[ ABSTRACT] AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein ( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms .METHODS:The RAW264.7 macropha-ges were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid ( PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h.The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively.The activities of lactate de-hydrogenase ( LDH) in the medium and caspase-3 in the cells were determined by detection kits .The protein levels of bec-lin-1 (a molecular marker of autophagy ), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein ( CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis ) were examined by Western blot .Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autoph-agy) was observed under laser scanning confocal microscope .RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability , and dramatic elevation in LDH leakage , cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autoph-agy inducer ) .ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap.Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL.Moreover , PBA ( endoplasmic reticulum stress inhibitor ) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granula-tion of LC3.CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages , and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression .

Objective: To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan. Methods: We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected. Results: We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot. Conclusions The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Immune checkpoint inhibitors are commonly used in clinical practice and have achieved good efficacy, but immune-related adverse events (irAEs) are often unpredictable and may lead to serious clinical consequences. Risk factors for irAEs include gender, body mass index, tumor class, drug type, pre-existing autoimmune disease, and tumor mutational burden; predictors are mainly immune cells, cytokines and chemokines, autoantibodies, genome, and gut microbes. By further investigating the mechanisms, risk factors and predictors for the occurrence of irAEs, guidance for the clinical prediction and management of irAEs is supposed to be provided.

Objective: To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay. Methods: Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E. coli BL21 to screen antibodies. Synthetic monkeypox virus A29_{17 ~ 49} polypeptide was used to immune BALB/c mice. Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques. Indirect ELISA was performed to test antibodies specificity and subtype. Results: A29, A27 and 162 proteins were highly expressed in E. coli and detected by Western blot. The three his-tagged proteins were purified using His-Bind affinity chromatography column. The purity of the proteins was all more than 90%. And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins. Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype. Conclusions Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.

Objective:To investigate the factors influencing the success of external cephalic version.Methods:Pregnant women who underwent an external cephalic version due to breech or transverse presentation by the same operator in the Third Affiliated Hospital of Zhengzhou University from July 2015 to July 2021 were selected as the study objects. Univariate analysis and logistic regression analysis were used to explore the clinical factors influencing the success of the external cephalic version. The receiver operating characteristic (ROC) curve was used to analyze the best cut-off value of gestational week and amniotic fluid index at the time of operation and to evaluate the predictive value of the influencing factors on the success of the external cephalic version.Results:(1) A total of 118 cases finally entered this study. Among the 118 cases,77 cases (65.3%) succeeded in the external cephalic version, among which the success rate was 49.1% (27/55) for primipara and 79.4% (50/63) for multipara. The vaginal delivery rate was 56.8% (67/118). (2) Complications occurred in 19 (16.1%) of the 118 cases. The main complications were abnormal fetal heart rate (13 cases, 11.0%), umbilical cord presentation, and fetal position reversion (two cases and 1.7% in each), and the serious complications were intrauterine fetal death and placental abruption (one case and 0.8% in each).The complication rate of patients with successful external cephalic version was 7.8% (6/77), which was lower than that of those who failed the external cephalic version [31.7%(13/41)] ( χ 2=11.33, P=0.001). (3) Multivariate analysis showed that gestational week at surgery before 38, amniotic fluid index >11.10 cm, and multipara were the factors affecting the success of the external cephalic version [ OR(95% CI)=0.561(0.351-0.897), 1.173(1.018-1.351) and 4.201(1.547-11.404), all P<0.05]. (4) The area under the ROC curve of the combination of the gestational week at surgery, amniotic fluid index, and parity was 0.744 (95% CI: 0.640-0.848, P<0.001), and the Youden index was 0.518, with a sensitivity of 70.0% and a specificity of 81.8%. Conclusion:Gestational weeks, amniotic fluid index, and multipara are related to the success of the external cephalic version, and the combination of the three has certain predictive power for the success of the surgery.

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K _D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively.

Objective: To investigate the cellular and humoral immune responses induced by combined immunization with the fusion protein of human papillomavirus type 18 (HPV18) and the recombinant vaccinia virus. Methods: Purified HPV18L2_31-600E7E6 fusion protein, expressed by prokaryotic expression system, were immunized in combination with the recombinant vaccinia virus vaccine expressing HPV18E7E6 fusion protein (rVV18E7E6) by using various prime-boost regiments in C57BL/6 mice. Cellular and humoral immune responses were analyzed by enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and pseudovirus neutralization assay. Results: Higher levels of cellular immune responses were induced in mice primed with the HPV18L2_31-600E7E6 fusion protein/adjuvant CpG and boosted with the recombinant vaccinia virus rVV18E7E6, than in other immunized mice. Higher binding antibody level was induced, and low level neutralizing antibody against pseudovirus was detected simultaneously. Conclusions Priming with HPV18L2_31-600E7E6 fusion protein/CpG and boosting with the recombinant vaccinia virus rVV18E7E6 could induce higher cellular and humoral immune response in immunized mice, which might be taken as vaccine candidate for treatment of HPV18 chronic infection and postoperative adjuvant treatment for cervical cancer.