Objective : To generate heterozygous TRAF2 knockout mice, the CRISPR/Cas9 technology was successfully employed. These mice were served as a valuable model to explore the pathological mechanisms underlying inflammatory and immune disorders mediated by abnormal TNF-α-TRAF2 signaling and to develop new therapeutic targets. Methods : A vector targeting the knockout of the TRAF2 gene was constructed. Lead RNA and Cas9 Mrna were introduced into the fertilized eggs of C57BL/6JGpt mice through microinjection to mediate the TRAF2 gene mutation in mice. The mouse tail protein was extracted and the genotype of the F0 generation was determined by PCR and Western blot. TRAF2+/- mice were successfully obtained. F0 generation mice were backcrossed with C57BL/6JGpt wild-type mice to obtain stable TRAF2+/- mice for propagation and subsequent experiments. The body weight of TRAF2+/- mice was detected; Western blot was used to detect the expression of TRAF2 in the spleen, liver and kidney tissues of TRAF2+/- mice. The development of spleen, liver and kidney tissues in TRAF2+/- mice was detected by HE staining. Results : PCR identification using specific primers demonstrated that TRAF2+/- mice exhibited a target band at 679 bp. Western blot analysis results indicated that, compared with the WT group, the expression of TRAF2 in the tail protein of TRAF2+/- mice was significantly reduced(P+/- mice had a lower body weight compared to their littermate WT mice(P+/- mice was decreased(P+/- mice and WT mice. Conclusion The successful construction of TRAF2+/- mice has provided an important animal model for exploring the role of TRAF2 in developmental regulation, revealing the mechanism of inflammatory immune diseases mediated by abnormal TNF-α-TRAF2 signaling, and screening related drug targets.

SARS-CoV-2 main protease (M^pro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as M^pro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed M^pro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the M^pro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to M^pro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit M^pro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; Foot-and-Mouth Disease Virus

Objective To investigate the effects of commonly used inductive agents on peripheral blood monocytic myeloid-derived suppressor cells (M-MDSCs) in renal transplantation recipients and to discuss their possible mechanism.Methods The enrolled patients received rabbit anti-thymocyte globulin (rATG) or basiliximab for induction therapy,with the maintenance immunosuppressive regimen of tacrolimus,mycophenolate mofetil and steroid.The number of CD11 b + CD33 + HLA-DR-CD14 + CD1 5-M-MDSCs and cytokine levels in peripheral blood,including interferon-γ(IFN-γ),interleukin-2 (IL-2),IL-4 and IL-6,were measured by flow cytometry before and 1 week,2 weeks,1 month,2 months,3 months after operation.Results A total of 47 recipients (29 given rATG 29,and 18 given basiliximab) were included in this study.Compared to the patients with basiliximab,asignificant increase in the frequency of M-MDSCs was observed in the rATG group at 2nd month after operation (5.5％ ± 2.8％ vs.3.8％ ± 1.6％,P＜0.001) and at 3rd month after operation (7.0 ％ ± 3.1％vs.4.1％ ± 2.3 ％,P＜ 0.001),while there was no significant difference in the cell number between the two groups.In the cytokine detection,levels of IL-2 and IL-4 in the rATG-treated recipients were significantly higher at 2nd weekpostoperation (Pr2 =0.032,and PIL-4 =0.019)and 1st month postoperation (PIL-2 =0.024,PIL-4 ＜0.001) than the basiliximab group.Conclusions ATG promotes the expansion of M-MDSCs,which is associated with the secretion of IL-2 and IL-4 due to the lymphocytes depletion.The synergistic immunosuppressive effect may contribute to the induction of immune tolerance.

Objective To compare the curative effects between recombinant human brain natriuretic peptide (rhBNP) and sodium nitroprusside in treatment of the acute attack of elderly patients with chronic heart failure (CHF), and probe the impacts of rhBNP on the heart function,serum B-type natriuretic peptide (BNP), norepinephrine (NE), endothelin 1 (ET-1) and antidiuretic hormone (ADH) levels. Methods The 89 patients aged 65-85 years at acute attack stage of CHF were randomized into two therapy groups: rhBNP group (n= 47) and sodium nitroprusside group (n=42). The clinical effects, heart function, serum BNP, NE, ET-1 and ADH changes were observed before and after the treatment. Results After 24 hours of treatment, the efficacy rate and total effective rate were higher in rhBNP group than in sodium nitroprusside group (51.1% vs. 26.2 %,95.7% vs. 66. 7%, respectively, P＜0. 05 and P＜0. 01), and non-efficacy rate in rhBNP group was lower (4.3% vs. 33. 3%, P＜0. 01). There was one death case in rhBNP group and three in sodium nitroprusside group. In rhBNP group, left ventricular ejection fraction values increased after 2 weeks of treatment [(46.2± 9.5)% vs. (38.1 ±6.0)%], P＜0.05. Serum BNP level significantly decreased in rhlBNP group than in sodium nitroprusside group after 2 weeks of treatment (P＜0.05).In rhBNP group, serum BNP and NE levels decreased 24 hours and 2 weeks after treatment (P＜0. 01) and the levels furtherly reduced after 2 weeks (P＜0.01). Serum ET-1 level decreased in rhBNP group than in sodium nitroprusside group 2 weeks after treatment (P＜0.05). In rhBNP group, there was no significant difference in serum ET-1 level between baseline and 24 hours after treatment (P＞ 0. 05), but the ET-1 level decreased 2 weeks after treatment as compared with 24 hours after treatment (P＜0.01). There were no significant differences between the two groups before and after treatment (P＞0.05). Incidences of headache and hypotension were lower in rhBNP group than in sodium nitroprusside group (4.3% vs. 19.0%, 14.9% vs. 23.8%, both P＜0.05),Conclusions RhBNP can be safely and effectively used for acute attack of CHF.

Objective To evaluate the efficiency of treatment of iliac-femoral venous thrombus(I-FVT)via double femoral veins.Methods Eighteen patients with I-FVT were treated with implantation of inferior vena caval filter,catheter-directed cut,balloon-directed extend and drag.Some patients were treated with ATD or OASIS catheter.After operation,the catheter was kept in iliac-femoral veins for one to six days.Meanwhile,strict anti-coagulation was necessary.Results The treatment was successful in seventeen patients,the iliac-femoral veins were patent after the opration.Swollen symptom of lower limb and pain disappeared in fifteen patients,swollen symptom of lower limb became better in two patients.The treatment was fail in one patient.Follow-up study showed no pulmonary embolization or recrudescence in all eighteen patients.Conclusion The treatment of I-FVT via double femoral veins is a safe and effective method,there are no serious complications.