ObjectiveTo explore the function of DANCR during the differentiation of human embryonic stem cells （hESC） toward definitive endoderm （DE）. MethodsThe in vitro DE differentiation system was established and its efficiency was verified. The correlation between the expression level of DANCR and DE differentiation process was detected. Using lentivirus system， we stably knocked down DANCR in hESC. The shDANCR hESC line was applied to DE differentiation， using qPCR and Western blot to detect the expression of DE marker genes SOX17 and FOXA2， and that of primitive streak marker genes Brachyury （T）， EOMES， MIXL1 and GSC. Dual luciferase reporter assay and qPCR were used to confirm the interaction between DANCR and the WNT pathway during DE differentiation. ResultsThe in vitro differentiation system mimicked DE differentiation efficiently. And the expression of DANCR was gradually downregulated during differentiation. DANCR was efficiently knocked down in the shDANCR hESC line （P < 0.001）. Compared with those in the control group， the expression levels of primitive markers Brachyury （T）， EOMES， MIXL1 and GSC， as well as DE markers SOX17 and FOXA2， were significantly decreased in shDANCR groups （P < 0.05）. Furthermore， the transcriptional activity of the WNT pathway in shDANCR groups was lower than that in the control group （P < 0.05）. And RNA levels of downstream genes of the WNT pathway， FZD5， FZD8， SFRP1， FRZB and ANKRD6， were significantly decreased in shDANCR groups （P < 0.05）. However， differences in protein levels of the TGFβ pathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups （P > 0.05）. Forced activation of β-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation. ConclusionsThe expression of DANCR decreases during DE differentiation. DANCR may promote DE differentiation through modulating the activity of the WNT pathway.

Strengthening Party building in public hospitals is a crucial guarantee politically and organizationally for the high-quality development.This study aims at promoting high-quality development in public hospitals.It employs literature analy-sis,research interviews,and questionnaires to access the new situations and requirements of such development.It examines the status of promoting the high-quality development of hospitals through Party building,identifies problems and breakthrough points in work,and explores the four strategic points of focus for Party building in public hospitals.This paper proposes four implemen-tation pathways centered around the keywords"closed-loop Party responsibility system","duties and tasks of the General Party Branch","medical humanities",and"co-construction in Party building".These pathways are intended to provide a fresh and sustainable impetus for the high-quality Party building that drives the high-quality development of public hospitals.

Objective:To evaluate the morphological characteristics of the fetal heart and the contractile function of the left and right ventricles in fetuses with pulmonary stenosis (PS) and pulmonary atresia (PA) using fetal heart quantitative analysis technology (fetal HQ), and to assess the impact of different degrees of right ventricular outflow tract obstruction (RVOTO) on the contractile function of the fetal left and right ventricles. To accumulate early data and explore parameters for constructing a predictive model and clinical decision-making tool for the progression of fetal PS and PA.Methods:A retrospective analysis was conducted on 42 cases of mild to moderate PS and 23 cases of severe PS or PA detected through fetal echocardiography in the Department of Ultrasound, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, from July 2020 to December 2021. A control group of 195 normal fetal cases matching gestational weeks was selected. The fetal HQ technique was employed to measure the global sphericity index (GSI), left ventricular ejection fraction (EF), left/right ventricular area change percentage (LVFAC, RVFAC), and left/right ventricular longitudinal strain (LVGLS, RVGLS). Additionally, 24-segment sphericity index (SI) and fractional shortening (FS) for both left and right ventricles were measured. Comparative analyses were performed between the case and control groups, as well as within the case group.Results:In comparison with the control group, the case group exhibited significantly decreased GSI, LVGLS, LVEF, LVFAC, RVGLS, and RVFAC.The differences were statistically significant in the mild to moderate PS group (all P<0.05) and highly significant in the severe PS/PA group (all P<0.01). In the mild to moderate PS group, the left ventricle′s 2nd segment, right ventricle′s 24th segment SI, and the left ventricle′s 1st-13th segments, right ventricle′s 1st-16th and 20th-24th segments FS showed statistically significant differences compared to the control group (all P<0.05). In the severe PS/PA group, the right ventricle′s 1st-22nd segment SI, and the left ventricle′s 6th-13th, 21st-24th segments, and the right ventricle′s 1st-14th segments FS were reduced, showing statistically significant differences compared to the control group (all P<0.05). The severe PS/PA group showed lower RVGLS, RVFAC, and SI for the right ventricle′s 1st to 17th segments when compared to the mild to moderate PS group, with statistically significant differences (all P<0.05). Conclusions:Quantitative indices derived by fetal HQ is capable of evaluating the cardiac morphology and function of fetuses with PS/PA, which may provide for reference information for comprehensive understanding of cardiac morphological and functional changes in such fetuses.

Pulmonary fibrosis (PF) is a pathological change caused by repeated injuries and repair dysfunction of the alveolar epithelium. Our previous study revealed that the residues Asn3 and Asn4 of peptide DR8 (DHNNPQIR-NH₂) could be modified to improve stability and antifibrotic activity, and the unnatural hydrophobic amino acids α-(4-pentenyl)-Ala and d-Ala were considered in this study. DR3penA (DHα-(4-pentenyl)-ANPQIR-NH₂) was verified to have a longer half-life in serum and to significantly inhibit oxidative damage, epithelial-mesenchymal transition (EMT) and fibrogenesis in vitro and in vivo. Moreover, DR3penA has a dosage advantage over pirfenidone through the conversion of drug bioavailability under different routes of administration. A mechanistic study revealed that DR3penA increased the expression of aquaporin 5 (AQP5) by inhibiting the upregulation of miR-23b-5p and the mitogen-activated protein kinase (MAPK) pathway, indicating that DR3penA may alleviate PF by regulating MAPK/miR-23b-5p/AQP5. Safety evaluation showed that DR3penA is a peptide drug without obvious toxicity or acute side effects and has significantly improved safety compared to DR8. Thus, our findings suggest that DR3penA, as a novel and low-toxic peptide, has the potential to be a leading compound for PF therapy, which provides a foundation for the development of peptide drugs for fibrosis-related diseases.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*

OBJECTIVE: To observe the effect of Shenbing Decoction Ⅲ for improving renal function and pathology in rats with 5/6 nephrectomy and analyze its therapeutic mechanism for renal fibrosis in chronic kidney disease using network pharmacology combined with molecular docking. METHODS: Forty male SD rats were randomized into two groups to receive two-staged 5/6 nephrectomy (n=30) or sham operation (n=10), and 2 weeks after the final operation, serum creatinine level of the rats was measured. The rats with nephrectomy were further randomized into Shenbing Decoction Ⅲ group, losartan group and model group for daily treatment with the corresponding drugs via gavage starting at 1 week after 5/6 nephrectomy. After 16 weeks of treatment, serum creatinine and urea nitrogen levels of the rats were measured, and HE staining and Western blotting were used to examine the changes in renal pathology and fibrosis-related factors. Network pharmacology combined with molecular docking study was performed to explore the therapeutic mechanism Shenbing Decoction Ⅲ against renal fibrosis in chronic kidney disease, and Western blotting was used to verify the expressions of the core targets. RESULTS: Compared with those in the model group, the rats receiving 5/6 nephrectomy and Shenbing Decoction Ⅲ treatment showed significantly reduced serum creatinine and urea nitrogen levels, lessened renal pathologies, and improvement of the changes in epithelial mesenchymal transition-related proteins. Network pharmacological analysis showed that the main active ingredients of Shenbing Decoction Ⅲ were acacetin, apigenin, eupatilin, quercetin, kaempferol and luteolin, and the key targets included STAT3, SRC, CTNNB1, PIK3R1 and AKT1. Molecular docking study revealed that the active ingredients of Shenbing Decoction Ⅲ had good binding activity to the key targets. Western blotting showed that in rats with 5/6 nephrectomy, treatment with Shenbing Decoction Ⅲ obviously restored the protein expression of STAT3, PI3K, and AKT in renal tissue. CONCLUSION Shenbing Decoction Ⅲ can reduce renal injury induced by 5/6 nephrectomy in rats, and its therapeutic effects are mediated possibly by its main pharmacologically active ingredients that alleviate renal fibrosis via modulating multiple targets including STAT3, PIK3R1, and AKT1.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Molecular Docking Simulation ; Network Pharmacology ; Creatinine ; Renal Insufficiency, Chronic/drug therapy* ; Fibrosis ; Urea

Objective:To study the changes of early (i.e., within post injury day (PID) 14) coagulation function in patients with severe burns.Methods:A retrospective case series study was conducted. From December 2018 to December 2019, 50 severe burn patients who met the inclusion criteria were admitted to Guangzhou Red Cross Hospital of Jinan University. According to the severity of burns, the patients were divided into severe burn group (17 cases, including 12 males and 5 females) and extremely severe burn group (33 cases, including 26 males and 7 females). The platelet count (PLT), and conventional coagulation indexe and thromboelastogram index levels of patients were collected at admission, post injury hour (PIH) 48 and 72, and on PID 7 and 14. The conventional coagulation indexes included prothrombin time (PT), thrombin time (TT), activated partial prothrombin time (APTT), and fibrinogen (FIB) and D-dimer levels. The thromboelastogram indexes included coagulation angle (i.e., α angle), coagulation composite index (CI), MA value, R value, and K value (reflecting maximum amplitude, coagulation reaction time, and blood agglutination time, respectively). Data were statistically analyzed with independent sample t-test, Wilcoxon rank sum test, and chi-square test. Verification of the mixed effect model was performed on each index data of patients in the two groups, while the repeated measures analysis of variance was performed on PLT. Pearson correlation analysis or Spearman correlation analysis were performed to analyze the correlation between the thromboelastogram index data (except CI) and the PLT and conventional coagulation index data, respectively. Results:At admission, PIH 48 and 72, and on PID 7 and 14, PLT of patients in severe burn group were (203±91), (148±70), (123±63), (203±62), (402±140)×10 9/L, respectively, PLT of patients in extremely severe burn group were (235±116), (145±71), (109±52), (235±106), (455±138)×10 9/L, respectively. In overall comparison, only the difference of the main effect of time factor was statistically significant ( F=92.55, P<0.05). In severe burn group, statistically significant differences were only identified in comparison of patients' PLT between PID 7 and the adjacent two time points (at PIH 72 and on PID 14, with both P values <0.05). The differences in PLT of patients between all the adjacent time points in extremely severe burn group were statistically significant ( P<0.05). In the overall comparison of PT, TT, and FIB level of patients in the two groups at each time point, only the difference of main effect of time factor was statistically significant (with F values of 6.04, 8.45, and 32.90, respectively, all P values <0.05), and APTT and FIB level of patients in extremely severe burn group within PID 14 were higher than those in severe burn group. There were statistically significant differences in MA value, α angle, K value, and CI of patients in the two groups at each time point (with F values of 18.82, 11.38, 9.11, and 9.42, respectively, all P values <0.05). MA value was moderately correlated with PLT ( r=0.69, P<0.05), weakly correlated with TT and FIB level (with r values of -0.29 and 0.30 respectively, P<0.05), and very weakly correlated with D-dimer level ( r=-0.15, P<0.05); α angle was moderately correlated with PLT ( r=0.58, P<0.05), and weakly correlated with FIB level and TT (with r values of 0.26 and -0.29, respectively, P<0.05); R value was weakly correlated with APTT and FIB level (with r values of 0.24 and 0.31, respectively, P<0.05), and very weakly correlated with PT and TT (with r values of 0.16 and 0.14, respectively, P<0.05); K value was moderately correlated with PLT ( r=-0.59, P<0.05), and weakly correlated with FIB and TT (with r values of -0.29 and 0.32, respectively, P<0.05), and very weakly correlated with D-dimer level ( r=-0.15, P<0.05). Conclusions:Severe burn patients are already characterized with coagulation function changes in early stage, including insufficiency of coagulation function, enhanced platelet aggregation ability and enhanced FIB function. There is a certain correlation between conventional coagulation indexes and thromboelastogram indexes, but they cannot replace each other.

Objective:To investigate the clinical significance of plasma thrombin-antithrombin complex (TAT) levels in patients with sepsis-induced cardiomyopathy(SIC).Methods:One hundred and seven sepsis patients who were admitted to intensive care units (ICU) of the 908th Hospital of Chinese PLA Logistical Support Force were enrolled in the study. Patients were divided into sepsis group ( n=79) and the sepsis-induced cardiomyopathy group ( n=28) according to whether the cardiac ultrasound examination in 2 hours after admission, and the differences of each indicators between the two groups were compared including acute physiological and chronic health score (APACHEⅡ), lactate, blood routine, liver and kidney function, cardiac troponin I, N-terminal?pro-brain?natriuretic?peptide (NT-pro BNP), conventional coagulation tests and molecular markers of coagulation [tissue plasminogen activator-inhibitor complex (t-PAIC), thrombomodulin (TM), TAT, plasmin-α2-plasmin inhibitor complex (PIC)].Logistical?regression?was?used?to analyze the?risk?factors?for sepsis-induced cardiomyopathy and the receiver operating characteristic (ROC) curve was to analyze their cut-off values. The effect of low-molecular-weight heparin anticoagulation therapy on sepsis patients with TAT>8.26 ng/ml was evaluated by Kaplan-Meier analysis. Results:Compared with the cardiac troponin I[0.02(0.01, 0.09) ng/ml], NT-proBNP [1 118.09 (333.25, 2687.00) pg/ml], lactate[1.35(0.90, 2.60) mmol/L], TAT[6.50(3.94, 12.14) ng/ml], PIC[1.256 (0.668, 2.045) μg/ml] and t-PAIC[10.50 (6.70, 21.30) ng/ml] in sepsis group, the cardiac troponin I [0.75(0.01, 6.02) ng/ml], NT-proBNP[12 125.14(4 185.89, 33 611.62) pg/ml], lactate[2.35(1.43, 4.34) mmol/L], TAT[19.85 (9.08, 45.78) ng/ml], PIC[2.115 (0.878, 4.114) μg/ml] and t-PAIC [22.03(15.61,33.20) ng/ml] levels in the sepsis-induced cardiomyopathy group were significantly increased ( P<0.05). Logistical regression showed that positive NT-pro BNP and elevated TAT levels were independent risk factors for sepsis-induced cardiomyopathy. ROC curve analysis showed that the area under the curve of plasma TAT level for predicting sepsis-induced cardiomyopathy was 0.78. The sensitivity and specificity at the cut-off value of plasma TAT level with 8.26 ng/ml were 0.82 and 0.63, respectively. Conclusions:The elevated TAT level was an independent risk factor for the development of sepsis-induced cardiomyopathy. Low-molecular-weight heparin anticoagulation therapy can improve the 28-day survival rate of sepsis patients with TAT>8.26 ng/ml.

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosor-bent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were suc-cessfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation be-tween serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias％ranged from-12.2％to-5.2％,precision ranged from-12.4％to-1.4％,and the relative standard deviation(RSD)was less than 6.6％and 8.7％,respectively.The total error was less than 20.4％.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

The antimicrobial peptide APKGVQGPNG (named YD), a natural peptide originating from Bacillus amyloliquefaciens CBSYD1, exhibited excellent antibacterial and antioxidant properties in vitro. These characteristics are closely related to inflammatory responses which is the central trigger for liver fibrosis. However, the therapeutic effects of YD against hepatic fibrosis and the underlying mechanisms are rarely studied. In this study, we show that YD improved liver function and inhibited the progression of liver fibrosis by measuring the serum transaminase activity and the expression of α-smooth muscle actin and collagen I in carbon tetrachloride-induced mice. Then we found that YD inhibited the level of miR-155, which plays an important role in inflammation and liver fibrosis. Bioinformatics analysis and luciferase reporter assay indicate that Casp12 is a new target of miR-155. We demonstrate that YD significantly decreases the contents of inflammatory cytokines and suppresses the NF-κB signaling pathway. Further studies show that transfection of the miR-155 mimic in RAW264.7 cells partially reversed the YD-mediated CASP12 upregulation, the downregulated levels of inflammatory cytokines, and the inactivation of the NF-κB pathways. Collectively, our study indicates that YD reduces inflammation through the miR-155–Casp12–NF-κB axis during liver fibrosis and provides a promising therapeutic candidate for hepatic fibrosis.