BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal ca...BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal cancer(CRC)with colitisassociated cancer(CAC).PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes.Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth.AIM To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.METHODS Genomic DNA from 32 colonic samples,including CAC(n=7),UC(n=10)and CRC(n=15),was sequenced for the rs10889677 mutation.The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector.The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line.CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium,then buparlisib was administered after 14 d.The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.RESULTS Luciferase activity decreased by 2.07-fold in the rs10889677 mutant,confirming the hypothesis that the variant disrupted miRNA binding sites,which led to an increase in IL23R expression and the activation of the PI3K signaling pathway.Furthermore,CAC-induced mice had a significantly higher disease activity index(P<0.05).Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice(P<0.05),reduced the percentage of proliferating cells by 5%,and increased the number of apoptotic cells.The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.CONCLUSION Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway,and buparlisib had the ability to prevent PI3K-non展开更多
Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial ...Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects.However,high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches.Here,we report that NanoLuc(Nluc)showed better performance than LuxCDABE in bacteria.However,the retention rate of plasmid constructs in bacteria was low.To construct stable Staphylococcus aureus reporter strains,a partner protein enolase(Eno)was identified by screening of S.aureus strain USA300 for fusion expression of Nluc-based luciferases,including Nluc,Teluc,and Antares2.Different substrates,such as hydrofurimazine(HFZ),furimazine(FUR),and diphenylterazine(DTZ),were used to optimize a stable reporter strain/substrate pair for BL imaging.S.aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S.aureus in vivo,with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys.USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics.The optimized S.aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.展开更多
Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role ...Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.展开更多
基金The Fundamental Research Grant Scheme,Ministry of Higher Education,Malaysia,No.FRGS/1/2018/SKK06/UKM/02/4.
文摘BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal cancer(CRC)with colitisassociated cancer(CAC).PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes.Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth.AIM To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.METHODS Genomic DNA from 32 colonic samples,including CAC(n=7),UC(n=10)and CRC(n=15),was sequenced for the rs10889677 mutation.The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector.The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line.CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium,then buparlisib was administered after 14 d.The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.RESULTS Luciferase activity decreased by 2.07-fold in the rs10889677 mutant,confirming the hypothesis that the variant disrupted miRNA binding sites,which led to an increase in IL23R expression and the activation of the PI3K signaling pathway.Furthermore,CAC-induced mice had a significantly higher disease activity index(P<0.05).Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice(P<0.05),reduced the percentage of proliferating cells by 5%,and increased the number of apoptotic cells.The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.CONCLUSION Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway,and buparlisib had the ability to prevent PI3K-non
基金supported by the National Natural Science Foundation of China (No.82072238 to W.S.and 82272341 to X.R.).
文摘Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects.However,high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches.Here,we report that NanoLuc(Nluc)showed better performance than LuxCDABE in bacteria.However,the retention rate of plasmid constructs in bacteria was low.To construct stable Staphylococcus aureus reporter strains,a partner protein enolase(Eno)was identified by screening of S.aureus strain USA300 for fusion expression of Nluc-based luciferases,including Nluc,Teluc,and Antares2.Different substrates,such as hydrofurimazine(HFZ),furimazine(FUR),and diphenylterazine(DTZ),were used to optimize a stable reporter strain/substrate pair for BL imaging.S.aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S.aureus in vivo,with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys.USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics.The optimized S.aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.
基金the grants from the National Basic Research Program of China (No. 2007CB507400)the National Natural Science Foundation of China (No. 30500082 and No.30671064)
文摘Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.
