Objective To gain insights into the biological characteristics of different populations of Culex tritaeniorhynchus within and around the Yellow Sea wetland from the perspective of the occurrence of resistance, we investigated the levels of resistance to deltamethrin and kdr gene mutation in the wetland and its peripheral areas. Methods Specimens were collected from Cx. tritaeniorhynchus populations at two monitoring sites in the Rare Bird National Nature Reserve and Tiaozi Ni Wetland Scenic Area, and also from two populations in Yancheng City and the Liuhe District of Nanjing, and the resistance of these mosquitoes to deltamethrin was determined using the CDC biotest bottle method. For each concentration of deltamethrin assessed, a random subset of exposed specimens was selected for amplification of the kdr gene fragment, followed by Sanger sequencing to identify and analyze resistance-associated mutations. Results The LC50 levels of deltamethrin among mosquitoes from the four populations in Luhe, Yancheng, the Rare Bird National Nature Reserve and the Tiaozi Ni Wetland Scenic Area were 2.048 5, 7.798 2, 3.473 3, and 17.695 5 mg/mL, respectively, with corresponding concentrations of deltamethrin ranging from 0.005 to 5.000,0.050 to 50.000,0.050 to 25.000 and 0.050 to 50.000 mg/mL, respectively. Furthermore, the ranges of the KT50 values were 11.76-107.43, 67.05-216.30,29.77-107.43 and 28.40-329.51 min; the 1-h knockdown rates were 34.58%-99.15%, 9.52%-43.80%, 55.09%-73.01%, and 10.09%-68.07%; and the 24-h mortality rates were 12.15%-67.52%,9.52%-79.56%,13.17%-82.21%, and 11.01%-78.99%, respectively. With respect to kdr gene mutation, we assayed a total of 63,70,59, and 57 mosquitoes for the four populations, for which we detected L1014F mutation frequencies of 14.29%, 35.00%, 20.34%, and 31.58%, respectively, with a majority of these mutations being heterozygous for resistance. In addition, five adult mosquitoes were identified has having synonymous mutations at site 1011[i. e. , AAT(asparagine)mutation to AAC(asparagine)]. Conclusions Our findings revealed the clear resistance of Cx. tritaeniorhynchus to deltamethrin in the Yancheng region of the Yellow Sea wetland, and the resistance phenotype and kdr frequency of Cx. tritaeniorhynchus in the wetland environment were comparable to those of Cx. tritaeniorhynchus in the wetland environment, thereby indicating that the resistance of different populations of Cx. tritaeniorhynchus was homogeneous under the pressure of different insecticide selection within and around the wetland. However, the underlying mechanisms need to be further studied.

OBJECTIVE: EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen, an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome. Its composition, anticoagulant and fibrinolytic activities, and relevant mechanisms have been confirmed through in vitro experiments. However, whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown. This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro. METHODS: The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl₃. Then, the protein bands of EPF3 incubated with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and homogenate of Caco-2 cells (HC2C) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability. Finally, the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer. RESULTS: EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid. It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl₃. EPF3 was stable in SIF and HC2C and unstable in SGF. The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent. The efflux ratio was less than 1.2 during transport, and the transport behavior was not affected by inhibitors. EPF3 could reversibly reduce the expression of tight junction-related proteins, including zonula occludens-1, occludin, and claudin-1 in Caco-2 cells. CONCLUSION EPF3 could play a thrombolytic and antithrombotic role in zebrafish. It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction. This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation. Please cite this article as: Zhong WL, Yang JQ, Liu H, Wu YL, Shen HJ, Li PY, Du SY. Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong (Pheretima vulgaris Chen) and its transport mechanism in Caco-2 monolayer through cell bypass pathway. J Integr Med. 2025; 23(4): 415-428.
Animals ; Zebrafish ; Humans ; Caco-2 Cells ; Fibrinolytic Agents/pharmacology* ; Thrombosis/drug therapy* ; Intestinal Absorption

Objective:To evaluate the application of modular teaching program in the clinical competence training for community general practitioners.Methods:This was a cross-sectional study. A 140 teaching hour-modular training program was designed by the authors on the basis of the previous training experience and conducted in clinical competence training for 20 general practitioners (study group) from all 10 community health service centers in Shanghai Huangpu district between September 2022 and April 2023; and 26 general practitioners who planned to participate in the next batch of training courses were enrolled as the control group. The clinical competence assessment was conducted in May 2023, including knowledge level tests and clinical skill assessment. By comparing the scores of clinical competence assessment and satisfaction survey between two groups, the training effectiveness was evaluated.Results:Among the 46 general practitioners, 33 (71.7%) were female, aged 27 to 47 (37±6), working in general practice for 2 to 25 (9.4±5.5) years, 31 (67.4%) had participated in standardized residency training for general practice and 31 (67.4%) were contracted family doctors. The total score of 46 general practitioners was 69.7±9.6, and 68.9±8.6 for knowledge level and 69.8±10.6 for clinical skills. The total scores and single scores of trained participants were all significantly higher than those of untrained(all P<0.05). The multiple linear regression model showed that after controlling for gender, age, educational background, professional title, years of working in general practice, participation in standardized residency training, and status as a contracted family doctor, the trained participants had significantly higher scores in history collection, pediatric reception, indwelling gastric tube and abdominal puncture (all P<0.05). There was no statistically significant difference in the scores in knowledge level test, nervous system physical examination, SOAP medical record writing, and tutorium between the trained and untrained groups (all P>0.05). Among 20 general practitioners who completed training, the overall satisfaction score of the training program was 4.3±0.7 points, including 4.3±0.6 for the training course, 4.3±0.7 for teachers, and 4.3±0.7 for the organization and arrangement; 85% of the participants considered the training to be very helpful or helpful for improving their competence, and 90% considered the training to be very helpful or helpful for their future work. Conclusion:Modular teaching program is helpful to improve the clinical competence of community general practitioners, especially in clinical skills.

Objective To establish a renal cell co-culture system to simulate the renal barrier system,and to test its responsiveness to different glucose concentrations,and to investigate the regulatory effect of miR-29b-3p on osteopontin(OPN)/transforming growth factor β(TGF-β)pathway and the changes of this pathway under high glucose condition.Methods The three-cell co-culture system consisting of human renal podocytes,human glomerular mesangial cells and human renal tubular epithelial cells was established to test the cell viability and glucose consumption value at glucose concentrations of 5,8,12 and 16 mmol/L.The content of TGF-β and OPN in cell supernatant was measured.The recombinant plasmid and siRNA of OPN were transfected,and the expressions of TGF-β and OPN were detected by Q-PCR and Western blot.Results The mRNA expressions of OPN,TGF-β and miR-29b were significantly increased at 12 mmol/L glucose conditions.Western blot results showed that the protein expression of OPN increased in high glucose conditions,while the protein expression of TGF-β did not change significantly.After adding miR-29b-3p activator,the mRNA levels of OPN and TGF-β in the cell supernatant were significantly increased.After adding miR-29b-3p inhibitor,the mRNA levels of OPN and TGF-β in the cell supernatant were significantly decreased.Western blot results showed that compared with 5 mmol/L glucose,the protein expressions of OPN and TGF-β were increased by miR-29b-3p activator,and the protein expressions of OPN and TGF-β were decreased by miR-29b-3p inhibitor.After transfection with OPN recombinant plasmid,the content of TGF-β in the cell supernatant was significantly increased,and the mRNA expressions of OPN and TGF-β in the cells were significantly increased.After transfection with OPN siRNA,the content of TGF-β in the cell supernatant was decreased,and the expression of OPN mRNA in the cells was significantly decreased,but the expression of TGF-β mRNA was not significantly increased.Conclusion The renal cell co-culture system can mimic the complex renal environment in vivo.When induced by high glucose,cell proliferation is inhibited,glucose consumption is increased,and the content of TGF-β in the cell supernatant is increased,and miR-29b-3p has a regulatory effect on OPN/TGF-β signaling pathway in the co-culture system.

Objective:To compare the breadth of condition coverage, accuracy of suggested conditions and appropriateness of urgency advice of the 8 symptom assessment mobile applications (APPs) available on the Chinese market.Methods:The APPs were assessed using 200 primary care vignettes and were measured against the vignettes′ standard. The primary outcome measures were proportion of conditions covered by an APP, proportion of vignettes with the correct primary diagnosis,and proportion of safe urgency advice.Results:For APPs assessed,condition-coverage was from 29.0%(58/200)to 99.5%(199/200), top-3 suggestion accuracy was from 8.5%(17/200) to 61.5%(123/200), the proportion of safe urgency advice was from 84.8%(167/197) to 99.5% (198/199).Conclusions:The APPs showed a wide range of coverage, safety performance and condition-suggestion accuracy. Symptom assessment APPs with good performance could be used by general practitioners as supporting tools. However, even symptom assessment APPs with excellent performance need to be further assessed in a real clinical environment.

Objective: To evaluate the long-term efficacy and safety of the implantable ventricular assist system EVAHEART I in clinical use. Methods: Fifteen consecutive patients with end-stage heart failure who received left ventricular assist device therapy in Fuwai Hospital from January 2018 to December 2021 were enrolled in this study, their clinical data were retrospectively analyzed. Cardiac function, liver and kidney function, New York Heart Association (NYHA) classification, 6-minute walk distance and quality of life were evaluated before implantation and at 1, 6, 12, 24 and 36 months after device implantation. Drive cable infection, hemolysis, cerebrovascular events, mechanical failure, abnormally high-power consumption and abnormal pump flow were recorded during follow up. Results: All 15 patients were male, mean average age was (43.0±7.5) years, including 11 cases of dilated cardiomyopathy, 2 cases of ischemic cardiomyopathy, and 2 cases of valvular heart disease. All patients were hemodynamically stable on more than one intravenous vasoactive drugs, and 3 patients were supported by preoperative intra aortic balloon pump (IABP). Compared with before device implantation, left ventricular end-diastolic dimension (LVEDD) was significantly decreased ((80.93±6.69) mm vs. (63.73±6.31) mm, P<0.05), brain natriuretic peptide (BNP), total bilirubin and creatinine were also significantly decreased ((3 544.85±1 723.77) ng/L vs. (770.80±406.39) ng/L; (21.28±10.51) μmol/L vs. (17.39±7.68) μmol/L; (95.82±34.88) μmol/L vs. (77.32±43.81) μmol/L; P<0.05) at 1 week after device implantation. All patients in this group were in NYHA class Ⅳ before implantation, and 9 patients could recover to NYHA class Ⅲ, 3 to class Ⅱ, and 3 to class Ⅰ at 1 month after operation. All patients recovered to class Ⅰ-Ⅱ at 6 months after operation. The 6-minute walk distance, total quality of life and visual analogue scale were significantly increased and improved at 1 month after implantation compared with those before operation (P<0.05). All patients were implanted with EVAHEART I at speeds between 1 700-1 950 rpm, flow rates between 3.2-4.5 L/min, power consumption of 3-9 W. The 1-year, 2-year, and 3-year survival rates were 100%, 87%, and 80%, respectively. Three patients died of multiple organ failure at 412, 610, and 872 d after surgery, respectively. During long-term device carrying, 3 patients developed drive cable infection on 170, 220, and 475 d after surgery, respectively, and were cured by dressing change. One patient underwent heart transplantation at 155 d after surgery due to bacteremia. Three patients developed transient ischemic attack and 1 patient developed hemorrhagic stroke events, all cured without sequelae. Conclusion: EVAHEART I implantable left heart assist system can effectively treat critically ill patients with end-stage heart failure, can be carried for long-term life and significantly improve the survival rate, with clear clinical efficacy.
Humans ; Male ; Adult ; Middle Aged ; Female ; Heart Failure/complications* ; Follow-Up Studies ; Retrospective Studies ; Heart-Assist Devices ; Quality of Life

Objective: To examine the application value of 3D Slicer software assisted domestic frameless stereotactic robot in biopsy of intracranial lesions. Methods: A retrospective analysis was performed on 80 patients who admitted consecutively and underwent intracerebral lesions biopsy with the domestic frameless stereotactic robot at Department of Neurosurgery, Aerospace Central Hospital from January 2019 to December 2021. There were 36 males and 44 females, with a mean age of (38.5±18.0) years (range: 6 to 71 years). Before surgery only enhanced T1-weighted three-dimensional magnetization prepared gradient echo sequences and diffusion tensor imaging scans were performed. Self-reconstruction of intracranial lesions, cerebral cortex and blood vessels was carried out using 3D Slicer software system after the DICOM format imaging data of 80 patients were collected. These imaging data were merged to the workstation of the domestic frameless stereotactic robot for preoperative surgical planning and the surgical puncture path was designed to avoid blood vessels in the brain functional area, cerebral cortex and sulcus. Results: All frameless stereotactic biopsy were successfully performed. Postoperative pathological diagnosis included 50 cases of diffuse astrocytic and oligodendroglioma, 15 cases of lymphoma, 5 cases of metastatic tumors, 5 cases of inflammatory demyelinating disease, 2 cases of inflammatory granuloma, 1 case of hemangioma, 1 case of acute lymphoblastic leukemia intracranial invasion and 1 case of seminoma. The positive diagnosis rate was 100% (80/80). Postoperative imaging confirmed that the puncture path and target were accurately implemented according to the preoperative planning, and the target error was (1.32±0.44) mm (range: 0.55 to 1.99 mm). One case of puncture-related bleeding occurred at the target after surgery and improved after treatment. Conclusion: The three-dimensional multimodal images reconstructed by the 3D Slicer software before operation could help the surgeons make the preoperative planning and reduce the risk of stereotactic brain biopsy.
Male ; Female ; Humans ; Child ; Adolescent ; Young Adult ; Adult ; Middle Aged ; Aged ; Brain Neoplasms/pathology* ; Diffusion Tensor Imaging ; Retrospective Studies ; Robotics ; Biopsy ; Software ; Stereotaxic Techniques

Objective To evaluate the inductive effect of interferon-γ(IFN-γ) combined with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on programmed necrosis of the human immortalized keratinocyte cell line HaCaT,and to explore its mechanisms.Methods In vitro cultured HaCaT cells were divided into several groups:negative control group receiving no treatment,IFN-γ group treated with 50 μg/L IFN-γ,TRAIL group treated with 4 μg/L TRAIL,and cytokine combination group treated with 50 μg/L IFN-γ and 4 μg/L TRAIL or zVAD combination group pretreated with 40 μmo/L zVAD for 1 hour followed by the treatment with 50 μg/L IFN-γand 4 μg/L TRAIL.After 48-hour treatment,the morphology of HaCaT cells were observed under a light microscope,methyl-thiazolyl-tetrazolium assay was performed to evaluate the inhibitory effect of the treatment on the proliferation of HaCaT cells,and double staining flow cytometry to detect the necrosis of HaCaT cells.Meanwhile,real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of receptor interaction protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL),Western blot analysis to determine the expression of RIP1,RIP3,MLKL proteins and their phosphorylated forms (pRIP1,pRIP3,pMLKL),immunofluorescent staining to observe the distribution of pRIP3 and pMLKL in HaCaT cells,and the level of reactive oxygen species (ROS) in HaCaT cells in the above groups was detected by the fluorescence probe DCFH-DA.Statistical analysis was carried out with SPSS 22 software by using one-way analysis of variance (ANOVA) for comparing indices among different groups,and least significant difference (LSD)-t test for multiple comparisons.Results After 48-hour treatment,HaCaT cells in the cytokine combination group and zVAD combination group showed necrosis-like morphologic features.Methyl-thiazolyl-tetrazoliumassay revealed significant differences in the survival rate of HaCaT cells among the IFN-γgroup,TRAIL group,cytokine combination group,zVAD combination group and negative control group (73.16％ ± 5.71％,81.46％ ± 4.68％,72.18％ ± 2.93％,69.67％ ± 3.24％ and 100％,respectively;F =24.34,P ＜ 0.001).The necrosis rate of HaCaT cells was notably higher in the cytokine combination group and zVAD combination group (9.86％ ± 1.31％,10.33％ ± 2.16％,respectively) than in the negative control group (5.26％ ± 0.91％,t =4.61,5.07,respectively,both P ＜ 0.05).qPCR revealed that the mRNA expression of RIP3 and MLKL significantly increased in the cytokine combination group and zVAD combination group compared with the negative control group (tRIP3 =0.99,1.84,tMLKL =1.51,2.17,respectively,all P ＜ 0.05).Western blot analysis suggested that the protein expression of RIP1,RIP3,MLKL,pRIP1,pRIP3 and pMLKL significantly increased in the cytokine combination group compared with the negative control group (all P ＜ 0.05),and the zVAD combination group showed significantly decreased caspase 8 expression and increased expression of the above proteins compared with the cytokine combination group.Fluorescence microscopy showed that enhanced green dot-like or clump-like fluorescent spots (representing pRIP3) could be observed in the cytoplasm,and red fluorescent spots (representing pMLKL) could be seen on the cell membrane in the cytokine combination group.The average fluorescence intensity of ROS was significantly higher in the cytokine combination group than in the negative control group (t =702.00,P ＜ 0.05).Conclusion IFN-γcombined with TRAIL can induce the programmed necrosis of HaCaT cells with increased level of ROS.

Objective: To establish a model for the injury of human neuroblastoma cell (SH-SY5Y) induced by sodium glutamate, and to observe the protective effect of syringaresinol on cell damage from Viscum liquidambaricolum hayataon, and to explore its mechanism. Method: Construction of SH-SY5Y cell injury model using sodium glutamate.The experiment was divided into normal cell group, injury model group (sodium glutamate 50 mmol·L^-1, sodium glutamate 50 mmol·L^-1 + DMSO),syringaresinol experimental group (6.25, 12.5, 25 μmol·L^-1), by cell counting, cell morphology observation, Annexin V-FITC/PI apoptosis detection, ROS reactive oxygen species detection, mitochondrial membrane potential, and Western blot, evaluation of syringaresinol on glutamate-induced neuronal excitability injury neuroprotective activity. Result: Compared with normal group, the cell survival rate of the model group was significantly decreased (P<0.01), ROS accumulation was significant (P<0.01), mitochondrial membrane potential was significantly decreased (P<0.01), and the expression of poly-ADP-ribose polymerase (PARP) and PARP1 protein was significantly decreased (P<0.01), the apoptotic rate of cells also increased significantly (P<0.01). Compared with the model group, the syringaresinol group (6.25, 12.5, 25 μmol·L^-1) showed a concentration-dependent increase in cells. Survival rate (P<0.01), decreased ROS accumulation (P<0.01), restored mitochondrial membrane potential(P<0.01), up-regulated PARP, PARP1 protein (P<0.01), decreased apoptosis rate (P< 0.01). Conclusion: Syringaresinol has significant protective activity against excitatory damage induced by sodium glutamate in SH-SY5Y neurons, the mechanism may be through anti-oxidative stress, repairing mitochondrial function and DNA damage to significantly reduce sodium glutamate-induced neuronal apoptosis.

Objective To screen stroke risks among middle-aged and elderly people in community outpatient clinics in Shanghai.Methods A stroke risk screening was conducted among people aged 45-75 years selected with convenient sampling method from 10 community health service centers in Putuo district,Yangpu district and Pudong New Area of Shanghai during January to July 2017.The questionnaire and clinical measurement were used for screening.Multivariate logistic regression analysis was used to analyze the risk factors in subjects with high stroke risk.Results In this study,1 094 individuals with high stroke risk were screened out from 1 750 participants (62.5％).The proportion of high risk cases was higher among men (66.7％,473/709) than that among women (59.7％,621/1 041),unmarried,divorced or widowed (75.0％,90/120)than married or cohabitants (61.6％,1 004/1 630),living alone (72.1％,70/97) than living with others (61.9％,1 024/1 653) (x5=8.969,8.571,4.081;P＜0.01).Compared with non-high-risk subjects,the high-risk subjects had higher BMI,systolic blood pressure,diastolic blood pressure,fasting blood glucose,glycated hemoglobin,total cholesterol,triglycerides,serum creatinine and homocysteine,and were more likely to have carotid plaques or stenosis,the difference was statistically significant (P＜0.05).Multiple logistic regression analysis showed that unmarried/divorce/widowed,high BMI,systolic blood pressure,fasting blood glucose,total cholesterol,and carotid plaque or stenosis were positively associated with high stroke risk(OR=2.015,1.173,1.013,1.456,1.139,1.026,2.103;P＜0.05).Conclusions The proportion of high stroke risk individuals among middle-aged and elderly people is higher in community general practice outpatient clinics in Shanghai.Patients with high BMI,systolic blood pressure,fasting blood glucose and total cholesterol,and carotid plaque or stenosis,as well as those unmarried/divorced/widower should be the subjects for stroke intervention.