目的:构建M AT 2A基因的小干扰RNA(siRNA)快速筛选体系,筛选出有效的小干扰RNA。方法:首先构建能产生M AT 2A基因小干扰RNA的质粒表达载体,细胞内转录合成4条小干扰RNA;然后构建M AT 2A基因与荧光素酶报告基因(luciferase)的融合质粒表...目的:构建M AT 2A基因的小干扰RNA(siRNA)快速筛选体系,筛选出有效的小干扰RNA。方法:首先构建能产生M AT 2A基因小干扰RNA的质粒表达载体,细胞内转录合成4条小干扰RNA;然后构建M AT 2A基因与荧光素酶报告基因(luciferase)的融合质粒表达载体p lucF-M AT 2A;采用脂质体转染法将p lucF-M AT 2A与产生siRNA的质粒共转染293T细胞,定量测量荧光素酶活性,构建M AT 2A基因的小干扰RNA筛选体系,初步筛选出抑制荧光素酶表达的有效siRNA;然后将有效的siRNA转染Bel-7402肝癌细胞,半定量RT-PCR检测M AT 2A mRNA表达,并检测转染后肝癌细胞M AT的活性,进一步证实siRNA对M AT 2A表达的抑制效果。结果:所合成的4条小干扰RNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞M AT 2A mRNA表达,降低了肝癌细胞中M AT活性。结论:通过荧光素酶报告体系来检测siRNA有效性的方法为RNA i研究提供了一个快速有效地筛选小干扰RNA的途径。展开更多
The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant ...The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains has revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 - 30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination.展开更多
The gut microbiota plays key roles in host health by shaping the host immune responses through their metabolites,like indole derivatives from tryptophan.However,the direct role of these indole derivatives in macrophag...The gut microbiota plays key roles in host health by shaping the host immune responses through their metabolites,like indole derivatives from tryptophan.However,the direct role of these indole derivatives in macrophage fate decision and the underlying mechanism remains unknown.Here,we found that bacterial indole-3-propionic acid(IPA)downregulates interleukin-1beta(IL-1β)production in M1 macrophages through inhibition of nuclear factor-kappa B(NF-κB)signaling.Mechanistically,IPA binds specifically with methionine adenosyltransferase 2A(MAT2A)to promote S-adenosylmethionine(SAM)synthesis,which facilitates the DNA methylation of ubiquitin-specific peptidase 16(USP16,a deubiquitinase),and in turn promotes Toll-like receptor 4(TLR4)ubiquitination and NF-κB inhibition.Furthermore,IPA administration attenuates sepsis in mouse models induced by lipopolysaccharides(LPS),showcasing its potential as a microbial-derived adjunct in alleviating inflammation.Collectively,our findings reveal a newly found microbial metabolite-immune system regulatory pathway mediated by IPA.展开更多
Colletotrichum gloeosporioides sensu lato has been associated with anthracnose in diverse commercial crops.It is now established that C.gloeosporioides sensu lato comprises 33 phylogenetic species and C.gloeosporioide...Colletotrichum gloeosporioides sensu lato has been associated with anthracnose in diverse commercial crops.It is now established that C.gloeosporioides sensu lato comprises 33 phylogenetic species and C.gloeosporioides sensu stricto is not a common pathogen of tropical fruits.In this study,we investigated the phylogenetic relationships of 85 Colletotrichum isolates associated with select tropical fruits and flowering plants from India.In the ApMat marker analysis,the 85 isolates clustered with 7 known Colletotrichum species(C.aotearoa,C.dianesei,C.endomangiferae,C.musae,C.siamense,C.theobromicola,Glomerella cingulata f.sp.camelliae)and six novel lineages.One of the novel lineages is described and illustrated in this paper as Colletotrichum communis sp.nov.,while new-host pathogen associations for C.aotearoa,C.endomangiferae,C.dianesei and C.theobromicola are reported from India.Out of the 85 isolates analysed in this paper,73 isolates clustered within the C.siamense species complex,indicating that C.siamense species complex,not C.gloeosporioides sensu stricto,is common on tropical fruits.In comparison with act,cal,gapdh,ITS and tub2 gene markers,we recommend the use of the ApMat marker for accurate identification of cryptic species within the C.siamense species complex.We believe that the ApMat marker,in combination with one or two similar‘phylogenetically superior’gene markers,is a better candidate for specieslevel classification of fungi that were traditionally identified as‘Colletotrichum gloeosporioides’.展开更多
Endophytic species of Colletotrichum associated with Mangifera indica(mango)are poorly understood.In this study,Colletotrichum species were isolated from mango in Pernambuco State,Brazil.There were significant differe...Endophytic species of Colletotrichum associated with Mangifera indica(mango)are poorly understood.In this study,Colletotrichum species were isolated from mango in Pernambuco State,Brazil.There were significant differences in isolation frequencies of Colletotrichum species among sites and plant tissues.Mature leaf blades were colonized by most Colletotrichum isolates at the majority of sites.Partial sequences of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)of 97 Colletotrichum isolates were amplified as an initial measure of genetic diversity.Phylogenetic analysis with a subset of 22 isolates were performed based on a multilocus dataset(ACT,TUB2,CAL,CHS-1,GAPDH,ITS)followed by Apn2/MAT IGS sequence-analysis for isolates within the C.gloeosporioides species complex.Molecular analysis associated with phenotypic characteristics revealed six previously described species[C.asianum,C.cliviae,C.dianesei(syn.C.melanocaulon),C.fructicola,C.karstii and C.tropicale]and one new species.This new species is introduced as C.endomangiferae.All species isolated were pathogenic on mango fruits but varied in their virulence.There was no distribution pattern of species among sites and plant tissues,although C.asianum was the most prevalent species at all sites and in all plant tissues studied.Five previously reported Colletotrichum species causing anthracnose in mango fruits in northeastern Brazil were also recovered as endophytes.展开更多
文摘目的:构建M AT 2A基因的小干扰RNA(siRNA)快速筛选体系,筛选出有效的小干扰RNA。方法:首先构建能产生M AT 2A基因小干扰RNA的质粒表达载体,细胞内转录合成4条小干扰RNA;然后构建M AT 2A基因与荧光素酶报告基因(luciferase)的融合质粒表达载体p lucF-M AT 2A;采用脂质体转染法将p lucF-M AT 2A与产生siRNA的质粒共转染293T细胞,定量测量荧光素酶活性,构建M AT 2A基因的小干扰RNA筛选体系,初步筛选出抑制荧光素酶表达的有效siRNA;然后将有效的siRNA转染Bel-7402肝癌细胞,半定量RT-PCR检测M AT 2A mRNA表达,并检测转染后肝癌细胞M AT的活性,进一步证实siRNA对M AT 2A表达的抑制效果。结果:所合成的4条小干扰RNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞M AT 2A mRNA表达,降低了肝癌细胞中M AT活性。结论:通过荧光素酶报告体系来检测siRNA有效性的方法为RNA i研究提供了一个快速有效地筛选小干扰RNA的途径。
文摘The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains has revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 - 30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination.
基金supported by the National Natural Science Foundation of China(32225047,U22A20510)Double First-Class Discipline Promotion Project(2023B10564001)+1 种基金National Center of Technology Innovation for Pigs(NCTIP-XD/B13)the Open Project Program of Sichuan Provincial Key Laboratory of Animal Disease-resistant Nutrition,Sichuan Agricultural University(SZ202301-02)。
文摘The gut microbiota plays key roles in host health by shaping the host immune responses through their metabolites,like indole derivatives from tryptophan.However,the direct role of these indole derivatives in macrophage fate decision and the underlying mechanism remains unknown.Here,we found that bacterial indole-3-propionic acid(IPA)downregulates interleukin-1beta(IL-1β)production in M1 macrophages through inhibition of nuclear factor-kappa B(NF-κB)signaling.Mechanistically,IPA binds specifically with methionine adenosyltransferase 2A(MAT2A)to promote S-adenosylmethionine(SAM)synthesis,which facilitates the DNA methylation of ubiquitin-specific peptidase 16(USP16,a deubiquitinase),and in turn promotes Toll-like receptor 4(TLR4)ubiquitination and NF-κB inhibition.Furthermore,IPA administration attenuates sepsis in mouse models induced by lipopolysaccharides(LPS),showcasing its potential as a microbial-derived adjunct in alleviating inflammation.Collectively,our findings reveal a newly found microbial metabolite-immune system regulatory pathway mediated by IPA.
基金The authors would like to thank CSIR-Institute of Microbial Technology,Chandigarh for the financial support,Dr.D.Ananthapadmanaban for his help in the microscopy and Mr.Deepak Bhatt for DNA sequencing assistance.Drs.Kevin D.Hyde,Lei Cai and Bevan Weir are thanked for the inspiration and useful discussions on Colletotrichum taxonomy.This work was supported by IMTECHOLP0071 project and CSIR-SRF fellowship awarded to GS.This is NIO contribution no.7636 and IMTECH communication no.IMT2014/21.
文摘Colletotrichum gloeosporioides sensu lato has been associated with anthracnose in diverse commercial crops.It is now established that C.gloeosporioides sensu lato comprises 33 phylogenetic species and C.gloeosporioides sensu stricto is not a common pathogen of tropical fruits.In this study,we investigated the phylogenetic relationships of 85 Colletotrichum isolates associated with select tropical fruits and flowering plants from India.In the ApMat marker analysis,the 85 isolates clustered with 7 known Colletotrichum species(C.aotearoa,C.dianesei,C.endomangiferae,C.musae,C.siamense,C.theobromicola,Glomerella cingulata f.sp.camelliae)and six novel lineages.One of the novel lineages is described and illustrated in this paper as Colletotrichum communis sp.nov.,while new-host pathogen associations for C.aotearoa,C.endomangiferae,C.dianesei and C.theobromicola are reported from India.Out of the 85 isolates analysed in this paper,73 isolates clustered within the C.siamense species complex,indicating that C.siamense species complex,not C.gloeosporioides sensu stricto,is common on tropical fruits.In comparison with act,cal,gapdh,ITS and tub2 gene markers,we recommend the use of the ApMat marker for accurate identification of cryptic species within the C.siamense species complex.We believe that the ApMat marker,in combination with one or two similar‘phylogenetically superior’gene markers,is a better candidate for specieslevel classification of fungi that were traditionally identified as‘Colletotrichum gloeosporioides’.
基金financed by Fundacao de Amparoà Ciência e Tecnologia do Estado de Pernambuc(FACEPE)Coordenacao de Aperfeicoamento de Pessoal de Nível Superior(CAPES)+1 种基金the Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)research fellowshipNRCT of Thailand-Colletotrichum 54201020003 for support.
文摘Endophytic species of Colletotrichum associated with Mangifera indica(mango)are poorly understood.In this study,Colletotrichum species were isolated from mango in Pernambuco State,Brazil.There were significant differences in isolation frequencies of Colletotrichum species among sites and plant tissues.Mature leaf blades were colonized by most Colletotrichum isolates at the majority of sites.Partial sequences of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)of 97 Colletotrichum isolates were amplified as an initial measure of genetic diversity.Phylogenetic analysis with a subset of 22 isolates were performed based on a multilocus dataset(ACT,TUB2,CAL,CHS-1,GAPDH,ITS)followed by Apn2/MAT IGS sequence-analysis for isolates within the C.gloeosporioides species complex.Molecular analysis associated with phenotypic characteristics revealed six previously described species[C.asianum,C.cliviae,C.dianesei(syn.C.melanocaulon),C.fructicola,C.karstii and C.tropicale]and one new species.This new species is introduced as C.endomangiferae.All species isolated were pathogenic on mango fruits but varied in their virulence.There was no distribution pattern of species among sites and plant tissues,although C.asianum was the most prevalent species at all sites and in all plant tissues studied.Five previously reported Colletotrichum species causing anthracnose in mango fruits in northeastern Brazil were also recovered as endophytes.
