Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid p...Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid phase separation(LLPS)creates specialized,membraneless physicochemical microenvironments capable of modulating enzymatic functions and overcoming mass transfer limitations.This study aims to investigate whether the gut microbiota-derived DgpB/C complex-a multienzyme system traditionally recognized for cleaving stable C-glycosidic bonds and facilitating isomerization-can undergo functional remodeling within phase-separated condensates.Our core objective is to elucidate the role of phase separation in expanding enzymatic catalytic plasticity and to provide a non-canonical,highly cost-effective biocatalytic mechanism for the direct utilization of free sugars in the synthesis of O-glycosylated natural products.Methods An artificial phase-separation platform was constructed utilizing the multivalent arginine-glycine-glycine motif(RGG)-repeat domain derived from the Caenorhabditis elegans LAF-1 protein.To ensure precise spatial compartmentalization,the DgpB/C complex was specifically recruited into the RGG condensates via a high-affinity SZ1/SZ2 heterodimerization tag system.Condensate formation and substrate partitioning were visualized using light and confocal fluorescence microscopy.The chemical structures and regioselectivity of the reaction products were rigorously characterized using high-performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)/MS systems.Furthermore,molecular docking and 20-ns molecular dynamics(MD)simulations were performed via the Hermite platform and Uni-GBSA pipeline to elucidate the structural and thermodynamic basis underlying the phase-transitioninduced functional shift.Results We observed that the condensates formed by RGG proteins selectively recruited and significantly enriched hydrophobic flavonoid substrates.Strikingly,within the LLPS microenvironment,the DgpB/C complex-which typically exhibits only degradative or isomerase activities-underwent a profound functional remodeling,transforming into an efficient O-glycosyltransferase.Diverging from canonical pathways that require high-energy donors,the sequestered DgpB/C complex directly utilized unactivated free glucose to catalyze the formation of new O-glycosidic bonds.This remodeled activity was found to be evolutionarily conserved across DgpB/C homologs from diverse gut microbiota strains,such as P581a and W974-1.LC-MS/MS analysis further revealed that the phase-separated environment reduced the regioselectivity constraints of the enzyme,enabling catalytic action on multiple hydroxyl positions of the flavonoid scaffold.MD simulations further indicated that the low-water-activity microenvironment of the condensates reconfigured the conformational dynamics of the catalytic pocket,favoring a spatial orientation highly conducive to dehydration condensation.Conclusion This study demonstrates that LLPS drives the functional remodeling of the gut microbiota enzyme DgpB/C through the reconfiguration of the physicochemical microenvironment.These findings challenge traditional perceptions regarding the functional boundaries of metabolic enzymes and reveal a novel biocatalytic pathway that bypasses the requirement for nucleotide-activated sugars.Consequently,this provides a highly promising artificial compartmentalization strategy for the green manufacturing of complex,high-value-added natural products.展开更多
Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-...Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-CoA reductase(LlccR),two double bond reductases(LIDBR1~2),three aldehyde hydrogenases(LlALDH1~3),two 4-coumaroyl:CoA ligases(Ll4CL1~2)and four phloretin glycosyltransferases(LlP4'GT,LlP2'GT1~3)were comprehensively investigated.The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved.Through successive catalysis of LlALDH1,Ll4CL2,and chalcone synthase 1(LlCHS1)or combined action of LlCCR and LlCHS1,phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde,which had been converted from initial precursor p-coumaroyl-CoA by LlCcR-mediated carboxylic acid reduction and LlDBR1-catalyzedα,β-double bond saturation.High accumulation of the DHCs in tender leaves of L.litseifolius was mainly driven by efficient catalysis of LlccR toward p-coumaroyl-CoA and highly expressed genes in the pathway,especially the LlP4'GT and LlP2'GT1 which contributed to biosynthesis of trilobatin and phlorizin,respectively.Antisense oligodeoxyribonucleotide treatments against the LICCR,LIDBR1,LlALDH1,Ll4CL2,LIP4'GT,and LIP2'GT1 significantly reduced transcripts of the target genes and content of DHCs,confirming these genes might be involved in the pathway.This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.展开更多
Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and ...Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.展开更多
为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防...为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防护(lightning location protection,LLP)系统、雷电定位和跟踪(lightning position and tracking system,LPATS)与SAFIR系统(system de surveillance et d’alerte foudre par interferometrie radioelectrique)的定位原理及技术发展,及其在日本各大电力公司与一般商用服务中的应用现状及探测站分布情况;并对3种雷电定位系统或雷电信息采集系统特性进行了对比分析。(2)调查分析了日本电力中央研究所关于Tokyo Skytree的雷击电流观测、雷击电磁波观测等方面的最新研究进展。由调研结果可知:雷击电流、电磁波以及上行先导的联合试验观测体系可以获取大量雷电活动规律与特征参数,非常有助于揭示雷电发展过程中的基本物理现象,探索并完善上行先导的机理与模型等;随着观测设备及技术的提高,可以获取更准确的数据、更精细的雷电发展过程参数,非常有助于进一步提高雷电定位系统的性能与定位精度,有效促进电网主动防雷技术的发展。展开更多
Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we charac...Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells, prpf4 deficiency perturbs Fgf, Wnt/β-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.展开更多
A series of blue long afterglow mixed halide-phosphate phosphors Sr5(PO4)3 FxCll-x:Eu2+,Gd3+were synthesized in air by traditional solid-state reaction routte.The crystal structures,photoluminescence,thermolurninescen...A series of blue long afterglow mixed halide-phosphate phosphors Sr5(PO4)3 FxCll-x:Eu2+,Gd3+were synthesized in air by traditional solid-state reaction routte.The crystal structures,photoluminescence,thermolurninescenee properties and afterglow proper-ties of the phosphors were characterized systematically using X-ray diffraction(XRD),luminescence spectrophotometer,microcom-puter thermoluminescence dosimeter and single photon counter,respectively.Under 280 nm excitation,the broadband emissions of Eu2+ions were observed at 445 nm(blue)due to the 4f7→4f65d transition.It was demonstrated that there existed the self-reduction of the Eu3+to Eu2+ions in this special halide-phosphate matrix in air condition.The addition of Gd3+ions obviously enhanced the after-glow properties of the single doped Eu2+ions in the halide-phosphate phosphors.And the content of the fluoride anions also had sig-nificant influence on the afterglow properties.All results indicated that Srs(PO4)3 FxCI1-x:Eu2+,Gd3+might be potential phosphors for long lasting phosphorescence(LLP)materials.展开更多
摘要Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid phase separation(LLPS)creates specialized,membraneless physicochemical microenvironments capable of modulating enzymatic functions and overcoming mass transfer limitations.This study aims to investigate whether the gut microbiota-derived DgpB/C complex-a multienzyme system traditionally recognized for cleaving stable C-glycosidic bonds and facilitating isomerization-can undergo functional remodeling within phase-separated condensates.Our core objective is to elucidate the role of phase separation in expanding enzymatic catalytic plasticity and to provide a non-canonical,highly cost-effective biocatalytic mechanism for the direct utilization of free sugars in the synthesis of O-glycosylated natural products.Methods An artificial phase-separation platform was constructed utilizing the multivalent arginine-glycine-glycine motif(RGG)-repeat domain derived from the Caenorhabditis elegans LAF-1 protein.To ensure precise spatial compartmentalization,the DgpB/C complex was specifically recruited into the RGG condensates via a high-affinity SZ1/SZ2 heterodimerization tag system.Condensate formation and substrate partitioning were visualized using light and confocal fluorescence microscopy.The chemical structures and regioselectivity of the reaction products were rigorously characterized using high-performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)/MS systems.Furthermore,molecular docking and 20-ns molecular dynamics(MD)simulations were performed via the Hermite platform and Uni-GBSA pipeline to elucidate the structural and thermodynamic basis underlying the phase-transitioninduced functional shift.Results We observed that the condensates formed by RGG proteins selectively recruited and significantly enriched hydrophobic flavonoid substrates.Strikingly,within the LLPS microenvironment,the DgpB/C complex-which typically exhibits only degradative or isomerase activities-underwent a profound functional remodeling,transforming into an efficient O-glycosyltransferase.Diverging from canonical pathways that require high-energy donors,the sequestered DgpB/C complex directly utilized unactivated free glucose to catalyze the formation of new O-glycosidic bonds.This remodeled activity was found to be evolutionarily conserved across DgpB/C homologs from diverse gut microbiota strains,such as P581a and W974-1.LC-MS/MS analysis further revealed that the phase-separated environment reduced the regioselectivity constraints of the enzyme,enabling catalytic action on multiple hydroxyl positions of the flavonoid scaffold.MD simulations further indicated that the low-water-activity microenvironment of the condensates reconfigured the conformational dynamics of the catalytic pocket,favoring a spatial orientation highly conducive to dehydration condensation.Conclusion This study demonstrates that LLPS drives the functional remodeling of the gut microbiota enzyme DgpB/C through the reconfiguration of the physicochemical microenvironment.These findings challenge traditional perceptions regarding the functional boundaries of metabolic enzymes and reveal a novel biocatalytic pathway that bypasses the requirement for nucleotide-activated sugars.Consequently,this provides a highly promising artificial compartmentalization strategy for the green manufacturing of complex,high-value-added natural products.
基金financially supported by the National Natural Science Foundation of China(grant 32272763)China Agriculture Research System of MOF and MARA.and the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding-Tea Plant(grant 2021C02067-6).
摘要Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-CoA reductase(LlccR),two double bond reductases(LIDBR1~2),three aldehyde hydrogenases(LlALDH1~3),two 4-coumaroyl:CoA ligases(Ll4CL1~2)and four phloretin glycosyltransferases(LlP4'GT,LlP2'GT1~3)were comprehensively investigated.The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved.Through successive catalysis of LlALDH1,Ll4CL2,and chalcone synthase 1(LlCHS1)or combined action of LlCCR and LlCHS1,phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde,which had been converted from initial precursor p-coumaroyl-CoA by LlCcR-mediated carboxylic acid reduction and LlDBR1-catalyzedα,β-double bond saturation.High accumulation of the DHCs in tender leaves of L.litseifolius was mainly driven by efficient catalysis of LlccR toward p-coumaroyl-CoA and highly expressed genes in the pathway,especially the LlP4'GT and LlP2'GT1 which contributed to biosynthesis of trilobatin and phlorizin,respectively.Antisense oligodeoxyribonucleotide treatments against the LICCR,LIDBR1,LlALDH1,Ll4CL2,LIP4'GT,and LIP2'GT1 significantly reduced transcripts of the target genes and content of DHCs,confirming these genes might be involved in the pathway.This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.
基金supported by the grants from National Key R&D Program of China(2021YFC2300702 and 2021YFC2300200)the Hubei Provincial Natural Science Foundation of China(2021CFB364)+1 种基金the National Natural Science Foundation of China(82130064,81825015,U22A20337 and 32000119)the Key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001).
摘要Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.
摘要为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防护(lightning location protection,LLP)系统、雷电定位和跟踪(lightning position and tracking system,LPATS)与SAFIR系统(system de surveillance et d’alerte foudre par interferometrie radioelectrique)的定位原理及技术发展,及其在日本各大电力公司与一般商用服务中的应用现状及探测站分布情况;并对3种雷电定位系统或雷电信息采集系统特性进行了对比分析。(2)调查分析了日本电力中央研究所关于Tokyo Skytree的雷击电流观测、雷击电磁波观测等方面的最新研究进展。由调研结果可知:雷击电流、电磁波以及上行先导的联合试验观测体系可以获取大量雷电活动规律与特征参数,非常有助于揭示雷电发展过程中的基本物理现象,探索并完善上行先导的机理与模型等;随着观测设备及技术的提高,可以获取更准确的数据、更精细的雷电发展过程参数,非常有助于进一步提高雷电定位系统的性能与定位精度,有效促进电网主动防雷技术的发展。
基金financially supported by grants from the National Natural Science Foundation of China (Nos.31522035,31371460 and 31590832)
摘要Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells, prpf4 deficiency perturbs Fgf, Wnt/β-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.
基金Project supported by Cooperation Project in Industry,Education and Research of Guangdong Province and Ministry of Education of China(2012B09110044)the Guangdong Provincial Natural Science Foundation of China(9151009001000052)
摘要A series of blue long afterglow mixed halide-phosphate phosphors Sr5(PO4)3 FxCll-x:Eu2+,Gd3+were synthesized in air by traditional solid-state reaction routte.The crystal structures,photoluminescence,thermolurninescenee properties and afterglow proper-ties of the phosphors were characterized systematically using X-ray diffraction(XRD),luminescence spectrophotometer,microcom-puter thermoluminescence dosimeter and single photon counter,respectively.Under 280 nm excitation,the broadband emissions of Eu2+ions were observed at 445 nm(blue)due to the 4f7→4f65d transition.It was demonstrated that there existed the self-reduction of the Eu3+to Eu2+ions in this special halide-phosphate matrix in air condition.The addition of Gd3+ions obviously enhanced the after-glow properties of the single doped Eu2+ions in the halide-phosphate phosphors.And the content of the fluoride anions also had sig-nificant influence on the afterglow properties.All results indicated that Srs(PO4)3 FxCI1-x:Eu2+,Gd3+might be potential phosphors for long lasting phosphorescence(LLP)materials.