The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization.However,the in vivo distribution and release of siRNA still cannot be effect...The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization.However,the in vivo distribution and release of siRNA still cannot be effectively monitored.In this study,based on the fluorescence resonance energy transfer(FRET)principle,a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds(Au-DR-siRNA),which were then wrapped with lipid nanoparticles(LNPs)for monitoring the release behaviour of siRNA in vivo.The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells,the fluorescence of Cy5 would change from quenched state to activated state,showing the location and time of siRNA release.Besides,the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds.Therefore,this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA,but also a siRNA delivery system for treating and diagnosing tumors.展开更多
COVID-19 has devastated numerous nations around the world and has overburdened numerous healthcare systems,which has also caused the loss of livelihoods due to prolonged shutdowns and further led to a cascading effect...COVID-19 has devastated numerous nations around the world and has overburdened numerous healthcare systems,which has also caused the loss of livelihoods due to prolonged shutdowns and further led to a cascading effect on the global economy.COVID-19 infections have an incubation period of 2–7 days,but 40 to 45%of cases are asymptomatic or show mild to moderate respiratory symptoms after the period due to subclinical lung abnormalities,making it more likely to spread the pandemic disease.To restrict the spread of the virus,on-site diagnosis methods that are quicker,more precise,and easily accessible are required.Rapid Antigen Detection Tests and Polymerase Chain Reaction tests are currently the primary methods used to determine the presence of COVID-19 viruses.These tests are typically time-consuming,not accurate,and,more importantly,not available to everyone.Hence,in this review and hypothesis,we proposed equipment that employs the properties of photonics to improve the detection of COVID-19 viruses by taking the advantage of typical binding of coronavirus with angiotensin-converting enzyme 2(ACE2)receptors.This hypothetical model would combine Surface-Enhanced Raman Scattering(SERS)and Fluorescence Resonance Energy Transfer(FRET)to provide great flexibility,high sensitivities,and enhanced accessibility.展开更多
Following the gradual maturation of synthetic techniques for nanomaterials,exciton-plasmon composites have become a research hot-spot due to their controllable energy transfer through electromagnetic fields on the nan...Following the gradual maturation of synthetic techniques for nanomaterials,exciton-plasmon composites have become a research hot-spot due to their controllable energy transfer through electromagnetic fields on the nanoscale.However,most reports ignore fluorescence resonance energy transfer(FRET)under electrostatic repulsion conditions.In this study,the FRET process is investigated in both electrostatic attraction and electrostatic repulsion systems.By changing the Au:quantum dot ratio,local-field induced FRET can be observed with a lifetime of ns and a fast component of hundreds of ps.These results indicate that the intrinsic transfer process can only elucidated by considering both steady and transient state information.展开更多
Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used ...Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.展开更多
Retraction note:Khan M,Rauf W,Habib F,Rahman M,Iqbal M.Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonan...Retraction note:Khan M,Rauf W,Habib F,Rahman M,Iqbal M.Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry.World J Hepatol 2020;12(11):976-992 PMID:33312423 DOI:10.4254/wjh.v12.i11.976.The online version of the original article can be found at https://www.wjgnet.com/1948-5182/full/v12/i11/976.htm.展开更多
BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiv...BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.展开更多
目的探讨棕榈酰化修饰调节非受体酪氨酸激酶Fyn活性的分子机制。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时检测细胞中的Fyn活性,并结合棕榈酰化位点缺失和共转染蛋白质酪氨酸激酶(C-terminal Src...目的探讨棕榈酰化修饰调节非受体酪氨酸激酶Fyn活性的分子机制。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时检测细胞中的Fyn活性,并结合棕榈酰化位点缺失和共转染蛋白质酪氨酸激酶(C-terminal Src kinase,CSK)表达质粒研究其分子机制。结果实验发现,(C3,C6)任一位点的棕榈酰化缺失能引起Fyn的高活性表达,且C6位点影响更显著。已知CSK激活后发生膜转移,FRET检测证实其对细胞中的Fyn活性有下调作用,但不能有效调控(C3,C6)棕榈酰化位点缺失的Fyn(GSS)活性。结论本文结果初步支持了Fyn活性受细胞内的物理空间定位分布的一种调控机制假设,即棕榈酰化缺失的Fyn(GSS)受细胞膜上CSK抑制性的调节作用被减弱,从而促进了组成性的高活性表达。展开更多
目的探索Jurkat T细胞中胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)活性动力学以及基质刚度对ERK活性的影响。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时观测Jurkat细胞中ER...目的探索Jurkat T细胞中胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)活性动力学以及基质刚度对ERK活性的影响。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时观测Jurkat细胞中ERK活性的变化,或细胞处于I型胶原基质胶中检测其影响。结果部分Jurkat细胞中存在ERK活性脉冲现象,频率约为3次/h,FRET振幅变化约为20%。在抗体激活T细胞抗原受体(T-cell receptor,TCR)的条件下,ERK脉冲依然存在,频率和振幅无显著变化。当细胞处于I型胶原水凝胶中,随着胶基质刚度增加,脉冲频率有所下调。结论Jurkat T细胞中存在自发的ERK活性脉冲现象,初步实验显示其频率受基质刚度影响。而该信号波动的生理意义和分子机制仍有待探索。展开更多
真菌毒素对人和动物具有剧毒和致癌性,且预防和控制其对食品造成的污染较为困难,因此人们对真菌毒素的关注度越来越高。检测食品中的真菌毒素十分必要,传统检测方法的检测结果准确、可靠,但所需设备昂贵、检测时间长,不符合快速检测真...真菌毒素对人和动物具有剧毒和致癌性,且预防和控制其对食品造成的污染较为困难,因此人们对真菌毒素的关注度越来越高。检测食品中的真菌毒素十分必要,传统检测方法的检测结果准确、可靠,但所需设备昂贵、检测时间长,不符合快速检测真菌毒素的要求。因此,需要开发出快速、灵敏、准确且经济的真菌毒素检测方法。基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)效应的荧光传感器由于操作简单、反应速度快、结果可靠且成本低而广泛应用于检测行业。本文主要介绍了基于FRET效应荧光传感器的检测机制,综述了该传感器在真菌毒素检测中的应用情况,提出了目前荧光传感器仍存在的问题并对其未来的发展趋势进行了展望,以期为新型荧光传感器的设计及真菌毒素检测时灵敏度的优化提供参考。展开更多
The structural and functional study of protein is a major topic of current functional genomics. Fluorescence resonance energy transfer (FRET) is one of few tools available for measuring nanometer scale distances and c...The structural and functional study of protein is a major topic of current functional genomics. Fluorescence resonance energy transfer (FRET) is one of few tools available for measuring nanometer scale distances and changes in distances in vivo . FRET is an ideal technology for detection of protein conformation and protein-protein interaction by using fluorescence protein, traditional organic dyes and other dyes as probes. It uses fluorescence protein, traditional organic dyes and other dyes as its probes. The application of FRET in the determination of intracellular events would be helpful for us to understand the structure and function of biology molecules. [展开更多
基金by the National Natural Science Foundation of China(81872812,82073800)the China Postdoctoral Science Fundation(2021TQ0111,2021M691040).
文摘The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization.However,the in vivo distribution and release of siRNA still cannot be effectively monitored.In this study,based on the fluorescence resonance energy transfer(FRET)principle,a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds(Au-DR-siRNA),which were then wrapped with lipid nanoparticles(LNPs)for monitoring the release behaviour of siRNA in vivo.The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells,the fluorescence of Cy5 would change from quenched state to activated state,showing the location and time of siRNA release.Besides,the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds.Therefore,this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA,but also a siRNA delivery system for treating and diagnosing tumors.
文摘COVID-19 has devastated numerous nations around the world and has overburdened numerous healthcare systems,which has also caused the loss of livelihoods due to prolonged shutdowns and further led to a cascading effect on the global economy.COVID-19 infections have an incubation period of 2–7 days,but 40 to 45%of cases are asymptomatic or show mild to moderate respiratory symptoms after the period due to subclinical lung abnormalities,making it more likely to spread the pandemic disease.To restrict the spread of the virus,on-site diagnosis methods that are quicker,more precise,and easily accessible are required.Rapid Antigen Detection Tests and Polymerase Chain Reaction tests are currently the primary methods used to determine the presence of COVID-19 viruses.These tests are typically time-consuming,not accurate,and,more importantly,not available to everyone.Hence,in this review and hypothesis,we proposed equipment that employs the properties of photonics to improve the detection of COVID-19 viruses by taking the advantage of typical binding of coronavirus with angiotensin-converting enzyme 2(ACE2)receptors.This hypothetical model would combine Surface-Enhanced Raman Scattering(SERS)and Fluorescence Resonance Energy Transfer(FRET)to provide great flexibility,high sensitivities,and enhanced accessibility.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.10904049 and 61575079)the Science and Technology Development Program of Jilin Province+4 种基金China(Grant No.20180101230JC)the Fundamental Research Funds for the Central Universities(Grant No.JCKYQKJC45)China Postdoctoral Science Foundation(Grant No.201003537)the Scientific Research Foundation for the Returned Overseas Chinese Scholars,the Ministry of EducationChina。
文摘Following the gradual maturation of synthetic techniques for nanomaterials,exciton-plasmon composites have become a research hot-spot due to their controllable energy transfer through electromagnetic fields on the nanoscale.However,most reports ignore fluorescence resonance energy transfer(FRET)under electrostatic repulsion conditions.In this study,the FRET process is investigated in both electrostatic attraction and electrostatic repulsion systems.By changing the Au:quantum dot ratio,local-field induced FRET can be observed with a lifetime of ns and a fast component of hundreds of ps.These results indicate that the intrinsic transfer process can only elucidated by considering both steady and transient state information.
基金supported in part by the National Natural Science Foundation of China(61871251 and 61871022)Sichuan Science and Technology Program(2019YFSY0048)
文摘Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.
文摘Retraction note:Khan M,Rauf W,Habib F,Rahman M,Iqbal M.Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry.World J Hepatol 2020;12(11):976-992 PMID:33312423 DOI:10.4254/wjh.v12.i11.976.The online version of the original article can be found at https://www.wjgnet.com/1948-5182/full/v12/i11/976.htm.
文摘BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.
文摘目的探索Jurkat T细胞中胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)活性动力学以及基质刚度对ERK活性的影响。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时观测Jurkat细胞中ERK活性的变化,或细胞处于I型胶原基质胶中检测其影响。结果部分Jurkat细胞中存在ERK活性脉冲现象,频率约为3次/h,FRET振幅变化约为20%。在抗体激活T细胞抗原受体(T-cell receptor,TCR)的条件下,ERK脉冲依然存在,频率和振幅无显著变化。当细胞处于I型胶原水凝胶中,随着胶基质刚度增加,脉冲频率有所下调。结论Jurkat T细胞中存在自发的ERK活性脉冲现象,初步实验显示其频率受基质刚度影响。而该信号波动的生理意义和分子机制仍有待探索。
文摘真菌毒素对人和动物具有剧毒和致癌性,且预防和控制其对食品造成的污染较为困难,因此人们对真菌毒素的关注度越来越高。检测食品中的真菌毒素十分必要,传统检测方法的检测结果准确、可靠,但所需设备昂贵、检测时间长,不符合快速检测真菌毒素的要求。因此,需要开发出快速、灵敏、准确且经济的真菌毒素检测方法。基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)效应的荧光传感器由于操作简单、反应速度快、结果可靠且成本低而广泛应用于检测行业。本文主要介绍了基于FRET效应荧光传感器的检测机制,综述了该传感器在真菌毒素检测中的应用情况,提出了目前荧光传感器仍存在的问题并对其未来的发展趋势进行了展望,以期为新型荧光传感器的设计及真菌毒素检测时灵敏度的优化提供参考。
文摘The structural and functional study of protein is a major topic of current functional genomics. Fluorescence resonance energy transfer (FRET) is one of few tools available for measuring nanometer scale distances and changes in distances in vivo . FRET is an ideal technology for detection of protein conformation and protein-protein interaction by using fluorescence protein, traditional organic dyes and other dyes as probes. It uses fluorescence protein, traditional organic dyes and other dyes as its probes. The application of FRET in the determination of intracellular events would be helpful for us to understand the structure and function of biology molecules. [
