Progress in the study of antiviral activity of cepharanthine against SARS-CoV-2 / 南方医科大学学报

As a member of the dibenzyl isoquinoline alkaloid family, cepharathine is an alkaloid from the traditional Chinese medicine cepharathine, which is mainly used for treatment of leukopenia and other diseases. Recent studies of the inhibitory effect of cepharathine against SARS-CoV-2 have attracted widespread attention and aroused heated discussion. As the original discoverer of the anti-SARS-CoV-2 activity of cepharanthine, here we briefly summarize the discovery of cepharanthine and review important progress in relevant studies concerning the discovery and validation of anti-SARS-CoV-2 activity of cepharathine, its antiviral mechanisms and clinical trials of its applications in COVID-19 therapy.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus (2019-nCoV) related coronavirus model / 中华医学杂志(英文版)

Background@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those without such facilities or 2019-novel coronavirus (2019-nCoV). This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV related coronavirus model.@*Methods@#A 2019-nCoV related pangolin coronavirus GX_P2V/pangolin/2017/ Guangxi was described. Whether GX_P2X uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA) -mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2X infection. The antiviral activities and antiviral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*Results@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs-cepharanthine (CEP), selamectin and mefloquine hydrochloride exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48±0.02]×10-4 vs. 1.00±0.12, t=150.38, P<0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP has potent antiviral activities against both viral entry (1.00±0.37 vs. 0.46±0.12, t=2.42, P<0.05) and viral replication (1.00±0.43 vs. [6.18±0.95]×10-4, t=3.98, P<0.05).@*Conclusions@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and clinical trial of CEP for treatment of 2019-nCoV infection is warranted.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model / 中华医学杂志(英文版)

BACKGROUND@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those institutions without such facilities or 2019-nCoV. This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV-related coronavirus model.@*METHODS@#A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described. Whether GX_P2V uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA)-mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection. The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*RESULTS@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs, including cepharanthine (CEP), selamectin, and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ± 0.02] × 10vs. 1.00 ± 0.12, t = 150.38, P < 0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP had potent anti-viral activities against both viral entry (0.46 ± 0.12, vs.1.00 ± 0.37, t = 2.42, P < 0.05) and viral replication ([6.18 ± 0.95] × 10vs. 1.00 ± 0.43, t = 3.98, P < 0.05).@*CONCLUSIONS@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin, and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and further study of CEP for treatment of 2019-nCoV infection is warranted.

Characterization of Highly Pathogenic Avian Influenza H5N1 Viruses Isolated from Domestic Poultry in China / 生物医学与环境科学（英文）

The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.

Prognostic analysis of carcinoma of duodenal papilla treated by pancreatoduodenectomy / 中国现代普通外科进展

Objective:To evaluate the prognostic factors for duodenalpapilla carcinoma (DPC)treated by pancreatoduodenectomy (PD).Methods:Clinicopathological data of 68 patients with duodenalpapilla carcinoma who undergone PD and finally diagnosed by surgery and histopathology from January 2001 to June 2010 were retrospectively analyzed.The patients were followed-up until 2015.Survival analysis was conducted using the Kaplan-Meier survival and Cox proportional hazards model analysis.The difference in survival curves was evaluated with a log-rank test.Results:The patients were followed-up with a median follow-up of 57 months (ranging from 4 months to 168 months).The univariate analysis showed that increased serum levels of total bilirubin were correlated with a poor prognosis,as well as a senior grade of infiltration depth,lymph node metastases,and TNM stage (P=0.043,0.003,0.004 0.002,respectively).Only increased serum levels of total bilirubin and a senior grade of TNM stage retained their significance when adjustments were made for other known prognostic factors in Cox multivariate analysis (RR=2.031,P=0.031 and RR=2.255,P=0.029).Conclusions:Increased serum levels of total bilirubin were correlated with a poor prognosis,as well as a senior grade of infiltration depth,lymph node metastases,and TNM stage.Only increased serum levels of total bilirubin and a senior grade of TNM stage can serve as independent prognosis indexes in the evaluation of patients with DPC after PD.

Utility of high throughput sequencing technology in analyzing the terminal sequence of caudovirales bacteriophage genome / 病毒学报

To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.

The curative effect and safety of collagen sponge with different pH and content of protein implanted into orthopedic bone defect / 生物医学工程学杂志

This paper presents a 3mm fracture model of radial bone of rabbits using operation method. The bone defect was treated with collagen sponge with different pH and different content. After the operation, the body temperature, blood routine measurement, serum antibody, X-ray examination and histological observation in each group were examined to evaluate and study the curative effect and safety of collagen sponge. Collagen sponge had a good result of safety, but there was slightly change in blood routine, serum antibody, and histological observation, etc, with the pH changing and different content of collagen. The results showed that there was no obvious influence of safety to tissues after treatment of the collagen sponge at different pH implanted into bone defect. Collagen sponge at lower pH could promote the healing of bone defect partly, while the safety of collagen sponge with lower content was better.

Construction of HCV-producing cell model based on self-cleaving ribozyme / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a stable HCV-producing cell model for anti-HCV drug research.</p><p><b>METHODS</b>The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.</p><p><b>RESULTS</b>HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.</p><p><b>CONCLUSION</b>We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.</p>

Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2 / 南方医科大学学报

<p><b>OBJECTIVE</b>To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.</p><p><b>METHODS</b>The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.</p><p><b>RESULTS</b>PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).</p><p><b>CONCLUSION</b>The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.</p>

Study on the indeterminate results of characterization and verification of HIV antibody from Western blot test / 中华流行病学杂志

Objective To study the serological characterization of indeterminate Western blot(WB)results of HIV antibody and to find a new way to verify the HIV antibody indeterminate results and provide references for editing"National Guideline for Detection of HIV/AIDS".Methods All of the 42 subjects who were confirmed as indeterminate HIV antibody in People'Libaretion Amry HIV Confirmation Laboratory from 2005 to 2006,were collected.Line immunoassay.HIV viral load test and HIV-1 p24 were tested and followed up for 3-6 months'to compare the changes of WB bands patterns.Results (1)For the 42 individuals with indeterminate HIV antibody.a total of 8 different patterns of bands were found in WB test including 45.2% of them were p24 monoband,30.9% were gp160 monoband,11.9% were gp160 with p24,2.4%(only one case)were gp160gp120 ±,gp41p24,p24p17,gp41 or gp120respectively.It was noticed that the most patterns of common bands with indeterminate results were p24 monoband.gpl60 monoband and gpl60 with p24.which composed 88.0% of the whole indeterminate WB band patterns.(2)Twenty three cases had been followed up for more than 3 months with 22 giving no WB band image change and were confirmed as HIV sero-negative.The other one with case gp160 and p24 had developed to more bands in the period of 77 days follow-up with more bands,including gpl60,gp120,p66,p31,p24 and p17,showed up and was confirmed as HIV primary infection.(3)Line immunoassay was applied to all of those 23 cases who had been followed up and the results showed that only one serological change was found and the case was confirmed to be HIV-positive.Among the other 22 cases without serological changes.16 cases were proved to be HIV-negative,6 cases were still indeterminate.The specificitv was 72.7%.P24 antigen test showed negative in all the 23 cases,including the case which later was confirmed as HIV-positive.Of all the 23 originally indeterminate cases,viral loads were tested in 7 eases.Positive result was found in the case which was proved later to be HIV-positive.No viral loads were detected in the other 6 cases(＜LDL).Conclusion The most common band patterns of indeterminate HIV antibody were mainly p24 monoband,gp160 monoband or with p24.Most of them (95.6%)were not infected by HIV,the bands showed up in WB test and demonstrated as non-specific reactions.Line immunoassay could determine about 70% of the indeterminate reactions.Results from viral load test also suggested that it was an efficient method to discriminate indeterminate results.With these two techniques,HIV serology could be diagnosed without 3 months'follow-up in primary infection which gave indeterminate WB results.

Recent Advance in Bacteriophage Therapy / 微生物学通报

At the time of phage’s discovery, phage therapy was regarded as a possible treatment method against bacterial infection. Although phage therapy was used to treat and prevent bacterial infection in the former Soviet Union and Eastern Europe, it was abandoned by the West in the 1940s with the arrival of the antibiotic era. However, the ongoing evolution of bacterial multidrug-resistance has recently motivated the Western scientific community to reevaluate phage therapy for bacterial infections that are incurable by conventional chemotherapy. With the indepth study of phages, it’s increasingly acknowledged that phages, as the medicine to cure bacterial infection, are convenient, safe and efficient therapeutics. This paper summarizes the recent years’ advanced researches in this area.

Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells / 中华外科杂志

<p><b>OBJECTIVES</b>To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.</p><p><b>METHODS</b>The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.</p><p><b>RESULTS</b>PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.</p><p><b>CONCLUSION</b>Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.</p>