genes and cell stemness was investigated in clinical ER+breast cancers from GEO data sets.Hyaluronidase(HAase)treatment was applied to remove the"HA coat",and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules.CCK-8 assays,flow cytometry(FCM),and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment.Results·IF results revealed that compared with MCF7 cells,the"HA coat"on the surface of MCF7/FulR cells was significantly thickened.IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues.ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells.Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER+breast cancer.IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules;concurrently,CCK-8 assays,FCM analysis,and Hoechst 33258 staining revealed that"HA coat"clearance reduced MCF7/FulR'tolerance to fulvestrant and increased apoptosis.Conclusion·Endocrine-resistant breast cancer cells develop an enriched"HA coat",which promotes stemness in fulvestrant-resistant tumors.Disruption of this HA coat through HAase treatment effectively reduces cell stemness,induces apoptosis,and re-sensitizes breast cancer cells to fulvestrant.

Objective·To develop a multi-algorithm collaborative computational biology strategy for constructing a predictive model of the peripheral morphine tolerance network and for screening high-confidence candidate targets.Methods·A murine model of morphine tolerance was established across multiple treatment time points.Bulk RNA sequencing was performed on harvested dorsal root ganglion(DRG)tissues.Using the expression matrix as a basis,a weighted gene co-expression network was constructed to identify co-expressed gene modules.Candidate genes were subsequently screened through the integration of differentially expressed genes(DEGs)with key weighted gene co-expression network modules.These candidates underwent functional annotation via Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses.A protein-protein interaction(PPI)network was established,and hub genes were systematically identified using the cytoHubba algorithm.Three distinct machine learning approaches,least absolute shrinkage and selection operator(LASSO)regression,support vector machine recursive feature elimination(SVM-RFE)model,and random forest(RF)model,were strategically integrated to screen characteristic signature genes.Finally,gene set enrichment analysis(GSEA)was implemented to functionally validate both the hub and signature genes.Results·Weighted gene co-expression network analysis(WGCNA)identified 8 297 key module genes,of which 177 candidate genes overlapped with DEGs.These genes were significantly enriched in biological processes including ion channel regulation and vascular smooth muscle contraction.A combination of PPI network analysis and machine learning revealed four signature genes[actin γ2,smooth muscle(Actg2),centriolar coiled-coil protein 110(Ccp110),neural cell adhesion molecule 2(Ncam2),and selenium binding protein 1(Selenbp1)]and six hub genes[actin α2,smooth muscle(Acta2),von Willebrand factor(Vwf),cellular communication network factor 2(Ccn2),integrin β4(Itgb4),integrin α11(Itga11),and TEK receptor tyrosine kinase(Tek)]closely associated with morphine tolerance.Conclusion·In this study,we successfully constructed a multi-algorithm collaborative peripheral nerve regulation network prediction model for morphine tolerance,and screened out 10 core genes with high confidence.

Objective·To study the regulatory effect of saikosaponin A(SSA)on the differentiation,apoptosis,and immunosuppressive function of myeloid-derived suppressor cells(MDSCs)in mice,and to explore their molecular mechanism.Methods·Recombinant mouse granulocyte-macrophage colony-stimulating factor(GM-CSF)was used to induce the differentiation of mouse bone marrow cells(BMCs)into MDSCs,or magnetic beads were used to sort MDSCs from tumor-bearing mice.After treating MDSCs with different concentrations(0,2.5,5.0 mg/L),flow cytometry(FCM)was used to detect the differentiation and apoptosis of MDSCs,as well as the expression levels of liver X receptor α(LXRα),arginase-1(Arg-1),and reactive oxygen species(ROS).At the same time,the effects of MDSCs on the proliferation function of T cells,and the effects on the nuclear factor κB(NF-κB),and signal transducer and activator of transcription 1(STAT1)signaling pathways were also detected.The mRNA levels of LXRα and Arg-1 were detected by quantitative real-time PCR(qPCR).Mice were given SSA by gavage(ig)or intraperitoneal injection(ip),and the mice were sacrificed after administration;and body mass,spleen weight,and spleen index were calculated.FCM was used to detect the proportion of immune cells in the spleen of mice.Results·SSA could up-regulate the expression level of LXRα in MDSCs,reduce the differentiation of M-MDSCs,induce apoptosis of MDSCs,reduce the expression levels of Arg-1 and ROS in MDSCs,and reduce the inhibitory effect of MDSCs on T cell proliferation.SSA inhibited the phosphorylation levels of NF-κB and STAT1 in MDSCs.The mice treated with SSA by gavage or intraperitoneal injection showed no significant changes in body weight and spleen index.Both modes of administration can reduce the proportion of MDSCs and their subset M-MDSCs in mice,but had different degrees of regulatory effects on other immune cells.Conclusion·SSA could regulate the differentiation and apoptosis of MDSCs,and inhibit their immunosuppressive function,which may be associated with the up-regulation of LXRα expression,and down-regulation of the NF-κB and STAT1 signaling pathways in MDSCs.

Recurrent spontaneous abortion(RSA)is a pregnancy complication with a complex etiology that seriously threatens the fertility and physical and mental health of women of childbearing age.As the main component of the maternal-fetal interface,the placenta plays a central role in maternal and fetal health during pregnancy,and its dysfunction is closely associated with the development of RSA.Iron homeostasis is essential for supporting maternal needs,placental function,and fetal development.In recent years,ferroptosis,a novel form of cell death triggered by iron overload and the accumulation of lipid peroxides,has garnered increasing attention regarding its regulatory mechanisms in the physiological and pathological processes of the female reproductive system.Ferroptosis has been confirmed to correlate with placental pathology and to influence the pathogenesis of RSA.The placenta plays a crucial role in regulating iron transport.This paper systematically reviewed the mechanisms of ferroptosis in placental cells and its involvement in the pathogenesis of RSA by affecting trophoblast cell function,causing decidualization disorders,inducing angiogenesis defects,and the potential of ferroptosis-related molecules in the treatment of RSA was analyzed,with the aim of providing research directions for improving the pregnancy outcomes in patients with RSA.

Objective·To investigate the mechanisms by which different subtypes of G protein-coupled receptor kinases(GRKs)regulate the biased signaling transduction mediated by the muscarinic acetylcholine receptor 1(M1 receptor),focusing on their molecular effects in modulating the binding of the M1 receptor to the downstream heterotrimeric G protein(Gαq-Gβ1-Gγ2)andβ-arrestin 2(βarr2).Methods·By establishing a highly sensitive protein interaction detection system based on bioluminescence resonance energy transfer(BRET),six M1 receptor agonists/allosteric modulators were selected to measure the dynamic interactions between the M1 receptor and four GRK subtypes(GRK2/3/5/6),βarr2,and the G protein under stimulation.All BRET data were statistically quantified using the area under the curve(AUC)of the time-response curves.First,concentration-effect curves were established by treatment with gradient concentrations of agonists/allosteric modulators and AUC fitting,to comprehensively analyze the differences in efficacy between each agonist/allosteric modulator and the endogenous agonist acetylcholine chloride(ACh)in promoting the interactions of M1 receptor with GRK3/5,βarr2,and the G protein;next,GRKs were divided into two groups based on subtypes:GRK2/3 and GRK5/6.The maximum AUC values for the interaction between the M1 receptor and the two GRK groups under high concentrations were calculated respectively,to further evaluate the regulatory propensity of different types of GRKs on the binding strength of the M1 receptor to βarr2 or the G protein.Results·All six agonists/allosteric modulators effectively induced the association of the M1 receptor with GRK3,while simultaneousey inducing dissociation of the M1 receptor from GRK5.The allosteric modulator BQCA not only activated the M1 receptor alone and triggered its binding to downstream signaling proteins,but also,when co-treated with ACh,caused a significant leftward shift of the concentration-effect curves in the M1-G protein and M1-βarr2 systems,suggesting that its potentiation effect on ACh was mainly achieved by reducing the half-maximal effective concentration.A moderate positive correlation was observed between the maximum AUC values of M1-βarr2 and M1-G protein interactions induced by the seven groups of drug treatments(r=0.722,P=0.067).Further analysis showed that the ratio of the maximum AUC for M1-GRK2/3 interaction to that for M1-GRK5/6 interaction was also positively correlated with the ratio of the maximum AUC for M1-βarr2 interaction to that for M1-G protein interaction(r=0.760,P=0.047).Conclusion·The M1 receptor may be pre-coupled with GRK5/6 under basal conditions,and they dissociate upon receptor activation,suggesting that GRK5/6 may be involved in M1 receptor inactivation or signal reprogramming.The relative efficiency of the M1 receptor's interaction with different GRK subtypes determines its preference for downstream signaling pathways.

Objective·To identify and evaluate novel and reliable non-invasive serological biomarkers for detecting pancreatic ductal adenocarcinoma(PDAC).Methods·Sixty-seven PDAC patients(Ruijin cohort Ⅰ)were recruited at Pancreatic Center,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,from May 2018 to December 2019.Global proteome profiling of 67 PDAC tumor tissues and 67 matched adjacent normal tissues was performed using mass spectrum.Bioinformatics analysis on the proteomics data was conducted to identify new biomarkers,and receiver operating characteristic(ROC)curves and the area under the curve(AUC)were used to evaluate their value of detecting PDAC.The proteomic and mRNA sequencing data were further downloaded and analysed from the Clinical Proteomic Tumor Analysis Consortium(CPTAC)cohort for validation.In addition,the Ruijin Cohort Ⅱ,consisting of 47 PDAC patients and 75 healthy individuals,was recruited for a case-control study from June 2021 to June 2022.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression level of new biomarkers in the serum of patients and healthy individuals to evaluate the serological diagnostic values of them.Results·Collagen type Ⅻ α1 chain(COL12A1)was identified as a candidate biomarker for PDAC diagnosis based on differential expression analysis on the proteomic data and was validated to be higher in tumor tissues than in adjacent normal tissues in the CPTAC cohort.In addition,COL12A1 protein levels were significantly higher in the sera of PDAC patients than in those of healthy controls,showing good diagnostic performance with an AUC of 0.82,a sensitivity of 81%,and a specificity of 83%.ROC analysis revealed that COL12A1 improved the performance of carbohydrate antigen 199(CA199)in distinguishing PDAC patients from healthy individuals(AUCCA199=0.91 vs AUCCA199+COL12A1=0.95,P<0.05).Furthermore,COL12A1 also showed excellent ability to distinguish early-stage PDAC patients(stage Ⅰ?Ⅱ)from healthy individuals(AUCCOL12A1=0.83),and significantly improved the AUC of CA199 in early-stage PDAC patients(AUCCA199=0.92 vs AUCCA199+COL12A1=0.97,P<0.05).Conclusion·COL12A1 is a potential serological diagnostic marker that complements CA199 in detecting early-stage PDAC.