AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridizat...AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.展开更多
BACKGROUND Sodium glucose cotransporter-2 inhibitors(SGLT-2i)are a class of drugs with modest antidiabetic efficacy,weight loss effect,and cardiovascular benefits as proven by multiple randomised controlled trials(RCT...BACKGROUND Sodium glucose cotransporter-2 inhibitors(SGLT-2i)are a class of drugs with modest antidiabetic efficacy,weight loss effect,and cardiovascular benefits as proven by multiple randomised controlled trials(RCTs).However,real-world data on the comparative efficacy and safety of individual SGLT-2i medications is sparse.AIM To study the comparative efficacy and safety of SGLT-2i using real-world clinical data.METHODS We evaluated the comparative efficacy data of 3 SGLT-2i drugs(dapagliflozin,canagliflozin,and empagliflozin)used for treating patients with type 2 diabetes mellitus.Data on the reduction of glycated hemoglobin(HbA1c),body weight,blood pressure(BP),urine albumin creatinine ratio(ACR),and adverse effects were recorded retrospectively.RESULTS Data from 467 patients with a median age of 64(14.8)years,294(62.96%)males and 375(80.5%)Caucasians were analysed.Median diabetes duration was 16.0(9.0)years,and the duration of SGLT-2i use was 3.6(2.1)years.SGLT-2i molecules used were dapagliflozin 10 mg(n=227;48.6%),canagliflozin 300 mg(n=160;34.3%),and empagliflozin 25 mg(n=80;17.1).Baseline median(interquartile range)HbA1c in mmol/mol were:dapagliflozin-78.0(25.3),canagliflozin-80.0(25.5),and empagliflozin-75.0(23.5)respectively.The respective median HbA1c reduction at 12 months and the latest review(just prior to the study)were:66.5(22.8)&69.0(24.0),67.0(16.3)&66.0(28.0),and 67.0(22.5)&66.5(25.8)respectively(P<0.001 for all comparisons from baseline).Significant improvements in body weight(in kilograms)from baseline to study end were noticed with dapagliflozin-101(29.5)to 92.2(25.6),and canagliflozin 100(28.3)to 95.3(27.5)only.Significant reductions in median systolic and diastolic BP,from 144(21)mmHg to 139(23)mmHg;(P=0.015),and from 82(16)mmHg to 78(19)mmHg;(P<0.001)respectively were also observed.A significant reduction of microalbuminuria was observed with canagliflozin only[ACR 14.6(42.6)at baseline to 8.9(23.7)at the study end;P=0.043].Adverse effects of SGLT-2i were as follows:genital thrush and urinary infection-20(8.8%)&17(7.5%)with dapagliflozin;9(5.6%)&5(3.13%)with canagliflozin;and 4(5%)&4(5%)with empagliflozin.Diabetic ketoacidosis was observed in 4(1.8%)with dapagliflozin and 1(0.63%)with canagliflozin.CONCLUSION Treatment of patients with SGLT-2i is associated with statistically significant reductions in HbA1c,body weight,and better than those reported in RCTs,with low side effect profiles.A review of large-scale real-world data is needed to inform better clinical practice decision making.展开更多
AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other...AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Barn HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits.RESULTS: The sequences of MTE5 and the 6 constructs ofrecombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the coinoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+69.1 mIU/mL vs 27.6+17.3 mIU/mL, P<0.01, t =-6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.展开更多
文摘AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
文摘BACKGROUND Sodium glucose cotransporter-2 inhibitors(SGLT-2i)are a class of drugs with modest antidiabetic efficacy,weight loss effect,and cardiovascular benefits as proven by multiple randomised controlled trials(RCTs).However,real-world data on the comparative efficacy and safety of individual SGLT-2i medications is sparse.AIM To study the comparative efficacy and safety of SGLT-2i using real-world clinical data.METHODS We evaluated the comparative efficacy data of 3 SGLT-2i drugs(dapagliflozin,canagliflozin,and empagliflozin)used for treating patients with type 2 diabetes mellitus.Data on the reduction of glycated hemoglobin(HbA1c),body weight,blood pressure(BP),urine albumin creatinine ratio(ACR),and adverse effects were recorded retrospectively.RESULTS Data from 467 patients with a median age of 64(14.8)years,294(62.96%)males and 375(80.5%)Caucasians were analysed.Median diabetes duration was 16.0(9.0)years,and the duration of SGLT-2i use was 3.6(2.1)years.SGLT-2i molecules used were dapagliflozin 10 mg(n=227;48.6%),canagliflozin 300 mg(n=160;34.3%),and empagliflozin 25 mg(n=80;17.1).Baseline median(interquartile range)HbA1c in mmol/mol were:dapagliflozin-78.0(25.3),canagliflozin-80.0(25.5),and empagliflozin-75.0(23.5)respectively.The respective median HbA1c reduction at 12 months and the latest review(just prior to the study)were:66.5(22.8)&69.0(24.0),67.0(16.3)&66.0(28.0),and 67.0(22.5)&66.5(25.8)respectively(P<0.001 for all comparisons from baseline).Significant improvements in body weight(in kilograms)from baseline to study end were noticed with dapagliflozin-101(29.5)to 92.2(25.6),and canagliflozin 100(28.3)to 95.3(27.5)only.Significant reductions in median systolic and diastolic BP,from 144(21)mmHg to 139(23)mmHg;(P=0.015),and from 82(16)mmHg to 78(19)mmHg;(P<0.001)respectively were also observed.A significant reduction of microalbuminuria was observed with canagliflozin only[ACR 14.6(42.6)at baseline to 8.9(23.7)at the study end;P=0.043].Adverse effects of SGLT-2i were as follows:genital thrush and urinary infection-20(8.8%)&17(7.5%)with dapagliflozin;9(5.6%)&5(3.13%)with canagliflozin;and 4(5%)&4(5%)with empagliflozin.Diabetic ketoacidosis was observed in 4(1.8%)with dapagliflozin and 1(0.63%)with canagliflozin.CONCLUSION Treatment of patients with SGLT-2i is associated with statistically significant reductions in HbA1c,body weight,and better than those reported in RCTs,with low side effect profiles.A review of large-scale real-world data is needed to inform better clinical practice decision making.
基金Supported by the National Natural Science Foundation of China,NO.39970677 and the Science Foundation of Guangdong Province,NO.99M04801G
文摘AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Barn HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits.RESULTS: The sequences of MTE5 and the 6 constructs ofrecombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the coinoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+69.1 mIU/mL vs 27.6+17.3 mIU/mL, P<0.01, t =-6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.
