Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion ...Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion (OME). Methods: The middle ear effusions from 20 children with OME were analyzed both by bacterial culture and by 16S rDNA-gene-targeted PCR assay, DGGE fingerprinting and sequencing analysis. Results: In bacterial culture assay, only three middle ear effusions (15%) showed bacterial growth. None of the samples were positive for anaerobic culture. The PCR assay with 16S rDNA-gene-targeted universal primers was positive in 10 (50%) cases. The subsequent DGGE fingerprinting and 16S rDNA sequencing analysis revealed that the most commonly encountered bacteria in the middle ear effusions of children with OME are Haemophilus influenzae, Alloiococcus otitidis and Bacteroides spp. Conclusions: The present study demonstrated the applicability of PCR-DGGE based assay and 16S rDNA sequencing for analyzing of bacterial diversity in the middle ear effusion of children OME. The results of our study may contribute to a better understanding of the etiology of OME.展开更多
The occurre nce of biologically active pharmaceuticals in aquatic enviro nments raised the potential risks to aquatic species.Among these marketed biological active pharmaceuticals,it has been estimated that40%of them...The occurre nce of biologically active pharmaceuticals in aquatic enviro nments raised the potential risks to aquatic species.Among these marketed biological active pharmaceuticals,it has been estimated that40%of them target G-protein-coupled receptors(GPCRs).We have illustrated pharmaceutical activities of GPCR targeted pharmaceuticals in English and Japanese wastewater by the in vitro transforming growth factor-α(TGFα)shedding assay.However,as the most important producer and consumer of pharmaceuticals,the occurrence of GPCR targeted pharmaceuticals in China had remained unclear.In this study,we investigated the pharmaceutical activities of GPCR targeted pharmaceuticals in secondary effluents of Chinese wastewater treatment plants.We discovered antagonistic activities against angiotensin(AT1)receptor at up to 7.2×102 ng-valsartan-equivalent quantity/L in Chinese wastewater for the first time as well as agonistic activities against dopamine(D2)receptor.Furthermore,in parallel with the assay,we determined concentrations of GPCR targeted pharmaceuticals in target wastewater by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).Through the comparison of predicted antagonistic activities calculated by concentrations and potency values from the assay,we found that the measured antagonistic activities against AT1 receptor from the assay were higher than the predicted AT1 activities from valsartan,irbesartan,and losartan,indicating the potential existence of other unknown AT1 antagonists in wastewater.展开更多
Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase ...Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.展开更多
基金supported by SF109870 from Estonian Science Foundation.
文摘Background: The aim of the study was to analyze the performance of PCR-DGGE based assay and its applicability as a tool for the identification of bacteria in the middle ear of children with otitis media with effusion (OME). Methods: The middle ear effusions from 20 children with OME were analyzed both by bacterial culture and by 16S rDNA-gene-targeted PCR assay, DGGE fingerprinting and sequencing analysis. Results: In bacterial culture assay, only three middle ear effusions (15%) showed bacterial growth. None of the samples were positive for anaerobic culture. The PCR assay with 16S rDNA-gene-targeted universal primers was positive in 10 (50%) cases. The subsequent DGGE fingerprinting and 16S rDNA sequencing analysis revealed that the most commonly encountered bacteria in the middle ear effusions of children with OME are Haemophilus influenzae, Alloiococcus otitidis and Bacteroides spp. Conclusions: The present study demonstrated the applicability of PCR-DGGE based assay and 16S rDNA sequencing for analyzing of bacterial diversity in the middle ear effusion of children OME. The results of our study may contribute to a better understanding of the etiology of OME.
基金The Fundamental Research Funds for the Central UniversitiesJapan Society for the Promotion of science(JSPS)for Grant-in-Aid for Scientific Research(B)(No.17H01907)+2 种基金Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research(No.K-CONNEX),established by Human Resource Development Program for Science and Technology,MEXTfinancial support from the National Natural Science Foundation of China(No.21806082)Key Technologies R&D Program of Tianjin(Nos.18YFZCNC01410,16YFZCSF00410)。
文摘The occurre nce of biologically active pharmaceuticals in aquatic enviro nments raised the potential risks to aquatic species.Among these marketed biological active pharmaceuticals,it has been estimated that40%of them target G-protein-coupled receptors(GPCRs).We have illustrated pharmaceutical activities of GPCR targeted pharmaceuticals in English and Japanese wastewater by the in vitro transforming growth factor-α(TGFα)shedding assay.However,as the most important producer and consumer of pharmaceuticals,the occurrence of GPCR targeted pharmaceuticals in China had remained unclear.In this study,we investigated the pharmaceutical activities of GPCR targeted pharmaceuticals in secondary effluents of Chinese wastewater treatment plants.We discovered antagonistic activities against angiotensin(AT1)receptor at up to 7.2×102 ng-valsartan-equivalent quantity/L in Chinese wastewater for the first time as well as agonistic activities against dopamine(D2)receptor.Furthermore,in parallel with the assay,we determined concentrations of GPCR targeted pharmaceuticals in target wastewater by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).Through the comparison of predicted antagonistic activities calculated by concentrations and potency values from the assay,we found that the measured antagonistic activities against AT1 receptor from the assay were higher than the predicted AT1 activities from valsartan,irbesartan,and losartan,indicating the potential existence of other unknown AT1 antagonists in wastewater.
基金National Natural Science Foundation of China(No.31100042)Guangdong Natural Science Foundation(No.S2011010001625)+1 种基金Guangzhou Pearl River Rising Star Program for Science and Technology(No.2013091)Guangdong Chinese Academy of Science Comprehensive Strategic Cooperation Project(No.2012B091100276)
基金supported by grants from the Ministry of Science and Technology of China(2013CB734001 and 2015CB150600)the National Natural Science Foundation of China(31370097 and 31571281)
文摘Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.