A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

The core protein allosteric modulator targets the core protein and inhibits hepatitis B virus(HBV)replication by regulating the formation of covalently closed circular DNA(cccDNA), which is expected to completely cure hepatitis B and overcome the drug resistance of nucleoside drugs. This paper reviews the replication process of HBV, the function of core proteins, the mechanism, classification and research progress of core protein allosteric modulators, lists 12 drugs, and summarizes their mechanisms, categories, chemical structures, safety, anti-HBV effects, combined drug use, etc. In addition, the advantages and problems of core protein allosteric modulators are discussed to provide references for the development of new anti-HBV drugs.

To explore the effect of down-regulation of growth arrest and DNA damage inducible protein 45β (GADD45β) on the PC9 lung adenocarcinoma cells.
 Methods: GADD45β gene siRNA sequence was designed and synthesized, which was transfected into PC9 lung adenocarcinoma cells through lentivirus transfection. Quantitative real-time PCR (qRT-PCR) and Western blot are used to examine the mRNA and protein levels of GADD45β in PC9 cells before and after the transfection. Annexin V-allophycocyanin (APC) double-staining flow cytometry was used to detect the apoptosis level after the transfection. The intracellular DNA content after transfection was detected by flow cytometry. The percentage of the cells at each period of cell cycle was calculated, and the effect of RNA interference on the cell growth were analyzed. The effects of RNA interference on the tumor-formation ability of cells were tested by counting the number of clones. MTT assay was used to test the half maximal inhibitory concentration (IC50) of PC9 cells for gefitinib. 
 Results: The 5'-AAATCCACTTCACGCTCAT-3' sequence was identified as the effective sequence for GADD45β gene RNA interference. The mRNA and protein expression levels of GADD45β were markedly decreased (both P<0.05) at 48 h after transfection of GADD45β-siRNA, which resulted in the increased apoptosis rate (P<0.05), decreased tumor clone number (P<0.05) and increased percentage of PC9 cell at the S stage and G2/M stage (P<0.05). The IC50 for gefitinib was decreased obviously (P<0.05).
 Conclusion: Down-regulation of GADD45β can reduce the colony-forming ability of PC9 cells, promote the cell apoptosis, and enhance the sensitivity of PC9 cells to gefitinib.
Adenocarcinoma of Lung ; Antigens, Differentiation ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Gefitinib ; pharmacology ; Humans ; RNA, Small Interfering

Objective: To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. Methods: Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3⁺ T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. Results: Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ²=11.73, P=0.008). Conclusion Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future.

Objective: To investigate the effects of proteasome beta 5 subunit (PSMB5) on proliferation and bortezomib (BTZ) chemo-sensitivity of multiple myeloma (MM) and its related molecular mechanisms. Methods: We used two MM cell lines, RPMI 8226 and BTZ drug-resistant cell line RPMI 8226/BTZ100 (hereinafter referred to as BTZ100) , as the research object. PSMB5 was overexpressed or knocked down in two myeloma cell lines via lentivirus transfection. CCK8 assay was used to detect the impact of PSMB5 on cell viability and bortezomib sensitivity in human myeloma cells; Using flow cytometry to test the effects of PSMB5 on apoptosis rate of human myeloma cells under the treatment of bortezomib; Apoptosis-related gene expression of Bax, Bcl-2, p-Akt and cleaved caspase-3 were detected by Western blot. Results: ①PSMB5 overexpression and knockdown were successfully constructed in RPMI 8226 and BTZ100 cells. ②PSMB5 expression was positively correlated with cell proliferation of RPMI 8226 and BTZ100 cells (P<0.05) . ③The cell viability was lower after PSMB5 knockdown in RPMI 8226 cells than control cells under the same concentration of BTZ[IC₅₀ at 24 h: (7.01±0.47) and (9.64±0.55) nmol/L respectively, t=6.289, P=0.003]. The cell viability was higher after PSMB5 overexpression in RPMI 8226 cells than control cells under the same concentration of BTZ[IC₅₀ at 24 h: (10.99±0.58) and (9.51±0.37) nmol/L respectively, t=3.724, P=0.020) . PSMB5 expression was negatively correlated with the sensitivity of RPMI 8226 cells to BTZ. The results of BTZ100 cells were similar. ④The expression of PSMB5 was negatively correlated with the apoptosis of RPMI 8226 and BTZ100 under the treatment of BTZ. ⑤Meanwhile, PSMB5 knockdown could increase the expression of pro-apoptosis gene Bax and cleaved caspase-3 and decrease the expression of anti-apoptotic gene Bcl-2 and p-Akt. PSMB5 over-expression has the opposite results. Conclusion PSMB5 knockdown could improve the bortezomib sensitivity of MM cells via activation of apoptosis signaling. PSMB5 may be a potential therapeutic target for MM.

Objective:To construct GST-tagged human NRP-1 fusion protein expression vector and induce its expression in Escherichia coli ( E.coli) ,then carry on inclusion body refolding and purification so as to obtain GST-NRP-1 fusion protein.Methods:NRP-1 gene was amplified by RT-PCR and inserted into pCR-blunt vector.Then the reconstructed plasmid was inserted into prokaryotic expression vector pGEX-4T-1.The constructed pGEX-4T-1-NRP-1 expression vector was transformed into BL21 cells and induced by i-sopropyl-β-D-thiogalactoside ( IPTG).Bacterial bodies were disrupted by sonication.Then the soluble fraction of fusion proteins were verified by Western blot and purified by Glutathione Sepharose 4B after inclusion body refolding.Results: The NRP-1 gene fragment was amplified by RT-PCR and inserted into pCR-blunt vector.Fusion protein expression vector pGEX-4T-1-NRP-1 was constructed suc-cessfully.After transformation, GST-NRP-1 expression vector was detected in BL21 cells and obtained purifying protein after refolding.Conclusion:The plasmid GST-NRP-1 was constructed successfully and laid basis for subsequent studies.

Objective To evaluate the clinical results of total laparoscopic hepatectomy for hepatolithiasis. Methods The clinical data of 72 patients with intrahepatic lithiasis receiving total laparoscopic hepatectomy in our hospital from July 2005 to April 2009 were retrospectively analyzed. Results The mean age of the 72 patients was (43. 8±21.7) yrs (16-65 yrs). For laparoscopic hepatectomy, it was anatomical left liver resection in 34 patients, anatomical resection of left lateral liver in 19 and resection of S6 in 16. The operative duration was (262.5± 115.5)min (125-320 min). The median intraoperative blood loss was 150 ml (50-400 ml). The occurring rate of postoperative complications was 12.50 %. Complications included bile duct infection in 8 patients, bile leakage in 6, gastroparesis in 1,postoperative early inflammatory ileus in 1 and subcapsular fluid collection of liver in 1. All the complications were cured by non-surgical means. Conclusion In the era of minimally invasive surgery, total laparoscopic hepatectomy has gradually become the prominent treatment for hepatolithiasis.

Objective To evaluate the histological response and the clinical value in laparescopic colorectal surgery combined with preoperative regional intra-arterial chemotherapy(PRAC). Methods In cases of rectal cancer and fiver metastases selective regional intra-arterial chemotherapy and iodinated oil embolism was carried out in 23 cases of colorectal carcinoma. After 1 to 11 days laparoseopic radical resection was done, specimens were sent for histopathological examination. We analyzed the correlation between tumor differentiation and TNM stage, compared the effect of PRAC with PRAC + embolism by the criteria of histological response of chemotherapy. χ2-test was used to compare interclass correlation.Results The histological effect in the 23 cases of PRAC level 0 was in 2 cases, level Ⅰ in 7 cases, level Ⅱ in 10 cases, and level Ⅲ in 2 cases. The overall effective rate was 91% (21/23). In the 15 cases with lymph node metastases, the effective rate was 87% (13/15). There was no significant statistic correlation between tumor differentiation or TNM stage and histological response. PRAC associated embolism had a better histological response compared to PRAC alone. Conclusions Preoperative regional intra-arterial chemotherapy had marked therapeutic effect on histological response to the colorectal carcinoma patients of various tumor differentiations and TNM stage, especially combining with the embolism to rectal cancer could improve the efficacy.

Objective To investigate the feasibility and effectiveness of laparoscopic splenoctomy (LS)in patients with idiopathic thrombocytopenic purpura(ITP). Methods Clinical data of 17 ITP cases undergoing LS between Augest 2003 and December 2006 were analyzed retrospectively. Remits LS was Successfully conducted in all 17 cases without converting to open surgery with an average intraoperative blood loss of 120 ml in each case.There was no postoperative bleeding,fistula and infection.The platelet count increased rapidly in one week.After stopping glucocorticoid treatment for one month.15 cases achieved complete response(88.2%)and 2 caSes had partial response(11.8%).Fbllow-up of 3～43 months found no recurrence. Conclusions Use of LS for ITP is safe,feasible and effective.

Objective To evaluate laparoscopic resection of gastric stromal tumors. Methods Clinical data of 20 patients undergoing laparoscopic resection of gastric stromal tumors from June 2003 to October 2007 were retrospectively analyzed. Result Laparoscopic wedge resection was completed successfully in all 20 patients with a mean operating time of(60±34) min, and without major complications. The mean hospital stay was (6.0±2.6) days. During a follow-up period from 10 to 22 months there was no recurrence. Conclusions Laparoscopic wedge resection is safe, effective, and minimally invasive for treating gastric stromal tumors.