Objectives:Aimed to establish a rapid, high-throughput, automated method for determining the creatine kinase (CK-MM) isoform levels.Methods:Magnetic beads labeled with anti-CK-MM antibodies were combined with alkaline phosphatase-based chemiluminescence detection. Clinical and diagnostic performance validation of the assay was determined by analysis of 998 and 75 dried blood spot samples from healthy newborns and Duchenne muscular dystrophy (DMD) patients, respectively, and the CK activity was also determined. The blank and detection limits, cross-reactivity, recovery rate of the method, intra-and inter-assay coefficient, and the hook effect were evaluated.Results:Blank and detection limits were 17.4 and 39.3 ng/ml, respectively. Cross-reactivity toward CK-MB and CK-BB isoforms was 0.2% and 0.02%, respectively. Intra-and inter-assay coefficients of variation were<1%. Mean recovery was 100.32%, with no hook effect in CK-MM levels<50 000 ng/ml. Overall, the mean CK-MM concentrations in newborns and DMD patients were (27.05±0.97) and (3 720±300.5) ng/ml, respectively. A significant positive correlation between the dried blood spot detected CK-MM levels and total CK enzyme activity, evaluated in corresponding serum samples from the 75 DMD patients, was observed ( r=0.91, P<0.001), ?which is in good agreement with the clinical. Conclusions:An assay for rapid quantitative determination of CK-MM that meets clinical newborn screening requirements was established. It had a good value for application.

Objective:To investigate the correlation between serum proprotein convertase subtilisin/Kexin type 9 (PCSK9) level and white matter hyperintensities (WMHs) in healthy population.Methods:Consecutive healthy individuals underwent routine physical examinations at the Department of Neurology, Jinling Hospital Affiliated to Medical School of Nanjing University (April 2023 to December 2023) and Hexi Branch of Nanjing First Hospital (March 2024 to April 2024) were included prospectively. Enzyme-linked immunosorbent assay was used to detect serum PCSK9 level. The Fazekas scale was used to assess the severity of WMHs (total score 0-6) and they were divided into no or mild WMHs group (0-2) and moderate to severe WMHs group (3-6). Multivariate logistic regression analysis was used to determine the independent correlation between the serum PCSK9 level and the severity of WMHs. Results:A total of 177 subjects were enrolled, including 110 males (62.1%), aged 66.7±10.1 years. The median serum PCSK9 level was 203.9 ng/L. According to the Fazekas score, there were 102 patients (51.6%) in the no or mild WMHs group, and 75 (42.4%) in the moderate to severe WMHs group. One way analysis of variance showed that serum PCSK9 level significantly increased with the increase of WMHs total score ( P=0.001). The serum PCSK9 level in the moderate to severe WMHs group was significantly higher than that in the no or mild WMHs group (437.2±260.4 ng/L vs. 217.9±141.7 ng/L; P=0.001). Multivariate logistic regression analysis showed that after adjusting for age, gender, and other confounding factors, there was a significant independent correlation between higher serum PCSK9 level and moderate to severe WMHs (odds ratio 3.201, 95% confidence interval 2.107-5.082; P=0.001). Conclusion:Higher serum PCSK9 level is an independent risk factor for moderate to severe WMHs in healthy individuals.

Objective:To investigate the efficacy and safety of dienogest (DNG) alone and gonadotropin-releasing hormone agonist (GnRH-a) combined with DNG sequential treatment to adenomyosis.Methods:The clinical data of 110 patients with adenomyosis attending the First Affiliated Hospital of Nanjing Medical University from December 2019 to March 2022 were retrospectively analyzed, including 40 patients treated with DNG (2 mg/day) alone (DNG group) and 70 patients treated with sequential DNG (2 mg/day) after 3-6 injections of GnRH-a (GnRH-a+DNG group). The clinical data before and after treatment were compared between the two groups.Results:(1) The dysmenorrhea visual analogue scale (VAS) scores, cancer antigen 125 (CA 125) and cancer antigen 19-9 (CA 19-9) levels at different time periods after treatment were significantly lower than before treatment in both groups (median before treatment: DNG group 70.0 mm, 68.55 kU/L, 22.45 kU/L respectively, GnRH-a+DNG group 80.0 mm, 151.50 kU/L, 20.44 kU/L respectively; all P<0.001). (2) The hemoglobin (Hb) levels of patients in both groups at different time periods after treatment were significantly higher than those before treatment (median: DNG group 102.00 g/L, GnRH-a+DNG group 94.00 g/L; all P<0.001). (3) Treatment with DNG alone did not have a significant effect on uterine volume in patients of DNG group ( P>0.05), and uterine volume decreased significantly in the 15th-24th months of GnRH-a+DNG group compared with that before treatment (median: 167.76 vs 227.77 cm 3; P<0.05). (4) There were no significant differences in hepatic and renal function and coagulation indexes between the two groups before and after treatment (all P>0.05), and no significant abnormal lesions were observed in breast tissue during the follow-up period. (5) The incidence of amenorrhea of GnRH-a+DNG group was higher than that of DNG group, and the incidences of irregular spotting bleeding and breakthrough hemorrhage were lower than those in DNG group. Conclusions:Whether DNG is used alone or in combination with GnRH-a in sequence, it could significantly relieve dysmenorrhea symptoms, improve the level of Hb, reduce the levels of CA 125 and CA 19-9 in patients with adenomyosis, with no adverse effects on coagulation and hepatic or renal function. GnRH-a sequential DNG therapy is superior to DNG alone in improving uterine bleeding patterns and controlling the growth of uterine volume in patients with adenomyosis.

Objective:To explore the different coagulation state in patients with adenomyosis and its clinical significance.Methods:Clinical data of the patients admitted to the First Affiliated Hospital of Nanjing Medical University from January 2017 to December 2021 were retrospectively analyzed. (1) Differential coagulation state between 25 healthy women and 25 patients with adenomyosis were compared during menstrual and non-menstrual periods. (2) The coagulation indexes of 145 patients with adenomyosis (observation group 1) and 129 patients with cervical intraepithelial neoplasia grade Ⅲ (control group 1) who underwent hysterectomy in non-menstrual period were compared. (3) The coagulation indexes of 154 patients with adenomyosis (observation group 2) and 147 women without myometrial lesions (control group 2) who underwent endometrial curettage during uterine bleeding period were compared. (4) Correlations of coagulation index with cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9) and uterine volume in patients with adenomyosis were analyzed. Results:(1) The coagulation state of each health women during the menstrual and non-menstrual period showed no significant differences (all P>0.05). For the 25 patients with adenomyosis, fibrinogen [FIB; 2.61 g/L(2.50-3.10 g/L)] and D-dimer [0.60 mg/L (0.40-1.00 mg/L)] in the menstrual period were significantly higher than those in the non-menstrual period [2.25 g/L (1.90-2.70 g/L) and 0.27 mg/L (0.20-0.40 mg/L), respectively; both P<0.01], while thrombin time [TT; 16.70 s (16.10-17.40 s)] in the menstrual period was significantly lower than that in the non-menstrual period [17.95 s (17.20-18.40 s); P<0.01]. (2) In the non-bleeding period, D-dimer [0.26 mg/L (0.20-0.40 mg/L)] and platelet count [257.0×10 9/L (212.0×10 9/L-308.5×10 9/L)] of observation group 1 were significantly higher than those of control group 1 (all P<0.01). Besides, FIB ( r=0.237, P=0.004) and D-dimer ( r=0.373, P<0.001) were positively correlated with CA 125, while prothrombin time (PT; r=-0.208, P=0.012) and internationalized normalized ratio of plasma prothrombin time (PT-INR; r=-0.201, P=0.015) were negatively correlated with CA 19-9. (3) In the bleeding period, PT [10.70 s (10.10-11.20 s)] and PT-INR [0.93 (0.90-1.00)] of observation group 2 were significantly lower than those of control group 2 (all P<0.01), while D-dimer [0.41 mg/L (0.20-0.80 mg/L)] was significantly higher than that in the control group 2 ( P<0.001). Furthermore, FIB ( r=0.252, P=0.038) and D-dimer ( r=0.321, P=0.008) were positively correlated with uterine volume, while activated partial thromboplastin time (APTT; r=-0.190, P=0.018) and TT ( r=-0.304, P=0.012) were negatively correlated with uterine volume. (4) During non-menstrual period and uterine bleeding period, APTT and TT in patients of observation group 1 and 2 combined with anemia were significantly lower than those of non-anemia patients (all P<0.05). Conclusion:Patients with adenomyosis have a tendency to hypercoagulability in both the uterine bleeding and non-bleeding periods, which may be related to enlarged uterine volume, increased serum CA 125 and anemia.

OBJECTIVE：To establish HPLC fingerprint of different products of suet oil-baked Epimedium brevicornum ，and to screen the optimal baking technology. METHODS ：HPLC method was adopted. Using icariin as reference ，HPLC fingerprints of 22 batches of samples were drawn. The similarity was evaluated by using Similarity Evaluation System of TCM Chromatographic Fingerprint（2012 edition），and common peaks were confirmed. HCA ，PCA and OPLS-DA analysis were performed by SIMCA 14.1 statistical software. Taking variable importance in the project ＞1 as criteria ，biomarkers affecting the quality difference of suet oil-baked E. brevicornum were screened ；using mass marker as index ，the baking technology was screened by baking technology. RESULTS：There were 18 common peaks in 22 batches of samples. The similarities were between 0.831 and 0.991. Totally 7 common peaks were identified ，i.e. epimedin A ，epimedin B ，epimedin C ，icariin，sagittatoside A ，sagittatoside B ，baohuoside Ⅰ. The 22 batches of samples were clustered into two categories ，S19-S22 were clustered into category Ⅰ and S 1-S18 were clustered into category Ⅱ. The category Ⅱ was sub-clustered into category Ⅱa（S15-S18），category Ⅱb（S10-S14），category Ⅱc （S1-S9）；the result of PCA analysis was consistent with above results. OPLS-DA showed that the biomarkers affecting the quality difference were icariin ，sagittatoside B and baohuoside Ⅰ. The results of kinetic studies showed that the content of icariin when baked at 180 ℃ for 25 min or 190 ℃ for 20 min，that of baohuoside Ⅰ when baked at 180 ℃ for 30 min or 190 ℃ for 15 min and that of epimedin B when baked at 210 ℃ for 18 min were the highest ；according to above results ，the optimal baking technology was baking at 180 ℃ for 25-30 min or 190 ℃ for 15-20 min. CONCLUSIONS ：Established fingerprint is stable ， reliable and reproducible. The multivariate statistical analysis can be used for the changes of chemical components in E. brevicornum under different baking condition and preliminary selection of baking technology.

Objective:To study the correlation between serum N-terminal pro-brain natriuretic peptide (NT-proBNP), homocysteine (Hcy) and SYNTAX score, and further clarify the prognostic significance of NT-proBNP and Hcy in patients with acute myocardial infarction (AMI).Methods:The clinical data of 303 patients with AMI from January 2014 to January 2016 in Lianjiang People′s Hospital of Guangdong Province were retrospectively analyzed. Patients were evaluated using version 2.11 of the SYNTAX score system. The SYNTAX score ≤ 22 scores was divided into the low risk group (103 cases), SYNTAX score 23 to 32 scores was divided into the medium risk group (85 cases), and the SYNTAX score ≥ 33 scores was divided into the high risk group (115 cases). The clinical data and laboratory examination results were collected. Univariate and multivariate Logistic regression analysis were used to analyze the influencing factors of SYNTAX score high risk. The optimal cutoff value of NT-proBNP and Hcy evaluating the SYNTAX score high risk was analyzed by receiver operating characteristic (ROC) curve. Kaplan-Meier survival curve was plotted to compare all-cause mortality of patients with different evaluation indicators. The influencing factors of prognosis in patients with AMI were analyzed by multivariate Cox risk regression analysis.Results:The patients were followed up for 0.1 to 4.5 (4.30 ± 0.76) years, The all-cause death was in 28 cases, and the all-cause mortality rate was 9.2% (28/303). There were statistical difference in age, smoking rate, diabetes mellitus rate, family history rate of coronary artery disease (CAD), left ventricular ejection fraction (LVEF), ST-elevation myocardial infarction (STEMI) rate, white blood cell, fasting blood glucose (FBG), estimation glomerular filtration rate (eGFR), troponin T (TnT), NT-proBNP and Hcy among the 3 groups ( P<0.01 or <0.05). SYNTAX score was positively correlated with NT-proBNP and Hcy ( r = 0.622 and 0.699, P<0.01). Multivariate Logistic regression analysis result showed that diabetes mellitus, LVEF, TnT, NT-proBNP and Hcy were the independent risk factors of SYNTAX score high risk in patients with AMI ( P<0.01 or <0.05). ROC curve analysis result showed that the area under curve (AUC) of NT-proBNP evaluating the SYNTAX score high risk was 0.807 (95% CI 0.757 to 0.850, P<0.05), the optimal cutoff value was 1 816 μg/L, with a sensitivity of 81.03% and a specificity of 82.26%; the AUC of Hcy evaluating the SYNTAX score high risk was 0.743 (95% CI 0.689 to 0.791, P<0.05), the optimal cutoff value was 17.55 μmol/L, with a sensitivity of 66.67% and a specificity of 79.84%. Kaplan-Meier survival curve analysis result showed that the all-cause mortality in high risk group was significantly higher than that in low risk group and medium risk group (17.4% vs. 2.9% and 5.9%), the all-cause mortality in patients with NT-proBNP> 1 816 μg/L was significantly higher than that in patients with NT-proBNP ≤ 1 816 μg/L (16.2% vs. 5.6%), the all-cause mortality in patients with Hcy>17.55 μmol/L was significantly higher than that in patients with Hcy ≤ 17.55 μmol/L (14.2% vs. 5.7%), and there were statistical differences ( P<0.01 or <0.05). Multivariate Cox risk regression analysis result showed that age, diabetes mellitus, SYNTAX score, TnT, NT-proBNP (>1 816 μg/L) and Hcy (>17.55 μmol/L) were the independent influencing factors of all-cause mortality in patients with AMI ( HR = 1.530, 1.368, 2.065, 1.414, 1.821 and 1.510; 95% CI 1.108 to 2.113, 1.012 to 3.485, 1.810 to 2.356, 1.248 to 2.714, 1.606 to 2.064 and 1.278 to 1.783; P<0.05 or <0.01). Conclusions:NT-proBNP>1 816 μg/L and Hcy>17.55 μmol/L can not only reflect the degree, severity and complexity of coronary atherosclerosis, but also predict the prognosis in patients with AMI.

OBJECTIVETo study polymorphisms of catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAO-B) genes among Chinese patients with Parkinson's disease.

METHODSGenotypes of the COMT and MAO-B genes of 1408 patients with Parkinson's disease was sequenced using Sanger method. And these patients were recruited by Chinese Parkinson Study Group from 29 research centers throughout the country.

RESULTSThe genotypic frequencies of COMT rs4680 AA, AG, GG were 8.9%, 42.0% and 49.1%. Those of rs4818 CC, CG, GG were 42.5%, 45.6% and 11.9%, respectively. The genotype frequencies of MAO-B rs1799836 A/AA, AG, G/GG were 74.4%, 14.1% and 11.5%, respectively. The haplotype formed by COMT rs4680 (GG) and MAO-B rs1799836 (A/AA) genotype has a frequency of 36.86%.

CONCLUSIONPolymorphisms of COMT and MAO-B genes has a unique characteristics among Chinese patients with Parkinson's disease. They may be related with differences in drug response in such patients.

Asian Continental Ancestry Group ; genetics ; Catechol O-Methyltransferase ; genetics ; Female ; Genotype ; Humans ; Male ; Monoamine Oxidase ; genetics ; Parkinson Disease ; genetics ; Polymorphism, Genetic

Objective To observe the influence of betahistine on the expression of histamine H3 receptor in the medial vestibular nucleus (MVN) following unilateral labyrinthectomy (UL).Methods Fifty-six healthy guinea pigs were randomly divided into three groups:the sham-operated group (group Ⅰ),the UL group [group Ⅱ,and UL + betahistine (BET) group (group Ⅲ)],BET was intraperitoneally injection at 2.17 mg · kg-1 · d-1 for 7 days.The expression of histamine H3 receptor was analyzed by immunohistochemistry at 1 day,3 days and 7 days after UL.Results H3 receptors were presented in the MVN and the expression of histamine H3 receptor were increased significantly in the ipsilateral MVN at 1 and 3 days after UL(P ＜0.05),the change turned into the normal value at 7 days(P ＞0.05).In the UL + BET group,the intensity of histamine H3 receptor was lower than that in the UL at 1 day and 3 days(4.25 ± 0.71,3.50 ± 0.92 vs 5.75 ± 0.71,5.50 ± 0.93,P ＜ 0.05).However,the changes turned into the normal values at 3 and 7 days (P ＞ 0.05).Conclusions The early stage of the vestibular compensation process may be associated with the change of H3 receptor expression in MVN.In the UL + BET group the histamine H3 receptor recovered quickly.

Objective:To study attachment and proliferation of human periodontal ligment cells(HPDLCs) on demineralized root surface of teeth with periodontitis following EDTA gel application. Methods:44 single-rooted teeth extracted because of severe periodontitis were treated by scaling and root planing(SRP). Then the fragments(5 mm?4 mm) of experimental surfaces were prepared. The fragments were randomly divided into four groups.The root surfaces in group 1,2,3 and 4 were treated by 240 g/L of EDTA gel for 2,3 and 4 min respectively or without treatment(control). Then 1?104 cells were seeded on the surface of each fragment and routinely cultured for 1,3 and 5 days respectively after seeding, the specimens were prepared for microscopic observasion, the cells on the root surfaces were counted. Moreover the other eight fragments were prepared for SEM observasion. Results:HPDLCs attached to and proliferated on all treated surfaces.Group 1, 2 and 3 presented significantly more cells on the root surfaces than the control group did at day 1,3 and 5. The cells on the root surfaces in group 2 and 3 were more than those in group 1 at day 1, 3 and 5. There was no evident difference between group 2 and 3 at any time. The root surfaces in group 2 and 3 had a fibrillar texture associated with an increased diameter of the exposed dentinal tubule openings.Smear layer were observed on root surfaces in group 1. Conclusion:EDTA gel(at 240 g/L) application may enhance attachment and proliferation on root surface of HPDLCs.