Porcine deltacoronavirus (PDCoV) is a major pathogen causing fatal diarrhea in suckling piglets, and there is currently a lack of effective vaccines and drugs to prevent and control the virus. The nonstructural protein 13 (NSP13) serves as a virus-coded helicase and is considered to be a crucial target for antiviral drugs, making it imperative to investigate the helicase activity of NSP13. In this study, the NSP13 gene of PDCoV was synthesized and integrated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-NSP13. NSP13 was successfully expressed in BL21 (DE3) and subsequently purified. The study also verified the helicase activity of the purified NSP13 and explored the factors that influence this activity. The results indicated that NSP13 from PDCoV was effectively expressed in the prokaryotic system and exhibited helicase activity, capable of unwinding double-stranded DNA with a tail at the 5' end. Additionally, NSP13 demonstrated an annealing function by promoting the complementary pairing of single-stranded nucleotide chains to form double strands. The helicase activity of NSP13 was affected by metal ions, but Mg²⁺concentrations in the range of 0.5-6.0 mmol/L had no significant effect on helicase activity of NSP13. When the solution pH was in the range of 4-9, there was no difference in helicase activity. ATP concentrations in the range of 0.25-6.00 mmol/L had a weak effect on helicase activity, and NSP13 concentration ≥80 nmol/L inhibited the helicase activity. We obtained the NSP13 of PDCoV and investigated its helicase activity. These findings provided a theoretical foundation for the further research on the regulatory mechanism of NSP13 in PDCoV replication and the development of anti-coronaviral drugs.
Viral Nonstructural Proteins/metabolism* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Swine ; Animals ; DNA Helicases/metabolism* ; Genetic Vectors/metabolism*

Objective To observe effect of TCM oral care solution in patients with gingival bleed-ing caused by oral administration of warfarin.Methods A total of 150 hospitalized patients in the car-diology department were selected as study objects,and were randomly divided into control group 1,control group 2 and experimental group,with 50 patients per group.Patients in the three groups were given medication care of warfarin and health guidance for mouth care.The control group 1 increased the use of normal saline gargle;the control group 2 increased the use of silver ion gargle,and the ex-perimental group was given the use of traditional Chinese medicine oral care solution gargle.After the intervention,the number of gingival bleeding,gingival sulcus bleeding index(SBI)and halitosis score in the three groups were compared.Results The number of gingival bleeding during gargling(10 d)and within 4 weeks after discharge in the experimental group were less than that in the control group 1 and control group 2,the difference was statistically significant(P＜0.05).The SBI of the experimen-tal group was(1.53±0.69),which was lower than(2.12±0.79)in the control group 1 and(2.09± 0.76)in the control group 2(P＜0.05).The score of halitosis during gargling in the experimental group was(1.70±0.68),which was lower than(2.28±0.74)in the control group 1 and(2.23± 0.79)in the control group 2(P＜0.05).Conclusion The TCM oral care solution for patients with gingival bleeding caused by oral administration of warfarin can significantly reduce the number of gingi-val bleeding,reduce SBI,alleviate bad breath,improve oral health and drug safety.

Objective To observe effect of TCM oral care solution in patients with gingival bleed-ing caused by oral administration of warfarin.Methods A total of 150 hospitalized patients in the car-diology department were selected as study objects,and were randomly divided into control group 1,control group 2 and experimental group,with 50 patients per group.Patients in the three groups were given medication care of warfarin and health guidance for mouth care.The control group 1 increased the use of normal saline gargle;the control group 2 increased the use of silver ion gargle,and the ex-perimental group was given the use of traditional Chinese medicine oral care solution gargle.After the intervention,the number of gingival bleeding,gingival sulcus bleeding index(SBI)and halitosis score in the three groups were compared.Results The number of gingival bleeding during gargling(10 d)and within 4 weeks after discharge in the experimental group were less than that in the control group 1 and control group 2,the difference was statistically significant(P＜0.05).The SBI of the experimen-tal group was(1.53±0.69),which was lower than(2.12±0.79)in the control group 1 and(2.09± 0.76)in the control group 2(P＜0.05).The score of halitosis during gargling in the experimental group was(1.70±0.68),which was lower than(2.28±0.74)in the control group 1 and(2.23± 0.79)in the control group 2(P＜0.05).Conclusion The TCM oral care solution for patients with gingival bleeding caused by oral administration of warfarin can significantly reduce the number of gingi-val bleeding,reduce SBI,alleviate bad breath,improve oral health and drug safety.

Using novel virtual reality (VR) technology to carry out the construction of clinical medical examination question bank, while deepening the reform of clinical medical course examination, it continues to innovate the medical professional evaluation system and improve the flexibility, diversity and scientificity of clinical medicine in teaching and assessment. It is of great and far-reaching significance to improve the teaching level and the quality of medical education in medical colleges and universities. This paper analyzes and discusses the necessity and feasibility of building a clinical medical examination question bank, and the advantages and prospects of integrating VR technology to carry out the construction of clinical medical examination question bank. At the same time, the exploration and practice of the examination question bank construction based on VR technology disscussed in detail would provide innovative thinking and reference for the clinical medical teaching and evaluation, medical personnel training and other aspects in China.

Objective To investigate the protective effect of FLPNF and the improvement of glucose-stimulated insulin secretion against dexamethasone-induced apoptosis of islet cells. Methods INS-1 cells were treated with oligopeptide FLPNF and dexamethasone, either separately or in combination. Proliferation of INS-1 cells in each group was assessed with CCK-8 assay and the insulin secretion stimulated by glucose was detected by ELISA. The apoptotic condition of the cells was observed and assessed with TUNEL and the apoptosis rate of each group was detected using flow cytometry. The expression of major target protein molecules related to apoptosis and Glut2 was detected by Western blotting analysis. Results Dexamethasone inhibited the growth of INS-1 cells in the group treated with dexamethasone. Cell damage was obvious with observable nuclear shrinkage and nuclear rupture. In addition, apoptosis rate was found to be 40.6％±2.4％. The expression of the apoptosis-related protein Bcl-2 and Glut2 was significantly reduced, whereas that of Bax and caspase-3 was significantly increased. After the combined treatment of oligopeptide FLPNF and dexamethasone, the results were reversed, and the apoptosis rate declined to 27.2％±2.0％ (P ＜ 0.001), cell morphology was improved, and the expression of apoptosis-related protein molecules of islet cells and protein Glut2 was significantly improved. Conclusion FLPNF has the ability of protecting islet cells from dexamethasone-induced apoptosis and improving the glucose-stimulated insulin secretion of islet cells.

Objective To analyze the clinical efficacy and safety of thalidomide combined with VAD regimen (vincristine+epirubicin+dexamethasone) or VAD regimen alone in treatment of multiple myeloma (MM) by using metaˉanalysis. Methods Thalidomide combined with VAD regimen was selected as the experimental group and VAD regimen alone was selected as the control group. The literatures were searched from CNKI, Wanfang and VIP database. And then the inclusive literatures were further searched. Randomized controlled trials eligible to the exclusive criteria were extracted, and the quality was evaluated. Metaˉanalysis was conducted by using Stata14.0 software. Results A total of 17 studies and 940 patients were included. The experimental group had a higher total effective rate compared with the control group , and the difference was statistically significant ( RR＝ 1.41, 95% CI 1.30-1.59, P< 0.01). Meanwhile, the experimental group had a better efficacy in improving hemoglobin and deducing βˉmacroglobulin (MG), M protein and myeloma cells compared with the control group; however, the experimental group also had a worse gastrointestinal reaction, and the difference was statistically significant ( RR＝ 1.36, 95% CI 1.04-1.78, P＝ 0.024). Conclusion Thalidomide combined with VAD regimen in treatment of MM has a better clinical efficacy compared with VAD regimen alone, but it also has a worse adverse reaction.

Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in hepatocytes in reduction of hepatic ischemia-reperfusion (I/R) injury by limb ischemic postconditioning in rats.Methods Forty-eight healthy male Sprague-Dawley rats,weighing 240-280 g,were randomly divided into 4 groups (n =12 each) using a random number table:sham operation group (group S);I/R group;limb ischemic postconditioning group (group LIPC);limb ischemic postconditioning and atractyloside group (group LIPC + APC).In I/R,LIPC and LIPC + APC groups,I/R injury was induced by 30 min occlusion of the hepatic artery and portal vein entering the left and middle lobes of the liver,followed by 120 min of reperfusion.The animals underwent 10 min of bilateral limb ischemia starting from 20 min after liver ischemia in group LIPC.In group LIPC+APC,the animals underwent 10 min of bilateral limb ischemia starting from 20 min after liver ischemia,and atractyloside 5 mg/kg was infused simultaneously via the penile vein.Blood samples were collected from inferior vena cava at the end of reperfusion to detect plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities.The rats were sacrificed,and the left lobe of the liver was removed to detect the degree of mPTP opening and to examine the ultrastructure of hepatocytes under electron microscope.Results Compared with group S,the plasma ALT and AST activities and degree of mPTP opening were significantly increased,and the damage to the ultrastructure of hepatocytes was obvious in the other three groups.Compared with group I/R,the plasma ALT and AST activities and degree of mPTP opening were significantly decreased,and the damage to the ultrastructure of hepatocytes was reduced in LIPC and LIPC + APC groups.Compared with group LIPC,the plasma ALT and AST activities and degree of mPTP opening were significantly increased in group LIPC+APC.Conclusion Limb ischemic postconditioning reduces hepatic I/R injury through inhibiting mPTP opening in hepatocytes of rats.

Objective To investigate the effects of propofol combined with remifentanil on he-patic ischemia-reperfusion injury in cirrhotic rats.Methods Sixty male SD rats of 260 to 300 grams were randomly divided into five groups(n=12):the sham-operated group(group S);the model con-trol group (group M);propofol group (group P);remifentanil group (group R);propofol combined with remifentanil group (group PR).In group M,P,R,PR,the hepatic arteries and veins of middle and left lobes were occluded for 20 min after 1 w hepatocirrhosis by using four principal factors,and group S went through an open surgery only.In groups P,R,and PR,porpofol (at a rate 20 mg·kg-1 ·h-1 for 1 h)、remifentanil (at a rate 1 μg·kg-1·min-1 for 1 h)and porpofol (at a rate 20 mg·kg-1· h-1 )combined with remifentanil(at a rate 1 μg·kg-1·min-1 for 1 h)was infused iv at 10 min before is-chemia,respectively.In group M,normal saline was infused iv at the same rate.Blood samples were taken at the end of 4 h reperfusion to determine serum AST,ALT activity.Meanwhile liver specimens were collected to assess Bcl-2 and Bax protein expression in liver cell and measuring the apoptosis in-dex(AI)of hepatic cell.The pathological change of liver was also examined.Results Compared with group S,the activity of AST,ALT,the expression of Bcl-2 and Bax and the apoptosis index(AI)of hepatic cell of the other groups all increased significantly(P <0.05);Compared with group M,the expression of Bcl-2 of groups P,R,PR significantly increased,and the activity of AST,ALT,the ex-pression of Bax and the apoptosis index(AI)of hepatic cell significantly decreased(P <0.05 );Com-pared with groups P and R,the expression of Bcl-2 of group PR significantly increased,and the activ-ity of AST,ALT,the expression of Bax and the apoptosis index(AI)of hepatic cell significantly de-creased(P <0.05);Microscopy and scanning electron microscopy showed that,for groups P,R,PR, pathological injury of liver tissue significantly reduced compared with group M.Conclusion Propofol combined with remifentanil can reduce the hepatic ischemia-reperfusion injury in cirrhotic rat by regu-lating Bcl-2 and Bax protein expression and inhibiting apoptosis.The effect is much more enhanced when they are used in combination than individuals.

Objective To study the impact of medicine retention time and the therapeutic effect to the chronic renal failure by retention enema with Chinese traditional Medicine at different hour.Methods Totals of 60 patients with chronic renal failure disease were randomly divided into observation group (n=30)and control group (n=30).The observation group adopted enema at 7:00-9:00, and the control group adopted enema at routine hour ( 19:00 -21:00 ) .Following items were observed and compared between two groups: subjective feeling, adverse reflection, defecating ratio before enema, the appetency of defecating after enema, medicine retention time after enema, the Scr before and after enema, the change of BUN and Ua.The observation period was 30 days ( one period of the treatment) .Results Compared with the control group, the adverse reflection, such as bellyache, abdominal distension, anus ache, downfallen pressure feeling at anus were significantly relieved in observation group (P<0.05).The times & severity of the appetency of defecating after enema in observation group was reduced apparently than that in control group (P<0.05).The average defecating ratio before enema in observation group was significantly improved (83.3%vs 20.0%;χ2 =79.452,P<0.01).The retention time in observation group was ( 324.12 ±129.47 ) min, and that in control group was ( 225.32 ± 104.21)min,and the difference was statistically significant (t=4.605,P<0.01).The blood BUN, Scr, Ua were down after enema in both groups, but the improvement in observation group was significantly better than that in the control group (P <0.05).Conclusions By practicing retention enema with Chinese traditional Medicine at 7:00-9:00, the medicine retention time in intestines can be extended, and the therapeutic effect to the chronic renal failure can be improved.

Objective To explore the characteristics of hemi-nested methylation specific polymerase chain reaction (hn-MSP) and to find out the possible relationship between patterns of methylation or deletion and the developmet of adult acute lymphoblastic leukemia(ALL).Methods hn-MSP and bisulfit-sequencing PCR (BSP) were designed and adopted to analyze p15 gene methylation or deletion patterns in 25 adult ALL patients,malignant hematopathy cell lines and normal lymphocytes. hn-MSP and BSP products were cloned and sequenced.The sensitivity and specificity of hn-MSP were also analized.Results The sequencing results of hn-MSP and BSP products were consistent, and the sensitivity of detection of p15 methylation was up to 1.0×10-5.17 adult ALL patients (68 ％) were p15 gene hypermethylation and 3 patients were with deletion of p15 gene exon 1.There were no hypermethylation or deletion in the 10 controls.Conclusions The detection rate of p15 methylation in many tumors,especially in adult ALL,is frequent high.hn-MSP is highly sensitive and specific in analyzing p15 methylation.