Objective:To develop a pre-processing workflow of real world data (RWD) derived from multicenter clinical laboratories so that the level of data standardization can be improved, and subsequently to produce more robust real world evidence (RWE).Methods:Purpose sampling was used to invite senior experts with experience in clinical research utilizing RWD, covering the fields of clinical laboratory, epidemiology, biostatistics, and clinical medicine. In-depth, semi-structured individual interviews were conducted and thematic analysis was used to analyze the collected data.Results:The in-depth interviews were completed in 16 experts. The experts unanimously agreed that pre-processing RWD derived from multicenter clinical laboratories is necessary prior to its application in research. Based on experts' insights, a comprehensive pre-processing workflow for RWD was constructed, comprising six key steps: ①developing a variable list based on research questions and distributing it to each clinical laboratory; ②conducting an initial quality assessment of RWD based on existing quality control results in clinical laboratories; ③cleaning the data; ④determining whether RWD (including categorical and continuous variables) is heterogeneity among different clinical laboratories; ⑤exploring potential sources of heterogeneity;⑥pre-processing RWD based on identified causes contributing to heterogeneity.Conclusion:The pre-processing workflow of RWD was established, to provide a methodological reference for controlling systematic errors in RWD derived from multicenter clinical laboratories, thereby enhancing the validity of RWE.

OBJECTIVE: To investigate the effect and mechanism of Hyssopus cuspidatus Boriss. extract (HCE) in ovalbumin (OVA)-induced allergic asthma. METHODS: Components identification of HCE was conducted using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry. Mice were sensitized with OVA to establish asthmatic model, and dexamethasone was used as positive control. Respiratory reactivity, white cells counting in bronchoalveolar lavage fluid and peripheral blood, cytokine level measurement in serum and lung tissue, and histologic examination were performed to evaluate the therapeutic effect of HCE on asthma. Network pharmacology approach was used for mechanism prediction. Western blotting and untargeted lipidomics method were applied for mechanism validation. RESULTS: Fifty-two compounds were identified in HCE, predominantly terpenoids and flavonoids. HCE markedly reduced airway resistance, the eosinophil infiltration in lung tissues, and the levels of immunoglobulin E, interleukin-4, interleukin-5, and interleukin-13. Network pharmacology analysis suggested phosphatidylinositol 3-kinases (PI3K), c-Jun N-terminal kinase (JNK), and p38 Mitogen-activated protein kinase (p38 MAPK) may be key proteins of HCE in the treatment of allergic asthma. Western blot results indicated that the levels of phosphorylated PI3K, JNK, and P38 were downregulated in HCE-treated group. Moreover, HCE significantly upregulated the levels of ceramide and sphingomyelin and downregulated the level of phosphatidylcholine. CONCLUSION HCE inhibited allergic asthma via PI3K/JNK/P38 signaling pathway and lipid homeostasis regulation.

OBJECTIVES: The aim of this study is to determine the effect of cannabinoid receptor (CB) 2 inhibitor on desmoglein 3 (DSG3) expression in HaCaT cells co-cultured with pemphigus serum. METHODS: Immunohistochemical staining was used to compare CB expression in pemphigus patients and normal individuals. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the concentration of CB2 in the serum of pemphigus patients and normal individuals. A correlation analysis was performed to examine the relationship between the serum CB2 and DSG of pemphigus patients. The CCK-8 assay was used to evaluate the inhibitory effect of AM630 on HaCaT cells, and the half-maximal inhibitory concentration (IC₅₀) value was utilized to determine the experimental concentration. Serum from normal individuals (negative control group) and pemphigus patients (pemphigus group) was co-cultured with HaCaT cells at a 1∶1 ratio. HaCaT cells cultured in complete medium were used as the control group. HaCaT cells in the pemphigus group treated with AM630 were employed as the AM630 group. Real-time polymerase chain reaction (PCR) and Western blot were conducted to assess the expression levels of CB2, DSG3, and β-catenin. Cell dissociation experiments were conducted to evaluate the effect of AM630 on the adhesion of HaCaT cells. RESULTS: Immunohistochemistry revealed significant differences in CB2 expression between pemphigus and normal mucosa (P<0.000 1), but no difference was found in CB1 expression. ELISA analysis revealed a statistically significant difference in the expression levels of CB2 in the serum between normal individuals and pemphigus patients (P<0.001). The expression of CB2 in the serum of pemphigus patients exhibited a significant positive correlation with that of DSG3 (r=0.831, P=0.003). The CCK-8 assay indicated that the IC₅₀ of AM630 on HaCaT cells was 0.55 μmol/L. Real-time PCR and Western blot showed that the expression levels of CB2 and DSG3 increased in the pemphigus group, while the expression level of β-catenin decreased compared with that in the AM630 groups (P<0.05). CONCLUSIONS CB2 is highly expressed in oral mucosal pemphigus. AM630 inhibits overexpression of CB2 and DSG3 and underexpression of β-catenin levels, which can provide new therapeutic targets for pemphigus.
Humans ; Pemphigus/pathology* ; Receptor, Cannabinoid, CB2/metabolism* ; Desmoglein 3/metabolism* ; Acantholysis/metabolism* ; Mouth Mucosa/pathology* ; HaCaT Cells ; Coculture Techniques ; beta Catenin/metabolism*

Objective: To investigate whether fibroblast growth factor 18(FGF18) can induce human gingival fibroblasts(HGFs) isolatedin vitroto differentiate into osteoblast-like cells, and to explore the mechanism of osteogenesis. Methods : HGFs were isolated, cultured and identified by tissue block method. The third generation of HGFs were divided into experimental group and control group. FGF18 and L-DMEM was added to the experimental group while L-DMEM was added to the control group.The effects of different concentrations of FGF18(0, 0.01, 0.02, 0.04, 0.06 mg/L) on proliferation of HGFs were detected by Methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay. Alkaline phosphatase(ALP) and alizarin red staining were used to detect the osteogenesis and mineralization ability of the cells after induction. RT-PCR, immunocytochemistry staining, and Western blot were used to detect the expression of genes and proteins related to osteogenesis and BMP2 in the BMP signaling pathway. Results: Compared with the control group, the experimental group could promote the proliferation of HGFs at 3, 5, 7, 9, and 11days(P<0.05),ALP activity and mineral salt deposition increased after induction at 14 and 21 days(P<0.05), and the expressions of ALP, OPN, OCN mRNA and BMP2 mRNA in BMP signaling pathway significantly increased(P<0.01). The expressions of OPN, OCN and BMP2 protein at 21 days were significantly higher than those at 14 days(P<0.01). Conclusion FGF18 can promote the proliferation of HGFs, and induce the differentiation of HGFs into functional osteoblasts. The osteogenic mechanism is related to the upregulation of BMP2.

Middle compartment defects,a common subtype of pelvic floor dysfunction(PFD),are primarily characterized by the prolapse of the uterus or vaginal vault.Magnetic resonance imaging(MRI)has emerged as a valuable diagnostic tool for PFD,offering superior soft tissue resolution while eliminating exposure to ionizing radiation.This review comprehensive summarizes current applications of MRI in the diagnosis and treatment of PFD,covering measurement methods,manifestations of three-level structural defects,postoperative efficacy evaluation,vaginal axial assessment,and evaluation of the mesh status.The authors suggest that MRI enables precise preoperative evaluation of three-level defects,thereby facilitating the development of personalized treatment plans.Additionally,MRI provides an accurate postoperative assessment of surgical outcomes and mesh status,offering a new basis for postoperative assessment.MRI demonstrates unique value in the diagnosis and treatment of middle compartment defects.

Objective:To assess the preliminary efficacy and safety of modified Société Internationale d′Oncologie Pédiatrique Epithelial Liver Tumor Study Group (SIOPEL)-4 protocol for pediatric hepatoblastoma (HB) with lung metastases.Methods:This prospective cohort study enrolled 27 newly diagnosed pediatric HB with lung metastases who received the modified SIOPEL-4 protocol at Shanghai Children′s Medical Center, Shanghai Jiao Tong University School of Medicine, and Shanghai Children′s Hospital between January 2020 to December 2023. Clinical characteristics, lung response rates to induction chemotherapy, treatment outcomes, prognostic factors and sever chemotherapy toxicities at different stages were analyzed. Survival analysis was performed by Kaplan-Meier method. Univariate prognostic analysis was conducted by Log-Rank test.Results:Of the 27 patients, there were 17 males and 10 females, with the age of 21 (15, 33) months. During the follow-up of 31 (12, 45) months for 17 continuous complete remission patients, 4 cases disease progression (2 cases death) and 6 cases relapse were observed. The 2-year event free survival (EFS) and overall survival (OS) rate was (58±11)% and (89±7)%, respectively. All the 27 patients had response to block 1-3 induction chemotherapy (cisplatin+doxorubicin), with 14 cases (52%) achieving complete response and 13 cases (48%) achieving partial response of lung metastatic lesions, the 2-year EFS rate was (81±12)% and (34±14)%, respectively ( χ 2=6.76, P=0.009), the 2-year OS rate was 100% and (79±13)%, respectively ( χ2=2.12, P=0.145). Patients with caudate lobe tumors or ≥10 pulmonary metastatic nodules had significantly lower EFS rates ( χ2=5.36, 7.84, P=0.021, 0.005, respectively). The incidence of grade 3/4 neutropenia after block 1-3 induction chemotherapy, CD (carboplatin+doxorubicin), and VI (vincristine+irinotecan) consolidation chemotherapy was 90% (73/81), 75% (58/77), and 31% (11/35), respectively. The incidence of grade 3/4 thrombocytopenia was 77% (62/81), 69% (53/77), and 14% (5/35), respectively. The incidence of grade 3/4 infections was 64% (52/81), 25% (19/77), and 20% (7/35), respectively. The differences between the groups were statistically significant ( χ2=43.51, 42.69, 33.00, all P<0.001). Two patients (10%) of the 20 evaluable patients for ototoxicity occurred grade 3 and higher hearing impairment, with 1 patient requiring a hearing aid. Conclusions:The modified SIOPEL-4 regimen shows good preliminary efficacy and safety in treating pediatric HB with lung metastases. The prognosis for patients with residual lesions in the lungs after induction chemotherapy needs to be improved. Attention should be given to the ototoxicity induced by high-dose cisplatin chemotherapy.

BACKGROUND:Studies have shown that different durations of pressure application on normal muscles can produce varying physiological responses.OBJECTIVE:To explore the expression levels of inflammatory factors tumor necrosis factor α and nuclear κB in skeletal muscle under different pressure durations.METHODS:Twenty healthy male SPF-grade Sprague-Dawley rats were randomly divided into four groups:control group,10-second pressure group,20-second pressure group,and 30-second pressure group.The right leg of each rat was used for the experiment.The control group received no intervention,while rats in each pressure group were anesthetized by intraperitoneal injection of 2%pentobarbital sodium(35 mg/kg),and the thin femoral muscle of the rats was pressed continuously at a constant pressure of 200 kPa using a homemade mechanical pressure device for 10,20,and 30 seconds,respectively.Muscle tissue at the pressing site of the right hind limb was collected immediately after pressure.Hematoxylin-eosin staining was used to observe the morphological changes of skeletal muscle tissues and changes in the cross-sectional area of muscle fibers,and immunohistochemistry was used to detect the expression levels of tumor necrosis factor α and nuclear factor κB in rat skeletal muscle.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results revealed that the pressure groups showed loosely arranged skeletal muscle fibers,reduced cross-sectional area and diameter,and enlarged intermuscular spaces.Compared with the control group,the cross-sectional area of muscle fibers was significantly reduced in the pressure groups(P<0.05),but there was no significant difference between the three pressure groups(P>0.05).The 10-second pressure group showed no significant presence of red blood cells in the interstitial spaces,while the 20-second pressure group exhibited a small amount of red blood cells,and the 30-second pressure group showed capillary dilation with red blood cells in the interstitial spaces.The expression level of tumor necrosis factor α in the 30-second pressure group was significantly higher than that in the control group(P<0.05).The expression level of nuclear factor κB in skeletal muscle showed no significant difference among groups(P>0.05).To conclude,skeletal muscle undergoes morphological changes and reduced cross-sectional area after pressure at 200 kPa,but there is no significant difference among the 10-,20-,and 30-second pressure groups.As the duration of pressure increases to 30 seconds,the inflammatory factor tumor necrosis factor α is activated,but nuclear factor κB remains unaffected,suggesting that inflammatory factors may express under short-term pressure,while transcription factors show no significant change.

Purpose To investigate the expression of LAMB3 and ITGB4 in esophageal squamous cell carcinoma(ESCC)and analyze their associations with clinicopathological features.Methods Bioinformatics was used to assess the expression of LAMB3 and ITGB4 in pan-cancer and ESCC.Immunohistochemistry was performed to evaluate their expression and distribution in ESCC tissues,and correlations clinicopathological features were analyzed.Follow-up data were collected to construct Kaplan-Meier survival curves,and Cox regression analysis was proformed to determine their prognostic significance.Results Bioinformatics analysis showed that LAMB3 and ITGB4 were highly expressed in ES-CC tissues(P＜0.001).Immunohistochemistry confirmed that both proteins were localized in the cytoplasm and membrane of tumor cells.High LAMB3 expression was significantly associated with poor differentiation(P＜0.001),lymph node metastasis(P=0.015),and T staging(P＜0.001).High ITGB4 expression was significantly correlated with poor differentiation(P=0.004)and advanced T staging(P=0.004).Cox regression analysis identified high ex-pression of LAMB3 and ITGB4 as independent prognostic factors for overall survival[HR=4.97(95％CI:2.73-9.02);HR=2.33(95％CI:1.36-3.99)].Conclusion LAMB3 and ITGB4 are highly expressed in ESCC.High LAMB3 expression is associated with tumor differentiation,lymph node metastasis,and T staging,while high ITGB4 expression is correlated with tumor differentiation and T staging.Patients with high expression of LAMB3 or ITGB4 have poorer overall survival,suggesting that these proteins may serve as prognostic biomarkers for unfavorable outcome in ESCC.

BACKGROUND:Studies have shown that different durations of pressure application on normal muscles can produce varying physiological responses.OBJECTIVE:To explore the expression levels of inflammatory factors tumor necrosis factor α and nuclear κB in skeletal muscle under different pressure durations.METHODS:Twenty healthy male SPF-grade Sprague-Dawley rats were randomly divided into four groups:control group,10-second pressure group,20-second pressure group,and 30-second pressure group.The right leg of each rat was used for the experiment.The control group received no intervention,while rats in each pressure group were anesthetized by intraperitoneal injection of 2%pentobarbital sodium(35 mg/kg),and the thin femoral muscle of the rats was pressed continuously at a constant pressure of 200 kPa using a homemade mechanical pressure device for 10,20,and 30 seconds,respectively.Muscle tissue at the pressing site of the right hind limb was collected immediately after pressure.Hematoxylin-eosin staining was used to observe the morphological changes of skeletal muscle tissues and changes in the cross-sectional area of muscle fibers,and immunohistochemistry was used to detect the expression levels of tumor necrosis factor α and nuclear factor κB in rat skeletal muscle.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results revealed that the pressure groups showed loosely arranged skeletal muscle fibers,reduced cross-sectional area and diameter,and enlarged intermuscular spaces.Compared with the control group,the cross-sectional area of muscle fibers was significantly reduced in the pressure groups(P<0.05),but there was no significant difference between the three pressure groups(P>0.05).The 10-second pressure group showed no significant presence of red blood cells in the interstitial spaces,while the 20-second pressure group exhibited a small amount of red blood cells,and the 30-second pressure group showed capillary dilation with red blood cells in the interstitial spaces.The expression level of tumor necrosis factor α in the 30-second pressure group was significantly higher than that in the control group(P<0.05).The expression level of nuclear factor κB in skeletal muscle showed no significant difference among groups(P>0.05).To conclude,skeletal muscle undergoes morphological changes and reduced cross-sectional area after pressure at 200 kPa,but there is no significant difference among the 10-,20-,and 30-second pressure groups.As the duration of pressure increases to 30 seconds,the inflammatory factor tumor necrosis factor α is activated,but nuclear factor κB remains unaffected,suggesting that inflammatory factors may express under short-term pressure,while transcription factors show no significant change.

Objective:To assess the efficacy and safety of Gongxuening Capsules in reducing post-abortion bleeding following artificial abortion.Methods:A multicenter, randomized, double-blind study was conducted. From May 31, 2022 to March 31, 2023, 484 women who underwent vacuum aspiration abortion for early intrauterine pregnancy were enrolled in 11 centers and randomly assigned to control group and the study group at a 1∶1 ratio using a center-block randomization method. Control group were administered a placebo of Gongxuening Capsules for 9 d, while the study group received the actual Gongxuening Capsules for the same duration. The outcomes measured included vaginal bleeding volume, duration of vaginal bleeding, endometrial thickness, time to menstrual recovery, and complications.Results:1) A total of 484 subjects were enrolled, and 472 completed the study. Totally 450 subjects were included in the efficacy analysis set, with 224 in control group and 226 in the study group; 468 subjects were included in the safety analysis set, with 236 in control group and 232 in the study group. The baseline characteristics of the two groups were comparable (all P>0.05). 2) The vaginal bleeding volume was lower in the study group [(13.30±12.14) mL] than in control group [(19.00±17.67) mL, P<0.001]. The proportion of subjects in the study group with bleeding days less than 4 d [29.65% (67/226)] was higher than that in control group [19.20% (43/224), P=0.010]. 3) No significant differences were observed between the two groups in terms of time to menstrual recovery and endometrial thickness (all P>0.05). 4) In the study group, 3 subjects experienced non-therapeutic-related complications, while 11 subjects in control group. The incidence of complications was lower in the study group [1.29% (3/232)] than in control group [4.66% (11/236), P=0.033]. Conclusion:The administration of Gongxuening Capsules to women following artificial abortion significantly reduced vaginal bleeding volume and was associated with good safety, with the treatment being well-tolerated by the subjects.