ObjectiveTo establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus (MHV), mouse parvovirus (MPV), and reovirus type 3 (Reo-3) in laboratory mice. MethodsRecombinant plasmid standards were constructed using conserved genomic regions of the three target viruses. Specific primers and probes were designed, and a triplex TaqMan quantitative PCR (qPCR) system was optimized through preliminary experiments. Sensitivity was evaluated using 10-fold serial dilutions (10¹-10⁷ copies/μL). Intra- and inter-assay repeatability were assessed via reproducibility experiments. ResultsA strong linear correlation (R ²>0.99) was observed between copy number and Ct value at template concentrations of 10²-10⁷ copies/μL. Intra-assay and inter-assay coefficients of variation (CV) were both below 5%. No cross-reactivity was observed with mouse cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), Sendai virus (SeV), or pneumonia virus of mice (PVM). ConclusionThe triplex TaqMan quantitative PCR method established in this study has high sensitivity, good repeatability, and strong specificity. It enables the rapid, specific, and sensitive detection of MHV, MPV, and Reo-3, allows the simultaneous detection of multiple pathogens in a single tube, and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations, thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.

ObjectiveTo analyze the composition of respiratory virus pathogen spectrum and epidemiological characteristics among adult influenza-like illness (ILI) cases in Putuo District of Shanghai from 2015 to 2024, to summarize the epidemic patterns of respiratory viruses across different time periods and populations, and to provide scientific evidence for the prevention and control of adult respiratory virus infections in Putuo District of Shanghai. MethodsSurveillance data of 2 273 adult ILI cases from sentinel hospitals under the comprehensive surveillance system for acute respiratory infections in Putuo District of Shanghai from 2015 to 2024 were retrospectively collected. Descriptive epidemiological methods were adopted to analyze the temporal and population distribution characteristics of respiratory viruses, including influenza virus (IFV), respiratory syncytial virus (RSV), adenovirus (ADV), and human metapneumovirus (HMPV). The mixed infection status among different age groups was compared, and the distribution of influenza virus subtypes was described. SPSS 22.0 and WPS Office software were used for statistical analyses. ResultsThe overall detection rate of respiratory viruses in 2 273 adult ILI cases was 43.51%. The detection rate in females (45.48%) was significantly higher than that in males (41.25%), with statistical significance (χ²=4.113, P=0.043). Individuals aged 60 years old and above had the highest detection rate (47.41%) (χ²=8.430, P=0.014). The detection rates in winter (50.41%) and summer (48.42%) were higher than those in other seasons (χ²=47.028, P<0.001). The overall viral detection rate decreased after 2020 (χ²=208.411, P<0.001). Detection rates of various viruses varied by year, with IFV and ADV showing high rates from 2015 to 2019 (all P<0.001), while human coronavirus (HCoV) and rhinovirus/enterovirus (HRV/EV) exhibited higher rates from 2020 to 2022 (all P<0.001). Co-infection with IFV and HCoV was the most common, followed by co-infection with IFV and ADV, together accounting for 50.00% of all mixed infections. During the surveillance period, the detection rate of IFV was significantly higher than that of other respiratory viruses. Influenza B/Victoria lineage predominated in 2021, followed by successive predominance of influenza A(H1N1), A(H3N2), and the co-circulation of the two subtypes from 2022 to 2023. Influenza A(H1N1) re-emerged as the predominant subtype in the first half of 2024. ConclusionFrom 2015 to 2024, respiratory virus infections in adults prevailed in winter and summer, and people aged 60 years old and above were the key high-risk population. The detection rates of respiratory viruses declined after 2020. IFV was the predominant pathogen detected, and different influenza virus subtypes circulated alternately each year.

Objective·To investigate the inhibitory effects of spermidine(SPD)on inflammatory osteolysis both in vivo and in vitro.Methods·The CCK-8 assay was used to assess the viability of RAW264.7 macrophages treated with various concentrations of SPD.The levels of intracellular reactive oxygen species(ROS)in lipopolysaccharide(LPS)-activated RAW264.7 cells were evaluated by staining with dichlorodihydrofluorescein diacetate(DCFH-DA)and dihydroethidium(DHE),respectively.Reverse transcription real-time fluorescence quantitative PCR(RT-qPCR)was utilized to determine the effects of SPD on the expression of pro-inflammatory genes in LPS-activated RAW264.7 cells.Tartrate-resistant acid phosphatase(TRAP)staining was used to evaluate the effect of SPD on the differentiation of mouse primary bone marrow-derived macrophages(BMMs)into osteoclasts.RT-qPCR was employed to further analyze the effect of SPD on the expression of genes related to osteoclast differentiation and functions after BMM-induced differentiation.An LPS-induced mouse calvarial osteolysis model was constructed,and the therapeutic efficacy of SPD on inflammatory osteolysis was assessed using Micro-CT analysis,hematoxylin-eosin(H-E)staining and TRAP staining of histological sections.Results·The CCK-8 assay showed that SPD,even at a concentration of 1 000 μmol/L,exhibited no significant cytotoxicity to RAW264.7 cells.ROS analysis revealed that SPD markedly inhibited LPS-induced elevation of intracellular ROS levels in macrophages.RT-qPCR results indicated that SPD suppressed the expression of pro-inflammatory genes induced by LPS.Both TRAP staining and RT-qPCR demonstrated that SPD effectively inhibited the differentiation of BMMs into osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL)and reduced the expression of genes associated with osteoclast differentiation and function.In the mouse calvarial osteolysis model,Micro-CT analysis showed that the bone volume fraction and bone mineral density in the SPD-treated groups were significantly higher than those in the LPS group.Histological staining revealed that SPD treatment reduced inflammatory cell infiltration,decreased osteoclast numbers,and alleviated tissue damage.Conclusion·SPD inhibits macrophage inflammatory responses and RANKL-induced osteoclast differentiation in vitro;in vivo,it alleviates LPS-induced inflammatory calvarial osteolysis in mice.

OBJECTIVE To investigate the effect and underlying mechanism of hydroxytyrosol(HT)on mouse chondrocyte injury induced by oxidative stress.METHODS Mouse chondrocytes were incubated with varying concentrations of HT 0-400 μmol L-1 for 24 h,and the viability of the mouse chondrocytes was assessed using CCK-8 kit.An oxidative stress model of chondrocytes was estab-lished by the addition of H2O2 200 μmol L-1.The experimental groups included the cell control group,H2O2 group,and H2O2+HT 10,50 and 250 μmol·L-1 groups.After 24 h,the mRNA expression levels of interleukin-6(IL-6),cyclooxygenase-2(COX-2),prostaglandin E2(PGE2),inducible nitric oxide synthase(iNOS),matrix metalloproteinase-3(MMP-3),MMP-13,a disintegrin and metalloproteinase with throm-bospondin motifs-4(ADAMTS-4),ADAMTS-5,SRY-box transcription factor-9(SOX-9)and aggrecan(ACAN)in mouse chondrocytes were detected by real-time quantitative PCR,the intracellular reactive oxygen species(ROS)level in chondrocytes was measured with 2,7-dichlorodihydrofluorescein diace-tate(DCFH-DA)staining,while the mitochondrial membrane potential was evaluated using JC-1 staining.After 48 h,the protein expression levels of iNOS,COX-2,MMP-13,and type Ⅱ collagen(Col-2)in mouse chondrocytes were detected using Western blotting.RESULTS HT at concentrations≤350 μmol·L-1 had no significant effect on the survival of mouse chondrocytes.Compared with the cell control group,after 24 h,the mRNA expression levels of IL-6,COX-2,PGE2,iNOS,MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 in the chondrocytes of mice in the H2O2 group were increased,while the mRNA expression levels of SOX-9 and ACAN were decreased.Additionally,there was an elevation in the ROS level and a significant loss of mitochondrial membrane potential in the chondrocytes of mice.Compared with the H2O2 group,after treatment with HT 10,50 and 250 μmol·L-1,there were significant decreases in mRNA expression levels of IL-6,COX-2,PGE2,iNOS,MM P-3,MMP-13,ADAMTS-4 and ADAMTS-5,the mRNA expressions of SOX-9 and ACAN were increased,the ROS level was lowered.After treatment with HT 50 and 250 μmol L-1,the loss of mitochondrial membrane potential was ameliorated.Compared to the cell control group,the protein expressions of iNOS,COX-2 and MMP-13 were upregulated in the H2O2 group,while the protein expression of Col-2 was downregulated after 48 h.Compared to the H2O2 group,treatment with HT at concentrations of 10,50 and 250 μmol·L-1 resulted in decreased protein expressions of iNOS,COX-2 and MMP-13 in mouse chondrocytes,but the protein expression of Col-2 increased following treatment with HT 50 and 250 μmol L-1.CONCLUSION HT can ameliorate H2O2-induced chondrocyte injury by reducing intracellular ROS levels and alleviating the loss of mitochondrial membrane potential,suppressing the release of inflammatory cytokines,inhibiting catabolic processes,and promoting anabolic activities.

OBJECTIVE To investigate the effect and underlying mechanism of hydroxytyrosol(HT)on mouse chondrocyte injury induced by oxidative stress.METHODS Mouse chondrocytes were incubated with varying concentrations of HT 0-400 μmol L-1 for 24 h,and the viability of the mouse chondrocytes was assessed using CCK-8 kit.An oxidative stress model of chondrocytes was estab-lished by the addition of H2O2 200 μmol L-1.The experimental groups included the cell control group,H2O2 group,and H2O2+HT 10,50 and 250 μmol·L-1 groups.After 24 h,the mRNA expression levels of interleukin-6(IL-6),cyclooxygenase-2(COX-2),prostaglandin E2(PGE2),inducible nitric oxide synthase(iNOS),matrix metalloproteinase-3(MMP-3),MMP-13,a disintegrin and metalloproteinase with throm-bospondin motifs-4(ADAMTS-4),ADAMTS-5,SRY-box transcription factor-9(SOX-9)and aggrecan(ACAN)in mouse chondrocytes were detected by real-time quantitative PCR,the intracellular reactive oxygen species(ROS)level in chondrocytes was measured with 2,7-dichlorodihydrofluorescein diace-tate(DCFH-DA)staining,while the mitochondrial membrane potential was evaluated using JC-1 staining.After 48 h,the protein expression levels of iNOS,COX-2,MMP-13,and type Ⅱ collagen(Col-2)in mouse chondrocytes were detected using Western blotting.RESULTS HT at concentrations≤350 μmol·L-1 had no significant effect on the survival of mouse chondrocytes.Compared with the cell control group,after 24 h,the mRNA expression levels of IL-6,COX-2,PGE2,iNOS,MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 in the chondrocytes of mice in the H2O2 group were increased,while the mRNA expression levels of SOX-9 and ACAN were decreased.Additionally,there was an elevation in the ROS level and a significant loss of mitochondrial membrane potential in the chondrocytes of mice.Compared with the H2O2 group,after treatment with HT 10,50 and 250 μmol·L-1,there were significant decreases in mRNA expression levels of IL-6,COX-2,PGE2,iNOS,MM P-3,MMP-13,ADAMTS-4 and ADAMTS-5,the mRNA expressions of SOX-9 and ACAN were increased,the ROS level was lowered.After treatment with HT 50 and 250 μmol L-1,the loss of mitochondrial membrane potential was ameliorated.Compared to the cell control group,the protein expressions of iNOS,COX-2 and MMP-13 were upregulated in the H2O2 group,while the protein expression of Col-2 was downregulated after 48 h.Compared to the H2O2 group,treatment with HT at concentrations of 10,50 and 250 μmol·L-1 resulted in decreased protein expressions of iNOS,COX-2 and MMP-13 in mouse chondrocytes,but the protein expression of Col-2 increased following treatment with HT 50 and 250 μmol L-1.CONCLUSION HT can ameliorate H2O2-induced chondrocyte injury by reducing intracellular ROS levels and alleviating the loss of mitochondrial membrane potential,suppressing the release of inflammatory cytokines,inhibiting catabolic processes,and promoting anabolic activities.

Objective·To investigate the inhibitory effects of spermidine(SPD)on inflammatory osteolysis both in vivo and in vitro.Methods·The CCK-8 assay was used to assess the viability of RAW264.7 macrophages treated with various concentrations of SPD.The levels of intracellular reactive oxygen species(ROS)in lipopolysaccharide(LPS)-activated RAW264.7 cells were evaluated by staining with dichlorodihydrofluorescein diacetate(DCFH-DA)and dihydroethidium(DHE),respectively.Reverse transcription real-time fluorescence quantitative PCR(RT-qPCR)was utilized to determine the effects of SPD on the expression of pro-inflammatory genes in LPS-activated RAW264.7 cells.Tartrate-resistant acid phosphatase(TRAP)staining was used to evaluate the effect of SPD on the differentiation of mouse primary bone marrow-derived macrophages(BMMs)into osteoclasts.RT-qPCR was employed to further analyze the effect of SPD on the expression of genes related to osteoclast differentiation and functions after BMM-induced differentiation.An LPS-induced mouse calvarial osteolysis model was constructed,and the therapeutic efficacy of SPD on inflammatory osteolysis was assessed using Micro-CT analysis,hematoxylin-eosin(H-E)staining and TRAP staining of histological sections.Results·The CCK-8 assay showed that SPD,even at a concentration of 1 000 μmol/L,exhibited no significant cytotoxicity to RAW264.7 cells.ROS analysis revealed that SPD markedly inhibited LPS-induced elevation of intracellular ROS levels in macrophages.RT-qPCR results indicated that SPD suppressed the expression of pro-inflammatory genes induced by LPS.Both TRAP staining and RT-qPCR demonstrated that SPD effectively inhibited the differentiation of BMMs into osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL)and reduced the expression of genes associated with osteoclast differentiation and function.In the mouse calvarial osteolysis model,Micro-CT analysis showed that the bone volume fraction and bone mineral density in the SPD-treated groups were significantly higher than those in the LPS group.Histological staining revealed that SPD treatment reduced inflammatory cell infiltration,decreased osteoclast numbers,and alleviated tissue damage.Conclusion·SPD inhibits macrophage inflammatory responses and RANKL-induced osteoclast differentiation in vitro;in vivo,it alleviates LPS-induced inflammatory calvarial osteolysis in mice.

This study aims to establish BHK-21 stable cell lines expressing APN from four species(human,pig,dog,and cat),the APN fragments were amplified from pEGFP-C1-APN plasmids of the four species stored in the laboratory to generate the recombinant plasmids pcDNA4.0-APN.Af-ter the recombinant plasmids were transfected into BHK-21 cells,the stable BHK-21 cell lines ex-pressing the APNs were selected by two rounds of limited dilution.The constructed BHK-21 cell lines were identified by indirect immunofluorescence assay(IFA),and their susceptibility to PD-CoV and TGEV was tested for these four cell lines.Virus infection experiments revealed that PD-CoV infected cells expressing human,pig,and dog APNs,while it did not infect cells expressing cat APN.Simultaneously,TGEV infected cells expressing pig,dog,and cat APNs,but did not infect cells expressing human APN.The results suggest that the risk of cross-species infection for different coronaviruses and the established cell line can be used effectively to evaluate the virus in-fection.The findings also revealed that PDCoV has the potential risk of cross-species infection of human and dog,and TGEV has the potential risk of cross-species infection of dog and cat.These results provide a basis for the prevention and control strategy of coronaviruses.

Through literature research,this study expounds the emergence of wholly foreign-owned hospitals(WFOHs)from both policy formulation and intrinsic demand perspectives.By accessing the current services landscape of WFOHs,the study systematically analyzes their developmental opportunities and challenges.Strategic recommendations are subsequently proposed across four key aspects:strengthening regulatory supervision,improving the medical insurance system,enhancing professional workforce development,and promoting Sino-foreign cooperation.These measures aim to facilitate WFOHs'high-quality develop-ment,thereby elevating institutional competitiveness,driving continuous healthcare service innovation,and ultimately delivering enhanced medical care that is efficient,accessible,and patient-centered.

Objective Predictive value of mismatch repair(MMR)protein expression in endometrial cancer(EC)tissues and blood immune-inflammatory marker levels on postoperative survival.Methods A total of 118 patients with EC who were treated at Jiamusi Central Hospital from January 2017 to January 2020 were selected as study subjects.The status of MMR proteins in endometrial cancer tissues was analyzed by immunohistochemistry,and based on the staining results,patients were divided into a MMR-deficient group(dMMR group,n=40)and a MMR-proficient group(pMMR group,n=78).The preoperative systemic immune-inflammation index(SII)was calculated.The pelvic lymph node status of the patients was observed.Enzyme-linked im-munosorbent assay(ELISA)was used to detect serum levels of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-6 and C-reactive protein(CRP)in the patients.Disease-free survival(DFS)and overall survival(OS)as well as their influencing factors were analyzed between the two groups.Results Comparison of age,FIGO stage,tumor histopathology,pelvic lymph node status and SII between the pMMR group and dMMR groups in patients,and the differences were statistically significant(Z/t/χ2=3.202～11.151,all P＜0.05).The average DFS of patients in pMMR group and dMMR group was 1 477 days and 756 days,the average OS was 1 588 and 826 days respectively,and the differences between the two groups were statistically significant(Log Rank χ2=24.804,30.411,all P<0.001).COX regression analysis showed that SII was an independent influencing factor of DFS in EC patients(Wald χ2=8.152,P<0.05).SII,pelvic lymph node status and MMR status were independent influencing factors of OS in EC patients(Wald χ2=7.066,5.936,6.971,all P<0.05).The ability of combining MMR status with SII to predict DFS and OS in EC patients was further improved,with AUC of 0.793 and 0.859,sensitivity of 72.4%and 75.0%,specificity of 78.3%and 80.0%,respectively.Conclusion MMR status and increased SII are independent influencing factors of poor postoperative prog-nosis of EC patients,and their combination is helpful to predict disease-free survival and overall survival of EC patients.

Objective Predictive value of mismatch repair(MMR)protein expression in endometrial cancer(EC)tissues and blood immune-inflammatory marker levels on postoperative survival.Methods A total of 118 patients with EC who were treated at Jiamusi Central Hospital from January 2017 to January 2020 were selected as study subjects.The status of MMR proteins in endometrial cancer tissues was analyzed by immunohistochemistry,and based on the staining results,patients were divided into a MMR-deficient group(dMMR group,n=40)and a MMR-proficient group(pMMR group,n=78).The preoperative systemic immune-inflammation index(SII)was calculated.The pelvic lymph node status of the patients was observed.Enzyme-linked im-munosorbent assay(ELISA)was used to detect serum levels of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-6 and C-reactive protein(CRP)in the patients.Disease-free survival(DFS)and overall survival(OS)as well as their influencing factors were analyzed between the two groups.Results Comparison of age,FIGO stage,tumor histopathology,pelvic lymph node status and SII between the pMMR group and dMMR groups in patients,and the differences were statistically significant(Z/t/χ2=3.202～11.151,all P＜0.05).The average DFS of patients in pMMR group and dMMR group was 1 477 days and 756 days,the average OS was 1 588 and 826 days respectively,and the differences between the two groups were statistically significant(Log Rank χ2=24.804,30.411,all P<0.001).COX regression analysis showed that SII was an independent influencing factor of DFS in EC patients(Wald χ2=8.152,P<0.05).SII,pelvic lymph node status and MMR status were independent influencing factors of OS in EC patients(Wald χ2=7.066,5.936,6.971,all P<0.05).The ability of combining MMR status with SII to predict DFS and OS in EC patients was further improved,with AUC of 0.793 and 0.859,sensitivity of 72.4%and 75.0%,specificity of 78.3%and 80.0%,respectively.Conclusion MMR status and increased SII are independent influencing factors of poor postoperative prog-nosis of EC patients,and their combination is helpful to predict disease-free survival and overall survival of EC patients.