ObjectiveTo analyze the epidemiological characteristics of 8 major respiratory pathogens in influenza-like illness (ILI) cases with acute respiratory infections at fever clinics in Huangpu District, Shanghai from 2015 to 2024, and to provide a scientific basis for the prevention and treatment of respiratory diseases. MethodsA retrospective study was conducted in Huangpu District. Individuals meeting the case definition of ILI from 2015 to 2024 was registered. Their nasopharyngeal swabs were collected for pathogen detection. A total of 8 respiratory viruses were tested, including Influenza A virus (Flu A), Influenza B virus (Flu B), adenovirus (ADV), enterovirus/human rhinovirus (EV/HRV), human parainfluenza virus (HPIV), human coronavirus (HCoV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV). ResultsFrom 2015 to 2019, a total of 344 ILI cases were tested, of which 192 out of 344 cases (55.81%) were tested positive for single respiratory pathogen. From 2023 to 2024, 1 557 ILI cases were tested, with 572 out of 1 557 cases (36.74%) being positive for single pathogen. From 2023 to 2024, the positive rate of single pathogen in ILI cases was significantly lower than that in 2015‒2019 (χ²=42.66, P<0.001). Specifically, the positive rate of Flu A (χ²=74.43, P<0.001) decreased, while that of HPIV (χ²=8.66, P=0.003) increased, both with statistically significant differences. According to the seasonal pattern, the epidemic intensity of Flu A decreased in summer, while that of HPIV increased in summer and autumn. Demographic results showed statistically significant differences in the positive rates of EV/HRV between genders (χ²=22.38, P<0.001), with males exhibiting a higher positive rate than females. No statistically significant differences were identified in the positive rates of single pathogen among different age groups (χ²=4.42, P=0.110). Nevertheless, statistically significant differences were noted when comparing the positive rates of EV/HRV, Flu A, Flu B and HPIV across different age groups (P<0.05). EV/HRV was more commonly detected in the 15‒<25 age group (10.93%), while Flu A and HPIV had the highest positive rates in the ≥60 age group (21.24% and 4.77%). Flu B had the highest positive rate in the 25‒<60 age group (11.26%). 52.63% of cases with co-infections occurred during winter, with the primary pathogens involved being EV/HRV (9 cases) and HCoV (6 cases). The most prevalent combination of co-infection was Flu A with EV/HRV. ConclusionThe prevalence of respiratory pathogens among ILI cases from 2023 to 2024 exhibited notable fluctuations compared to that from 2015 to 2019. Therefore, influenza surveillance should be strengthened, and attention should also be paid to the prevalence of respiratory pathogens such as HPIV. These findings have profound implications for future research, surveillance, vaccine planning, and public health policy making.

Background: Disorders of the blood-brain barrier (BBB) arising from diabetes mellitus are closely related to diabetic encephalopathy. Previous research has suggested that neuron-glia antigen 2 (NG2)-glia plays a key role in maintaining the integrity of the BBB. However, the mechanism by which NG2-glia regulates the diabetic BBB remains unclear. Methods: Type 2 diabetes mellitus (T2DM) db/db mice and db/m mice were used. Evans-Blue BBB permeability tests and transmission electron microscopy techniques were applied. Tight junction proteins were assessed by immunofluorescence and transmission electron microscopy. NG2-glia number and signaling pathways were evaluated by immunofluorescence. Detection of matrix metalloproteinase-9 (MMP-9) in serum was performed using enzyme-linked immunosorbent assay (ELISA). Results: In T2DM db/db mice, BBB permeability in the hippocampus significantly increased from 16 weeks of age, and the structure of tight junction proteins changed. The number of NG2-glia in the hippocampus of db/db mice increased around microvessels from 12 weeks of age. Concurrently, the expression of MMP-9 increased in the hippocampus with no change in serum. Sixteen- week-old db/db mice showed activation of the Wnt/β-catenin signaling in hippocampal NG2-glia. Treatment with XAV-939 improved structural and functional changes in the hippocampal BBB and reduced MMP-9 secretion by hippocampal NG2-glia in db/db mice. It was also found that the upregulation of β-catenin protein in NG2-glia in the hippocampus of 16-week-old db/db mice was significantly alleviated by treatment with XAV-939. Conclusion The results indicate that NG2-glia can lead to structural and functional disruption of the diabetic BBB by activating Wnt/β-catenin signaling, upregulating MMP-9, and degrading tight junction proteins.

Background/Aims: Lenvatinib resistance remains a critical barrier in advanced hepatocellular carcinoma (HCC) therapy. However, the underlying mechanisms and strategies for reversing resistance remain incompletely understood. Methods: Integrated transcriptomics of lenvatinib-resistant patient tumors and an acquired-resistance murine model identified a novel macrophage subpopulation. Functional validation employed CRISPR-SAM screening, conditioned medium (CM) assays, subcutaneous/orthotopic xenografts, patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). Mechanistic studies included ChIP-qPCR, co-immunoprecipitation, and pharmacologic targeting. Clinical relevance was assessed in a retrospective cohort. Results: Resistant HCC exhibited significant enrichment of a COLEC12high TAM subset , which correlated with poor survival and treatment response. These TAMs secreted neuregulin-1 (NRG1) , activating HER2/HER3-AKT signaling in tumor cells to drive cancer stemness and lenvatinib resistance. Mechanistically, in TAMs COLEC12 sequestered STAT1 in the cytoplasm, preventing its phosphorylation, and thereby derepressing STAT3-mediated NRG1 transcription. Depletion of NRG1 reversed the stemness phenotypes and resensitized tumors to lenvatinib both in vitro and in vivo. Clinically, high NRG1 expression predicted an inferior lenvatinib response and shorter survival. Crucially, the bispecific anti-HER2/HER3 antibody zenocutuzumab restored lenvatinib efficacy in PDOs, PDXs, and murine models. Conclusions Our work establishes the COLEC12high TAM/NRG1 axis as a master regulator of therapeutic resistance and identifies NRG1 as a predictive biomarker, providing a clinically actionable strategy to overcome lenvatinib resistance in HCC.

BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

Objective: To explore the prospective association between multidimensional sleep indicators and the risk of newlyonset dental caries, providing a reference for childrens oral healthrelated sleep intervention. Methods: In October 2021, 1 417 students in grades 1 to 4 (aged 6 to 11) from two elementary schools in Bengbu, Anhui Province, were selected by cluster sampling method. Surveys and followup visits were conducted at baseline (T1), November 2022 (T2), May 2023 (T3), and November 2023 (T4), respectively, including parental questionnaires, oral health and physical examination. Bedtime, sleep duration, sleep midpoint, social jet lag, weekend catchup sleep, and sleep habits were collected and calculated. A multifactorial Cox proportional risk regression model was used to analyze the association between multidimensional sleep indicators and newlyonset caries in schoolaged children after 2 years. Results: The prevalence of dental caries in children was 65.1% at baseline, and the prevalence was 59.0% at the end of the 2year followup. Cox proportional risk regression model showed that for every 1point increase in the childrens bedtime resistance, nocturnal awakenings, parasomnias, and daytime sleepiness scores, the risk of newlyonset caries increased by 12% (HR=1.12, 95%CI=1.08-1.15), 22% (HR=1.22, 95%CI=1.15-1.29), 12% (HR=1.12, 95%CI=1.08-1.17), and 15% (HR=1.15, 95%CI=1.12-1.19), respectively; the risk of newlyonset caries increased by 23% for each 1 h increase in the length of weekend catchup sleep (HR=1.23, 95%CI=1.14 -1.33); compared with children who went to bed before 21:00 on school days, those who went to bed later than 22:00 had a 57% higher risk of newlyonset caries (HR=1.57, 95%CI=1.22-2.03). Compared to children who slept adequately (≥9 h/d), those with insufficient sleep had a 67% higher risk of new caries (HR=1.67, 95%CI=1.43-1.95) (P<0.01). Conclusions These findings suggest a significant association between sleep patterns/sleep disorders and the development of childhood dental caries. Incorporating sleep behavior optimization and sleep quality improvement into comprehensive caries prevention and oral health management protocols may represent a promising intervention strategy to enhance childrens oral health outcomes.

ObjectiveTo explore the process and guidelines for ethical review in decentralized drug clinical trials， promote clinical trial progress， and ensure drug development progress. MethodsThe key points of the ethical review were summarized by studying the relevant laws and regulations on decentralized drug clinical trials， analyzing the advantages and challenges of decentralized drug clinical trials， and combining the experience of the ethics committee of the institution in reviewing decentralized drug clinical trials. ResultsRelevant laws and regulations were the basis for the ethical review， and the ethics committee should adopt appropriate review methods based on regulations and hospital ethical standard operating procedures. The ethics committee should focus on the feasibility， applicability， and rationality， the adequacy of informed consent， the protection of rights and interests and privacy of subjects， as well as the qualification and standard operating procedures of electronic platforms for conducting decentralized drug clinical trials. ConclusionDecentralized drug clinical trials are in their early stages and urgently require guidance from relevant laws and regulations. Ethical review is also constantly being refined through exploration. It is necessary to supervise the implementation of responsibilities by all parties， pay attention to the rights and interests of subjects， and gradually promote the implementation of decentralized drug clinical trials.

The catalytic activity of 3-mercaptopyruvate (3MP) sulfurtransferase (MPST) converts 3MP to hydrogen sulfide (H₂S). However, the regulatory mechanisms governing MPST and its impact on the brain remain largely unexplored. Our study reveals the neuroprotective role of endothelial MPST-generated H₂S, regulated by protein phosphatase 2A (PP2A). Bioinformatics analysis and RNA sequencing demonstrated that endothelial PP2A is associated with neurodegenerative disease pathways. Cerebral ischemic mice exhibited significant inactivation of endothelial PP2A, evidenced by the reduction of PP2Acα in the brain endothelium. Mice with endothelium-specific null PP2A (PP2A^EC-cKO) exhibited neuronal loss, cognitive dysfunction, and long-term potentiation deficits. Postnatal inactivation of endothelial PP2A also contributes to cognitive dysfunction and neuronal loss. However, regaining endothelial PP2A activity by overexpressing Ppp2ca rescued neuronal dysfunction. Mechanistically, PP2A deficiency is intricately linked to the MPST-H₂S signaling pathway. A robust reduction in endothelial MPST-dependent H₂S production followed PP2A deficiency. Exogenous H₂S treatment and AAV-mediated overexpression of MPST in brain endothelial cells significantly mitigated neuronal dysfunction in PP2A^EC-cKO mice. Furthermore, PP2A deficiency promotes an increase in calcium influx and calpain2 phosphorylation, subsequently leading to MPST degradation. The PP2A activator (FTY720) and MPST activator (3MP sodium) both remarkably restored endothelial MPST-dependent H₂S production, subsequently rescuing ischemia-induced neurological deficits. In conclusion, our study demonstrates that endothelial PP2A deficiency leads to MPST degradation by activating calpain2, thus damaging neuronal function.

Acetaminophen (APAP) is the primary cause of drug-induced acute liver failure. Ovarian tumor deubiquitinase 6A (OTUD6A), a recently discovered deubiquitinase of the OTU family, has been primarily studied in tumor contexts. However, its role in APAP-induced liver injury (AILI) remains unclear. Therefore, this study aimed to investigate the involvement of OTUD6A in the pathogenesis of AILI. Our findings demonstrated a substantial upregulation of OTUD6A in both the liver tissue and isolated hepatocytes of mice following APAP stimulation. OTUD6A knockout exacerbated APAP-induced inflammation, hepatocyte necrosis, and liver injury, whereas OTUD6A overexpression alleviated these pathologies. Mechanistically, OTUD6A directly interacted with the enhancer of zeste homolog 2 (EZH2) and selectively removed K48-linked polyubiquitin chains from EZH2, enhancing its stability. This resulted in increased protein levels of EZH2 and H3K27me3, as well as reduced endoplasmic reticulum (ER) stress and cell death in hepatocytes. Collectively, our research uncovers a novel role for OTUD6A in mitigating APAP-induced liver injury by promoting EZH2 stabilization.

OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*