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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19, and no effective antiviral agents and vaccines are available. SARS-CoV-2 is classified as a biosafety level-3 (BLS-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2-GFP/{Delta}N trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
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OBJECTIVE:To observe the effects of preoperative application of tirofiban on related indexes of patients with acute myocardial infarction. METHODS:In retrospective analysis,128 patients with acute myocardial infarction selected from our hospital during Jan. 2015-Jun. 2016 were divided into observation group (76 cases) and control group (52 cases) according to whether or not the tirofiban was used before PCI. Control group was given Aspirin enteric-coated tablets 300 mg,po+ Clopidogrel sulfate tablets 600 mg,po,before PCI,and given Heparin sodium injection 100 U/kg ,iv,during PCI. Observation group was ad-ditionally given Tirofiban hydrochloride injection 0.2 μg/(kg·min),iv,before PCI,on the basis of control group. ST-segment depres-sion and chest pain remission of 2 groups were observed after PCI. CK-MB level before surgery,CK-MB peak value,the time of CK-MB reaching the peak value,duration after surgery as well as TIMI blood flow grading after surgery were also observed in 2 groups. The levels of von Willebrand factor(vWF),ET-1 and NO were observed before and after surgery;the occurrence of ADR was recorded. RESULTS:After PCI,ST-segment depression rate(89.74%)of observation group was significantly higher than that (67.31%)of control group,the chest pain remission rate(89.47%)was significantly higher than that of control group(75.00%), the patients with TIMI blood flow grading grade 2-3 in observation group was more than control group,with statistical significance (P0.05). After PCI,CK-MB peak value,the time of CK-MB reaching the peak value,duration in observation group were significant-ly lower or shorter than control group;the levels of vWF and ET-1 significantly decreased,while NO levels increased;the improve-ment of vWF,ET-1 and NO in observation group was significantly better than control group,with statistical significance (P0.05). CONCLUSIONS:The application of tirofiban before PCI can relieve clinical symptom,improve cardiac function,protect vascular endothelial and restore coronary artery perfusion. The attention should be paid to the risk of bleeding.
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Objective To study the influence factors on detection coagulation factors Ⅸ activity in human prothrombin complex concentrates(PCC).MethodsUsing Chinese Pharmacopoeia(2010)as reference,factor Ⅸ deficient plasma from different manufacturers,different types of instruments,different methods of heparin neutralization,different sample pretreatment methods were used to determine the activity of coagulation factor Ⅸ in PCC.The influence on the results of coagulation factor Ⅸ was analyzed.ResultsThere was a significant difference between factor Ⅸ deficient plasma from different manufacturers for the activity of coagulation factors Ⅸ(P<0.05).There was no significant difference between the results of coagulation factor Ⅸ measured by different types of Automatic Coagulation Analyzer.When the dilution ratio was more than 60 times,the neutralization of heparin had little effect on the coagulation factor Ⅸ activity.When the dilution ratio is less than 60 times,the detection results of coagulation factor Ⅸ by neutralization of heparin are higher than no neutralization of heparin,and the difference is significant(P<0.05).Samples for pre-temperature or not and whether dissolution for 15minutes or not,the coagulation factor Ⅸ activity showed no significant difference.Conclusion The coagulation factor Ⅸ activity in PCC was affected by factor Ⅸ deficient plasma from different manufacturers,different dilution times of heparin neutralization method and sample pre-treatment methods.It must be paid attention to in the detection process.Strengthen the quality control of the factor deficient plasma and the standardization of operation process are necessary.The External Quality Assessment for the detection of coagulation factors in PCC products should be edtablished.
