Objective Based on the"preventive treatment of disease"theory and real-world data,this study aimed to evaluate the clinical efficacy of Yangyin Shugan Formula in patients with ovarian dysfunction characterized by the syndrome of kidney deficiency and liver depression at different stages and to provide evidence for optimizing the timing of traditional Chinese medicine(TCM)in premature ovarian insufficiency(POI)management.Methods Ovarian dysfunction patients under 40 years old characterized by syndrome of kidney deficiency and liver depression,registered in the outpatient electronic medical records of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine from January 2020 to December 2023,were stratified into three groups:subclinical POI,POI,and premature ovarian failure(POF)groups.The therapeutic outcomes of the three groups of patients were compared,including TCM syndrome total response rates,menstrual total response rates/normalization(cured)rates,base follicle-stimulating hormone(FSH)total response rate/normalization rates and anti-Müllerian hormone(AMH)levels.Results A total of 307 patients meeting the research criteria were included(97 cases in subclinical POI group,112 cases in POI group,and 98 cases in POF group).The TCM syndrome total response rates ranked as subclinical POI group(98.97%,96/97)>POI group(95.54%,107/112)>POF group(83.67%,82/98),with the POF group was lower than that of the subclinical POI and POI groups(P<0.017);while the difference of the subclinical POI group with the POI group was not significant.Menstrual total response rates ranked as subclinical POI group(97.22%,70/72)>POI group(72.32%,81/112)>POF group(51.02%,50/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017);normalization rates ranked as subclinical POI group(90.28%,65/72)>POI group(54.46%,61/112)>POF group(26.53%,26/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017).FSH total response rates ranked as subclinical POI group(89.69%,87/97)>POI group(81.25%,91/112)>POF group(60.20%,59/98),while POF group was lower than that of the other two groups(P<0.017),and the difference of the subclinical POI group with the POI group was not significant.Normalization FSH rates ranked as subclinical POI group(71.13%,69/97)>POI group(53.57%,60/112)>POF group(16.33%,16/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017).Compared with before treatment,the FSH levels in all three groups decreased after treatment(P<0.01).The reduction of FSH in the POF and POI groups were better than that in the subclinical P OI group(P<0.017).No significant difference was observed in the reduction of FSH between the POI and POF groups.Post-treatment AMH elevation observed in the subclinical POI and POI groups(P<0.01).Conclusion Yangyin Shugan Formula improves the menstrual conditions and TCM syndromes across all the stages.It also improves elevated FSH levels across all the stages and has a specific effect on normalizing FSH and menstruation.Yangyin Shugan Formula increases the AMH levels of patients with subclinical POI and POI,highlighting the applicability of TCM in staged preventive management.

Objective:To analyze the epidemiological characteristics and genetic evolution of human coronavirus OC43（HCoV-OC43）in Hangzhou from 2018 to 2023，and provide molecular epidemiological basis for the prevention and control of HCoV-OC43 infection.Methods:A total of 11 904 samples of cases with acute respiratory tract infection were collected from two sentinel hospitals in Hangzhou. The collection period was from January 2018 to December 2023. HCoV-OC43 was detected by real-time fluorescent quantitative RT-PCR. The positive samples were amplified by PCR using specific primers，and the amplified products were sequenced using third-generation nanopore technology for the whole genome. The genetic evolution features were analyzed.Results:A total of 265 HCoV-OC43 positive samples were detected，with a positive detection rate of 2.23%（265/11 904）. There was no significant difference in detection rate between males and females（χ 2=0.042， P>0.05）；the detection rate varied significantly among different age groups（χ 2=60.104， P<0.01），with higher detection rates observed in children under five years old and individuals over 65 years old. The infection peaks in 2018-2019 and 2023 occurred in spring and summer，while the infection peak in 2020-2022 was in summer and autumn. A total of 85 complete HCoV-OC43 whole genome sequences were obtained through high-throughput sequencing. The evolutionary analysis indicated that the K genotype was predominant in Hangzhou from 2018 to 2023. In 2019，both J and K genotypes co-existed. In 2023，a new genotype，temporarily named genotype L，was discovered，which originated from genotypes J and K. The K genotype had 22 specific amino acid variation sites，most of which were located in the important domain of S protein. The J genotype had seven specific amino acid variation sites，most of which were located in non-structural proteins. The new genotype L had 10 specific amino acid mutation sites，which were distributed across six proteins，and these mutation sites had the most extensive coverage. Conclusions:HCoV-OC43 is an important pathogen causing respiratory diseases in children and the elderly in the Hangzhou area，and there are multiple genotypes in the Hangzhou area，with a new recombinant genotype L discovered in 2023.

OBJECTIVES: To investigate the regulatory effect of LINC00837/miR-671-5p/SERPINE2 functional axis on pathological processes of fibroblast-like synovial cells (FLS) in rheumatoid arthritis (RA). METHODS: RA-FLS were transfected with a LINC00837 overexpression plasmid (pcDNA3.1-LINC00837), a LINC00837 interference plasmid (siRNA-LINC00837), or their respective negative control plasmids (pcDNA3.1-NC and siRNA-NC). Dual luciferase was used to verify the targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. RT-qPCR was used to detect the expression levels of LINC00837, miR-671-5p and SERPINE2 in normal FLS or the transfected cells, whose proliferation and migration abilities were assessed using Edu assay and scratch healing assay and by detecting the expression levels of Ki-67, PCNA, E-cadherin and N-cadherin with Western blotting. The changes in cellular secretion of the inflammatory cytokines (TNF‑α, IL-17, IL-4 and IL-10) were examined using ELISA. RESULTS: Dual luciferase reporter gene assay showed that LINC00837 was capable of binding to the 3'-UTR of miR-671-5p, and the latter bound to the 3-UTR of SERPINE2 at specific binding sites between them. Compared with normal FLS, RA-FLS showed significantly increased expressions of LINC00837 and SERPINE2, lowered miR-671-5p expression and enhanced proliferation and migration abilities with increased expressions of pro-inflammatory cytokines and reduced expressions of anti-inflammatory cytokines. Transfection of RA-FLS with pcDNA-LINC00837 further enhanced cell proliferation and migration and the changes in the inflammatory cytokines, while transfection with si-LINC00837 produced the opposite changes. CONCLUSIONS RA-FLS have a LINC00837/miR-671-5p/SERPINE2 functional axis, which regulates cell proliferation, migration and secretion of inflammatory factors, and interventions targeting LINC00837 may provide a potential strategy to regulate the pathological processes in RA-FLS.
Arthritis, Rheumatoid/metabolism* ; MicroRNAs/metabolism* ; Humans ; Cell Proliferation ; Cell Movement ; Synovial Membrane/pathology* ; RNA, Long Noncoding/genetics* ; Fibroblasts/metabolism* ; Synoviocytes/metabolism* ; Cells, Cultured ; Transfection

Objective Based on the"preventive treatment of disease"theory and real-world data,this study aimed to evaluate the clinical efficacy of Yangyin Shugan Formula in patients with ovarian dysfunction characterized by the syndrome of kidney deficiency and liver depression at different stages and to provide evidence for optimizing the timing of traditional Chinese medicine(TCM)in premature ovarian insufficiency(POI)management.Methods Ovarian dysfunction patients under 40 years old characterized by syndrome of kidney deficiency and liver depression,registered in the outpatient electronic medical records of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine from January 2020 to December 2023,were stratified into three groups:subclinical POI,POI,and premature ovarian failure(POF)groups.The therapeutic outcomes of the three groups of patients were compared,including TCM syndrome total response rates,menstrual total response rates/normalization(cured)rates,base follicle-stimulating hormone(FSH)total response rate/normalization rates and anti-Müllerian hormone(AMH)levels.Results A total of 307 patients meeting the research criteria were included(97 cases in subclinical POI group,112 cases in POI group,and 98 cases in POF group).The TCM syndrome total response rates ranked as subclinical POI group(98.97%,96/97)>POI group(95.54%,107/112)>POF group(83.67%,82/98),with the POF group was lower than that of the subclinical POI and POI groups(P<0.017);while the difference of the subclinical POI group with the POI group was not significant.Menstrual total response rates ranked as subclinical POI group(97.22%,70/72)>POI group(72.32%,81/112)>POF group(51.02%,50/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017);normalization rates ranked as subclinical POI group(90.28%,65/72)>POI group(54.46%,61/112)>POF group(26.53%,26/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017).FSH total response rates ranked as subclinical POI group(89.69%,87/97)>POI group(81.25%,91/112)>POF group(60.20%,59/98),while POF group was lower than that of the other two groups(P<0.017),and the difference of the subclinical POI group with the POI group was not significant.Normalization FSH rates ranked as subclinical POI group(71.13%,69/97)>POI group(53.57%,60/112)>POF group(16.33%,16/98),the differences among the three groups and in the post-hoc comparisons were significant(P<0.01,P<0.017).Compared with before treatment,the FSH levels in all three groups decreased after treatment(P<0.01).The reduction of FSH in the POF and POI groups were better than that in the subclinical P OI group(P<0.017).No significant difference was observed in the reduction of FSH between the POI and POF groups.Post-treatment AMH elevation observed in the subclinical POI and POI groups(P<0.01).Conclusion Yangyin Shugan Formula improves the menstrual conditions and TCM syndromes across all the stages.It also improves elevated FSH levels across all the stages and has a specific effect on normalizing FSH and menstruation.Yangyin Shugan Formula increases the AMH levels of patients with subclinical POI and POI,highlighting the applicability of TCM in staged preventive management.

Objective:To analyze the epidemiological characteristics and genetic evolution of human coronavirus OC43（HCoV-OC43）in Hangzhou from 2018 to 2023，and provide molecular epidemiological basis for the prevention and control of HCoV-OC43 infection.Methods:A total of 11 904 samples of cases with acute respiratory tract infection were collected from two sentinel hospitals in Hangzhou. The collection period was from January 2018 to December 2023. HCoV-OC43 was detected by real-time fluorescent quantitative RT-PCR. The positive samples were amplified by PCR using specific primers，and the amplified products were sequenced using third-generation nanopore technology for the whole genome. The genetic evolution features were analyzed.Results:A total of 265 HCoV-OC43 positive samples were detected，with a positive detection rate of 2.23%（265/11 904）. There was no significant difference in detection rate between males and females（χ 2=0.042， P>0.05）；the detection rate varied significantly among different age groups（χ 2=60.104， P<0.01），with higher detection rates observed in children under five years old and individuals over 65 years old. The infection peaks in 2018-2019 and 2023 occurred in spring and summer，while the infection peak in 2020-2022 was in summer and autumn. A total of 85 complete HCoV-OC43 whole genome sequences were obtained through high-throughput sequencing. The evolutionary analysis indicated that the K genotype was predominant in Hangzhou from 2018 to 2023. In 2019，both J and K genotypes co-existed. In 2023，a new genotype，temporarily named genotype L，was discovered，which originated from genotypes J and K. The K genotype had 22 specific amino acid variation sites，most of which were located in the important domain of S protein. The J genotype had seven specific amino acid variation sites，most of which were located in non-structural proteins. The new genotype L had 10 specific amino acid mutation sites，which were distributed across six proteins，and these mutation sites had the most extensive coverage. Conclusions:HCoV-OC43 is an important pathogen causing respiratory diseases in children and the elderly in the Hangzhou area，and there are multiple genotypes in the Hangzhou area，with a new recombinant genotype L discovered in 2023.

Objective:To investigate the characteristics of cortical morphology in children with attention defi-cit hyperactivity disorder(ADHD)and those with oppositional defiant disorder(ODD)from both categorical and dimensional analyses.Methods:A total of 72 children were enrolled,including 16 children with ADHD and ODD,20 children with ADHD without ODD,and 36 age-gender-matched normal children.The diagnoses were made ac-cording to the Diagnostic and Statistical Manual of Mental Disorders,Fourth Edition(DSM-Ⅳ)criteria.The Chi-nese Wechsler Intelligence Scale for Children(C-WISC)was used to access intelligence quotient.All subjects par-ticipated in the magnetic resonance imaging(MRI)scan.The features of cortical morphology were determined using FreeSurfer software.Results:Children with ADHD and ODD[(6 528.1±857.5)mm3 vs.(7 591.2±657.3)]and children with ADHD only[(6 867.2±41.3)mm3 vs.(7 591.2±657.3)mm3]had smaller volume in the left later-al superior temporal gyrus(P＜0.05)than controls.No difference was found between ADHD with ODD group and ADHD without ODD group.There was no correlation between the cortical volume of left lateral superior temporal gyrus and ODD symptoms.Conclusion:The reduced cortical volume of left lateral superior temporal gyrus may be an important indication of the abnormal brain structure of ADHD in children.And comorbid status of ODD dose not change this structural variation.

Objective:To investigate the co-infection and phylogenetic analysis of human rhinovirus (HRV) in 2019 novel coronavirus (2019-nCoV) positive samples.Methods:Ten common respiratory viruses, including HRV were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR) in 7 213 samples of 2019-nCoV positive cases and the co-infection characteristics were analyzed. The VP4/VP2 gene fragment of HRV was amplified and sequenced.Phylogenetic trees were constructed.Results:HRV accounted for 1.34% of the 2019-nCoV positive samples (97/7 213), followed by common coronavirus (0.50%, 36/7 213). The co-infection rate of HRV in 2019-nCoV positive samples was significantly different from that of other viruses ( χ2=318.09, P<0.001). There was significant difference in HRV co-infection rate among different age groups ( χ2=36.77, P<0.001), the peak was in<18 years age group. The co-infection rate of HRV had no significant difference in different seasons. The VP4/VP2 gene fragments of 39 HRV strains (40.21%, 39/97) were successfully sequenced and made phylogenetic analysis. There were 10 strains of HRV-A, 9 strains of HRV-B and 20 strains of HRV-C. Seventeen subtypes were identified, of which B6 (66.67%, 6/9) and C15 (70%, 14/20) were the most prevalent and other subtypes were scattered. Conclusions:The co-infection rate of HRV in patients with 2019-nCoV infection was the highest. The highest co-infection rate was in<18 years age group. Group A, B, and C of HRV were found in 2019-nCoV positive samples, and serotypes present diversity.

Objective To investigate the relationship between nestin expression and microvessel density(MVD)and its value as a potential prognostic marker of glioblastoma(GBM).Methods A retrospective trial was conducted on 50 GBM patients undergoing craniotomy under general an-esthesia in the Third Affiliated Hospital of Chongqing Medical University from March 2020 to March 2023.According to the median nestin H score(Nestin staining intensity × percentage of positive cells),they were divided into H score ≥80.0 group(28 cases)and H score＜80.0 group(22 cases).After 2 years of follow-up,they were also divided into a death group(23 cases)and a survival group(27 cases).The expression of nestin was determined by immunohistochemical staining,and MVD was quantified by von Willebrand factor(vWF)and CD105 immunohistochem-ical staining.Spearman rank test was used to analyze the correlation between nestin expression level and MVD formation,multivariate Cox proportional hazards regression analysis was em-ployed to identify the influencing factors for overall survival in the GBM patients,and Kaplan-Meier survival curve was plotted to analyze the 2-year survival rate of the patients.Results The nestin H score was positively correlated with vWF-MVD(Rho=0.701,P＜0.01)and CD105-MVD(Rho=0.753,P＜0.01).There were no significant differences between the H score ≥80.0 group and the H score＜80.0 group in terms of gender,age,isocitric dehydrogenase-1 mutation,tumor site,tumor location,brain necrosis,tumor volume,edema volume,preoperative Karnofsky Performance Status score,and extent of tumor resection(P＞0.05).The survival group had larger proportions of isocitrate dehydrogenase-1 mutation and maximal resection,and lower nestin H score than the death group(P＜0.05,P＜0.01).Cox proportional hazards regression showed that partial resection/biopsy(HR=4.781,95％CI:2.066-11.060,P=0.001)and increased nestin H score(HR=1.007,95％CI:1.001-1.013,P=0.032)were independent influencing factors for worsening overall survival in the GBM patients.Kaplan-Meier survival curve analysis indicated that the cumulative survival rate was lower in the H score ≥80.0 group than the H score＜80.0 group(log rank x2=8.147,P=0.004).Conclusion Nestin overexpression is associated with poor prognosis in GBM patients,indicating more microangiogenesis,which may be a therapeutic target for GBM patients.

Objective:To investigate the role of CD8 + T cells in lethal Plasmodium yoelii 17XL ( Py 17XL) infection. Methods:BALB/c mice and C57BL/6 mice were infected with Py 17XL-infected red blood cells (1×10 6 cells/0.1 ml) through intraperitoneal injection to establish the mouse models of Py 17XL infection. Parasitemia (the percentage of erythrocytes infected with Py 17XL) and the survival rates of the mice was observed dynamically. Flow cytometry was used to detect the number of effector T cells (T EFF) and central memory T cells (T CM) of CD8 + T cell subpopulations, the expression of IFN-γ and granzyme B (GB) levels, and the expression of surface chemokine receptors CXCR3, CXCR6 and CX3CR1. FTY720 blocking experiment was conducted on Py 17XL-infected C57BL/6 mice to analyze the impact of CD8 + T cell migration on Py 17XL infection. Results:The parasitemia of BALB/c mice increased rapidly 5 d after infection and reached the peak on 8 d [(79.57±3.82)%]. Besides, the parasitemia was higher in BALB/c mice than in C57BL/6 mice 5-8 d after infection ( P<0.000 1). All BALB/c mice died on 9 d. The parasitemia of C57BL/6 mice reached the peak on 14 d [(48.19±3.19)%] and then decreased to 0 on 26 d. There was statistically significant difference in the survival rate between the two groups ( P<0.000 1). Flow cytometry results showed that compared with the BALB/c mice, the absolute number of CD8 + T cells in spleen and liver tissues and the number of CD8 + T EFF and CD8 + T CM cells in spleen and lymph nodes of C57BL/6 mice increased significantly ( P<0.05). Compared with the BALB/c mice, the levels of GB, IFN-γ and chemokines expressed by CD8 + T cells in spleen and liver tissues of C57BL/6 mice increased significantly ( P<0.05). The FTY720 blocking experiment showed that the survival rate, the absolute number of CD8 + T cells in liver and spleen, and the number of CD8 + CXCR3 + T cells decreased significantly in the experimental group ( P<0.05). Conclusions:CD8 + T EFF and CD8 + T CM cells contribute to resistance against Py 17XL infection by secreting GB and IFN-γ. The chemokine receptor CXCR3 plays an important role in mediating the chemotaxis of CD8 + T cells to spleen and liver.

Objective To establish a reference interval for the pepsinogen(PG)Ⅰ,PGⅡ and PGⅠ/PGⅡ in healthy adult subjects in Zhejiang Province.Methods The data of 45 504 healthy adult subjects were collected,and the levels of serum PGⅠ and PG Ⅱ were detected by chemiluminescence microparticle immunoassay.The reference range of PGs were determined according to the CLSI-C28-A3 file.Results The median serum PGⅠ concentration in male was 132.62μg/L,PGⅡ concentration was 8.10μg/L,and PGⅠ/PGⅡ was 15.9.For females,they were 107.44μg/L,6.96μg/L and 15.0,respectively.PGI,PGⅡ concentration and PGⅠ/PGⅡ ratio were significantly higher in males than in females(P<0.001).The levels of PGⅠ and PGⅡ increased with age(P<0.001).Serum PGⅠ reference interval for male:59.79-234.97μg/L for 19-39 years old,63.33-294.62μg/L for 40-59 years old,64.25-333.61μg/L for≥60 years old,PGⅡ reference interval:3.33-22.60μg/L for 19-39 years of age,3.79-33.89μg/L for 40-59 years of age,4.15-42.08μg/L for≥60 years of age,PGⅠ/PGⅡ reference interval:those aged 19-39 years ranged from 7.3 to 31.4,those aged 40-59 years ranged from 5.8 to 30.9,and those aged≥60 years ranged from 3.9 to 30.7.The reference intervals of female PGⅠ were 48.79-215.68μg/L,52.10-276.01μg/L,and 64.34-317.20μg/L,respectively.The reference intervals of female PgⅡwere:2.87-23.93μg/L,3.41-33.31μg/L and 3.88-39.16μg/L,respectively.The reference intervals of of female PGⅠ/PGⅡ are 6.6-28.1,5.2-27.9 and 3.6-26.2.Conclusion This study determined the reference range of serum PGs deficiency in healthy subjects of different sex and age in Zhejiang Province.