BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

ObjectiveTo explore the relationship between negative life events and depression severity in adolescent patients with depressive disorder， as well as the chain mediating role of insomnia symptoms and quality of life. Methods374 outpatient patients and hospitalized patients with adolescent depressive disorders were enrolled. The Adolescent Life Event Scale （ASLEC）， the Insomnia Severity Index （ISI）， the World Health Organization Quality of Life Questionnaire Short Form （WHOQOL-BREF）， and the Center for Epidemiology Depression Scale （CES-D） were used to evaluate the negative life event situation， insomnia symptoms， quality of life level and depression severity of the subjects， respectively. In addition， the PROCESS 4.0 macroprogram was used to analyze the chain mediating effect of insomnia symptoms and quality of life between negative life events and depression severity in patients with adolescent depressive disorder. ResultsThe results of correlation analysis showed that there was a significant correlation between negative life events and insomnia symptoms， quality of life， and depression severity （all P<0.05）. In addition， the results of chain mediation showed that negative life events had a significant direct effect on depression severity， with an effect size of 0.12 （P<0.001）. Insomnia symptoms and quality of life played a mediating role in the relationship between negative life events and depression severity in patients with adolescent depressive disorders， with indirect effect sizes of 0.062 （95%CI： 0.040-0.087） and 0.091 （95%CI： 0.059-0.123）， respectively. It could also play a chain mediation role， and the effect size was 0.039 （95%CI： 0.024-0.057）. ConclusionNegative life events experienced by patients with adolescent depressive disorder not only directly affect the severity of depressive symptoms， but may also indirectly exacerbate depression through insomnia symptoms and quality of life.

Objective: To establish a method for the genotyping of fetal M blood group antigen by extracting cell-free fetal DNA (cff-DNA) from maternal plasma, so as to guide the management of M antigen-negative pregnant women with IgG anti-M antibody during pregnancy. Methods: A realtime fluorescent quantitative PCR (realtime PCR) method was established. The specificity and sensitivity of the method were validated by dilution of genomic DNA. Subsequently, a total of 12 M antigen-negative pregnant women were enrolled. The cff-DNA was extracted from maternal plasma, and fetal M antigen genotyping was performed by realtime PCR. Fetuses were classified as M-positive or M-negative according to the presence or absence of amplification curve. The accuracy of the method was validated by comparing fetal M antigen genotyping results with the serological results using the cord or peripheral blood of the neonate at birth. Results: Among the 12 M antigen-negative pregnant women, anti-M was detected in five cases, of which four cases had IgG anti-M, and one case had fetal anemia. The results of fetal M antigen genotyping showed that 9 cases were M-positive (9/12, 75%) and 3 cases were M-negative (3/12, 25%). Serological results of blood samples collected after birth from four M-positive fetuses and one M-negative fetus were consistent with the genotyping results. Conclusion: We have, for the first time, established a non-invasive prenatal genotyping method for fetal M antigen using maternal plasma cff-DNA, and preliminarily demonstrated the feasibility of this method.

BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

Objective: To conduct screening for rare blood types within important blood group systems for the Chinese population, such as Rh, Duffy, Kidd, P1Pk, Diego, and MNS, in the Guangzhou region, and to establish a corresponding rare blood type database and physical repository. Methods: The saline medium microplate method was used to screen blood donors with the ccDEE phenotype combined with either Jk(a-) or Jk(b-). The polybrene microplate method was employed to screen for donors with Fy(a-), s(-), Lu(b-), Di(b-), k(-), and p phenotypes. The urea lysis microplate method was applied to screen for the Jk(a-b-) phenotype. A high-resolution melting (HRM) curve method was established for screening some donors with the Di(b-) phenotype. Subsequently, expanded phenotyping of antigens in the Rh, Kidd, MNS, Duffy, P1Pk, Lewis, Kell, and Lutheran blood group systems was performed on identified rare blood type donors using monoclonal antibodies. The test results are entered into the Rare Blood Type Bank Management System of the Guangzhou Blood Center, enabling functions such as confirmation reminders and cryopreservation storage when the donor donates again. Red blood cells of rare blood types are processed into frozen red blood cells for long-term storage. Results: Among voluntary blood donors, 16 cases of the ccDEE combined with Jk(a-) phenotype were identified (0.221 7%, 16/7 216); 10 cases of the ccDEE combined with Jk(b-) phenotype (0.138 6%, 10/7 216); 78 cases of the Fy(a-) phenotype (0.169 5%, 78/46 012); 39 cases of the Lu(b-) phenotype (0.138 2%, 39/28 214); 31 cases of the s(-) phenotype (0.081 8%, 31/37 913); 22 cases of the Di(b-) phenotype (0.029 9%, 22/73 691); 30 cases of the Jk(a-b-) phenotype (0.010 1%, 30/298 250); and 1 case of the k(-) phenotype (0.001 3%, 1/77 382), which was further identified as KELnull phenotype (K0). No p phenotype donors were identified (0/88 528). A total of 228 units of frozen red blood cells were prepared. The screening results were compared and analyzed with rare blood type data from other regions. Conclusion: This study, through a combination of different screening methods, significantly improved the efficiency of rare blood type screening while remaining cost-effective. By conducting large-scale screening and performing data informatization processing, a database and physical repository of rare blood types in the Guangzhou region were successfully established. This provides a strong guarantee for the timely supply of blood to patients with difficult-to-match and rare blood types in the region, effectively enhances the level of transfusion safety in the region, and offers a practical paradigm for constructing a comprehensive blood transfusion support system.

Objective:To construct a risk warning model for severe mycoplasma pneumoniae pneumonia(SMPP)children based on clinical data,laboratory indicators and imaging indicators.Methods:162 Mycoplasma pneumoniae pneumonia(MPP)children who were admitted in Foshan Women and Children Hospital from January 2021 to December 2023 were selected,64 SMPP children were included in severe group,the remaining 98 children were included in mild group.The general data,laboratory indicators and imaging indicators of the children were collected.The influencing factors for the occurrence of SMPP were analyzed by univariate and multivariate logistic regression models,and a risk warning model for the occurrence of SMPP children was constructed based on multivariate logistic regression model.The predictive value of the risk warning model for the occurrence of SMPP were analyzed by receiver operating characteristic(ROC)curve.Results:The proportion of 3 years old ≤ age＜6 years old,course of disease,body temperature,fever course,C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),lactate dehydrogenase(LDH),cyanosis of lips,positive triconcave sign,pleural effusion,lesion site was the lower lobe,abnormal electrocardiogram and extrapulmonary manifestations in severe group were significantly higher than those in mild group(P＜0.05),there were no significant differences in gender,white blood cell count(WBC),neutrophil ratio and procalcitonin(PCT)between the two groups(P＞0.05).Multivariate logistic regression analysis model showed that,3 years old ≤age＜6 years old,high body temperature,long fever course,CRP elevated,ESR elevated,LDH elevated,cyanosis of lips,positive triconcave sign,pleural effusion,lesion site was the lower lobe,abnormal electrocardiogram and extrapulmonary manifestations were risk factors for the occurrence of SMPP(P＜0.05).ROC curve analysis showed that,the area under the curve(AUC)of the risk warning model was 0.829,the sensitivity was 84.82％,and the specificity was 78.15％,the actual prediction curve of the risk warning model was in good agreement with the prediction curve,the decision curve showed that,the threshold probability range of the model was 4.61％～88.14％.Conclusion:The risk warning model based on clinical multi parameters such as general data,laboratory indicators and imaging indicators has certain predictive value for the occurrence of SMPP.

Objective By systematically reviewing the development of policies related to rural doctors and their evolution patterns,and by quantitatively analyzing the policy tools and their effectiveness levels,it aim to provide a strategic basis and reference for the high-quality development of China's rural doctor workforce.Methods 60 policies related to rural doctors enacted at the national level from March 2009 to May 2024 were collected,and a three-dimensional analysis framework based on"policy tool-policy period-policy effectiveness"was adopted to analyse the development of rural doctors in China.Results Among the 245 coded policy instruments,demand-type policy instruments accounted for the highest proportion (38.38％),followed by supply-type (31.02％) and environment-type is the least (30.61％).The types of policies are mainly circular,and the number of policies issued fluctuates significantly,with the number of policies issued during the period of high-quality development being the highest in the four periods,and the policies issued during the period of deepening and consolidation being the most specific.Conclusion It is necessary to scientifically grasp the law of evolution of policy orientation and the trend of evolution of policy instruments,rationally adjust the internal structure of policy instruments,and enhance the relevance and synergy of the policy system in order to improve the cohesion of policy effectiveness.

Aim To investigate the effects of m6A demethylase ALKBH5 on cardiomyocytes pyroptosis in mice with myocardial infarction(MI).Methods The MI model of left anterior descending coronary artery ligation surgery was established by knocking down ALKBH5 using adeno-associated virus,and the hypox-ia model of mouse cardiomyocytes(HL-1)was estab-lished by knocking down small interfering RNA.The effects of ALKBH5 on the pyroptosis of MI mice and hypoxic HL-1 cells were observed.Subsequently,mechanism studies were conducted at the cellular lev-el,and the binding of ALKBH5 and IGF2BP2 to NL-RP3 mRNA was detected through RNA pull down and RNA immunoprecipitation(RIP)experiments.The MeRIP-qPCR method was used to determine the effects of ALKBH5 on the mRNA m6A level of NLRP3.Acti-nomycin D for RNA stability experiments were conduc-ted to detect the effects of ALKBH5 and IGF2BP2 on the stability of NLRP3 mRNA.Results Knocking down ALKBH5 in vivo and in vitro both inhibited NL-RP3 inflammasome activation and alleviated pyroptosis in MI mice and hypoxic HL-1 cells.Mechanistically,the results showed that NLRP3 mRNA could bind to ALKBH5 protein in HL-1 cells;knocking down ALK-BH5 could increase the m6A level of NLRP3 and re-duce the stability of NLRP3 mRNA;subsequently,it was confirmed that NLRP3 mRNA and IGF2BP2 pro-tein bound to each other;knocking down IGF2BP2 in-creased the mRNA stability of NLRP3.The Rescue ex-periment showed that knocking down IGF2BP2 re-versed the decrease in NLRP3 mRNA expression caused by knocking down ALKBH5.Conclusions ALKBH5 mediated m6A modification of NLRP3 pro-motes cardiomyocytes pyroptosis in mice with myocardi-al infarction.

Aim To investigate the effects of m6A demethylase ALKBH5 on cardiomyocytes pyroptosis in mice with myocardial infarction(MI).Methods The MI model of left anterior descending coronary artery ligation surgery was established by knocking down ALKBH5 using adeno-associated virus,and the hypox-ia model of mouse cardiomyocytes(HL-1)was estab-lished by knocking down small interfering RNA.The effects of ALKBH5 on the pyroptosis of MI mice and hypoxic HL-1 cells were observed.Subsequently,mechanism studies were conducted at the cellular lev-el,and the binding of ALKBH5 and IGF2BP2 to NL-RP3 mRNA was detected through RNA pull down and RNA immunoprecipitation(RIP)experiments.The MeRIP-qPCR method was used to determine the effects of ALKBH5 on the mRNA m6A level of NLRP3.Acti-nomycin D for RNA stability experiments were conduc-ted to detect the effects of ALKBH5 and IGF2BP2 on the stability of NLRP3 mRNA.Results Knocking down ALKBH5 in vivo and in vitro both inhibited NL-RP3 inflammasome activation and alleviated pyroptosis in MI mice and hypoxic HL-1 cells.Mechanistically,the results showed that NLRP3 mRNA could bind to ALKBH5 protein in HL-1 cells;knocking down ALK-BH5 could increase the m6A level of NLRP3 and re-duce the stability of NLRP3 mRNA;subsequently,it was confirmed that NLRP3 mRNA and IGF2BP2 pro-tein bound to each other;knocking down IGF2BP2 in-creased the mRNA stability of NLRP3.The Rescue ex-periment showed that knocking down IGF2BP2 re-versed the decrease in NLRP3 mRNA expression caused by knocking down ALKBH5.Conclusions ALKBH5 mediated m6A modification of NLRP3 pro-motes cardiomyocytes pyroptosis in mice with myocardi-al infarction.

Objective:To construct a risk warning model for severe mycoplasma pneumoniae pneumonia(SMPP)children based on clinical data,laboratory indicators and imaging indicators.Methods:162 Mycoplasma pneumoniae pneumonia(MPP)children who were admitted in Foshan Women and Children Hospital from January 2021 to December 2023 were selected,64 SMPP children were included in severe group,the remaining 98 children were included in mild group.The general data,laboratory indicators and imaging indicators of the children were collected.The influencing factors for the occurrence of SMPP were analyzed by univariate and multivariate logistic regression models,and a risk warning model for the occurrence of SMPP children was constructed based on multivariate logistic regression model.The predictive value of the risk warning model for the occurrence of SMPP were analyzed by receiver operating characteristic(ROC)curve.Results:The proportion of 3 years old ≤ age＜6 years old,course of disease,body temperature,fever course,C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),lactate dehydrogenase(LDH),cyanosis of lips,positive triconcave sign,pleural effusion,lesion site was the lower lobe,abnormal electrocardiogram and extrapulmonary manifestations in severe group were significantly higher than those in mild group(P＜0.05),there were no significant differences in gender,white blood cell count(WBC),neutrophil ratio and procalcitonin(PCT)between the two groups(P＞0.05).Multivariate logistic regression analysis model showed that,3 years old ≤age＜6 years old,high body temperature,long fever course,CRP elevated,ESR elevated,LDH elevated,cyanosis of lips,positive triconcave sign,pleural effusion,lesion site was the lower lobe,abnormal electrocardiogram and extrapulmonary manifestations were risk factors for the occurrence of SMPP(P＜0.05).ROC curve analysis showed that,the area under the curve(AUC)of the risk warning model was 0.829,the sensitivity was 84.82％,and the specificity was 78.15％,the actual prediction curve of the risk warning model was in good agreement with the prediction curve,the decision curve showed that,the threshold probability range of the model was 4.61％～88.14％.Conclusion:The risk warning model based on clinical multi parameters such as general data,laboratory indicators and imaging indicators has certain predictive value for the occurrence of SMPP.