Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especiall...Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.展开更多
Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of tran...Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.展开更多
Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this meth...Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.展开更多
Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished f...Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.展开更多
A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB inser...A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.展开更多
Multidrug-resistant(MDR)Enterobacteriaceae critically threaten duck farming and public health.The phenotypes,genotypes,and associated mobile genetic elements(MGEs)of MDR Enterobacteriaceae isolated from 6 duck farms i...Multidrug-resistant(MDR)Enterobacteriaceae critically threaten duck farming and public health.The phenotypes,genotypes,and associated mobile genetic elements(MGEs)of MDR Enterobacteriaceae isolated from 6 duck farms in Zhejiang Province,China,were investigated.A total of 215 isolates were identified as Escherichia coli(64.65%),Klebsiella pneumoniae(12.09%),Proteus mirabilis(10.23%),Salmonella(8.84%),and Enterobacter cloacae(4.19%).Meanwhile,all isolates were resistant to at least two antibiotics.Most isolates carried tet(A)(85.12%),blaTEM(78.60%)and sul1(67.44%)resistance genes.Gene co-occurrence analysis showed that the resistance genes were associated with IS26 and integrons.A conjugative IncFII plasmid pSDM004 containing all the above MGEs was detected in Proteus mirabilis isolate SDM004.This isolate was resistant to 18 antibiotics and carried the blaNDM-5 gene.MGEs,especially plasmids,are the primary antibiotic resistance gene transmission route in duck farms.These findings provide a theoretical basis for the rational use of antibiotics in farms which are substantial for evaluating public health and food safety.展开更多
Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurri...Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC<sub>50</sub> for lycopene was determined at 112 μg/mL which was almost partial to IC<sub>50</sub> of standard antioxidant L-ascorbic acid. The D<sub>10</sub> value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC<sub>50</sub> concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals.展开更多
Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is u...Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.展开更多
Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they wer...Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they were not discovered in the two unicellular genera Gloeocapsa and Gloeothece, and no further report was there on them ever since. Some species of these two genera having the ability to fix atmospheric nitrogen aerobically carry展开更多
QuickChange mutagenesis is the method of choice for site-directed mutagenesis(SDM) of target se-quences in a plasmid.It can be applied successfully to small plasmids(up to 10 kb).However,this method cannot efficiently...QuickChange mutagenesis is the method of choice for site-directed mutagenesis(SDM) of target se-quences in a plasmid.It can be applied successfully to small plasmids(up to 10 kb).However,this method cannot efficiently mutate bigger plasmids.Using KOD Hot Start polymerase in combination with high performance liquid chromatography(HPLC) purified primers,we were able to achieve SDM in big plasmids(up to 16 kb) involving not only a single base change but also multiple base changes.Moreover,only six polymerase chain reaction(PCR) cycles and 0.5 μl of polymerase(instead of 18 PCR cycles and 1.0 μl of enzyme in the standard protocol) were sufficient for the reaction.展开更多
This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of t...This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of the drug resistant plasmids pFD10 and pFD11 with R144 as a control. The results obtained by this method are identical with that by the recombination method. However, the former is simple and more convenient. When the pFD10 plasmid coexisted with the F′lac pro plasmid, the fre-展开更多
Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,lead...Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,leading to the failure of triple-negative breast cancer(TNBC)immunotherapy.Therefore,depleting CAFs may enhance the effect of immunotherapy(such as PD-L1 antibody).Relaxin(RLN)has been demonstrated to significantly improve transforming growth factor-β(TGF-β)induced CAFs activation and tumor immunosuppressive microenvironment.However,the short half-life and systemic vasodilation of RLN limit its in vivo efficacy.Here,plasmid encoding relaxin(pRLN)to locally express RLN was delivered with a new positively charged polymer named polymeric metformin(PolyMet),which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before.In order to improve the stability of pRLN in vivo,this complex was further formed lipid poly-γ-glutamic acid(PGA)/PolyMetpRLN nanoparticle(LPPR).The particle size of LPPR was 205.5±2.9 nm,and the zeta potential was+55.4±1.6 mV.LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro.In vivo,it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment,which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration.Thus,LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse,and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody(aPD-L1).Altogether,this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.展开更多
Chloroplast is a discrete,highly structured,and semi-autonomous cellular organelle.The small genome of chloroplast makes it an up-and-coming platform for synthetic biology.As a special means of synthetic biology,chlor...Chloroplast is a discrete,highly structured,and semi-autonomous cellular organelle.The small genome of chloroplast makes it an up-and-coming platform for synthetic biology.As a special means of synthetic biology,chloroplast genetic engineering shows excellent potential in reconstructing various sophisticated metabolic pathways within the plants for specific purposes,such as improving crop photosynthetic capacity,enhancing plant stress resistance,and synthesizing new drugs and vaccines.However,many plant species exhibit limited efficiency or inability in chloroplast genetic transformation.Hence,new transformation technologies and tools are being constantly developed.In order to further expand and facilitate the application of chloroplast genetic engineering,this review summarizes the new technologies in chloroplast genetic transformation in recent years and discusses the choice of appropriate synthetic biological elements for the construction of efficient chloroplast transformation vectors.展开更多
Conjugative transfer of antibiotic resistance genes(ARGs)by plasmids is an important route for ARG dissemination.An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of A...Conjugative transfer of antibiotic resistance genes(ARGs)by plasmids is an important route for ARG dissemination.An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs,highlighting potential challenges for controlling this type of horizontal transfer.Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance.Although such inhibitors are rare,they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood.Here,we studied the effects of dihydroartemisinin(DHA),an artemisinin derivative used to treat malaria,on conjugation.DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene(mcr-1)by more than 160-fold in vitro in Escherichia coli,and more than two-fold(IncI2 plasmid)in vivo in a mouse model.It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla_(NDM-5)by more than twofold in vitro.Detection of intracellular adenosine triphosphate(ATP)and proton motive force(PMF),in combination with transcriptomic and metabolomic analyses,revealed that DHA impaired the function of the electron transport chain(ETC)by inhibiting the tricarboxylic acid(TCA)cycle pathway,thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer.Furthermore,expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure,indicating that the transfer apparatus for conjugation may be inhibited.Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.展开更多
An extensively drug-resistant(XDR)Escherichia coli strain 258E was isolated from an anal swab sample of a chicken farm of Anhui province in China.Genomic analyses indicated that the strain 258E harbors an incompatibil...An extensively drug-resistant(XDR)Escherichia coli strain 258E was isolated from an anal swab sample of a chicken farm of Anhui province in China.Genomic analyses indicated that the strain 258E harbors an incompatibility group N(IncN)plasmid pEC258-3,which co-produces bla_(CTX-M-3),bla_(KPC-2),bla_(TEM-1B),qnrS1,aac(6')-Ib-cr,dfrA14,arr-3,and aac(6')-Ib3.Multiple genome arrangement analyses indicated that pEC258-3 is highly homologous with pCRKP-1-KPC discovered in Klebsiella pneumoniae from a patient.Furthermore,conjugation experiments proved that plasmid pEC258-3 can be transferred horizontally and may pose a significant potential threat in animals,community and hospital settings.展开更多
Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant ...Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.展开更多
Objective:To compare the targeting effects of lactosarninated alginate(AlgNP)、polyethylene glycol - coated hydroxyapatite- poly- L- lysine nanoparticles (PLL- PCHNP)and relative nonlactosaminated ones load ed with ex...Objective:To compare the targeting effects of lactosarninated alginate(AlgNP)、polyethylene glycol - coated hydroxyapatite- poly- L- lysine nanoparticles (PLL- PCHNP)and relative nonlactosaminated ones load ed with exogenous gene on liver via peripheral intravenous route. Methods:Preparation of AlgNP based on control of gelification phenomenon of algiante by calcium ions and HA- PLLNP with collosol - gel method, both further modified with lactosaminated - poly- L - lysine synthesized by reductive lactosamination . We used pEGFPCl as the reporter gene to establish receptor- mediated and positive liver targeting nanoparticles- gene model. The potential of adsorbing DNA on nanoparticles was analysed by electrophoresis and spectrophotometer. Then different complexes were transferred into the rat's body by peripheral intravenous route and their targeting characteristics in liver were investigated by using radioisotope tracing assay. Results: PCHNP presented as needle - like particles with a diameter of 20nm by TEM and could be effectively combined with PLL. The diameter of AlgNP was 280nm. Agarpse gel electrophoresis showed both nanoparticles could effectively combine with DNA and the optimal proportion of PLLPCHNP and DNA was 30:1 (w/w); DNA mixed ratio of AlgPLL was 68.3 % by spectrophotometer. The radioactivities in liver for the two lactosaminated nanoparticles were higher than the nonlactosaminated ones. No statistic difference between AlgNP and AlgLacNP could be found . Conclusions: Lactosaminated naroparticles can target to liver more effectively by peripheral intravenous route than nonlactosaminated ones, which is closely concerned with the characteritics of the nanopartide complex.展开更多
基金the Wellcome Trust,BBSRC,and the National Natural Science Foundation of China(81802065,102908/Z/13/Z).
文摘Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.
文摘Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.
文摘Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.
文摘Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.
基金the National Natural Science Foundation of China and Microbial Resource Project of the Ministry of Science and Technology of China (Grant No. 2005DKA21208-6)
文摘A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.
基金supported by the National Natural Science Foundation of China(32172188)Science and Technology Cooperation Project of ZheJiang Province(2023SNJF058-3)。
文摘Multidrug-resistant(MDR)Enterobacteriaceae critically threaten duck farming and public health.The phenotypes,genotypes,and associated mobile genetic elements(MGEs)of MDR Enterobacteriaceae isolated from 6 duck farms in Zhejiang Province,China,were investigated.A total of 215 isolates were identified as Escherichia coli(64.65%),Klebsiella pneumoniae(12.09%),Proteus mirabilis(10.23%),Salmonella(8.84%),and Enterobacter cloacae(4.19%).Meanwhile,all isolates were resistant to at least two antibiotics.Most isolates carried tet(A)(85.12%),blaTEM(78.60%)and sul1(67.44%)resistance genes.Gene co-occurrence analysis showed that the resistance genes were associated with IS26 and integrons.A conjugative IncFII plasmid pSDM004 containing all the above MGEs was detected in Proteus mirabilis isolate SDM004.This isolate was resistant to 18 antibiotics and carried the blaNDM-5 gene.MGEs,especially plasmids,are the primary antibiotic resistance gene transmission route in duck farms.These findings provide a theoretical basis for the rational use of antibiotics in farms which are substantial for evaluating public health and food safety.
文摘Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC<sub>50</sub> for lycopene was determined at 112 μg/mL which was almost partial to IC<sub>50</sub> of standard antioxidant L-ascorbic acid. The D<sub>10</sub> value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC<sub>50</sub> concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals.
文摘Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.
文摘Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they were not discovered in the two unicellular genera Gloeocapsa and Gloeothece, and no further report was there on them ever since. Some species of these two genera having the ability to fix atmospheric nitrogen aerobically carry
文摘QuickChange mutagenesis is the method of choice for site-directed mutagenesis(SDM) of target se-quences in a plasmid.It can be applied successfully to small plasmids(up to 10 kb).However,this method cannot efficiently mutate bigger plasmids.Using KOD Hot Start polymerase in combination with high performance liquid chromatography(HPLC) purified primers,we were able to achieve SDM in big plasmids(up to 16 kb) involving not only a single base change but also multiple base changes.Moreover,only six polymerase chain reaction(PCR) cycles and 0.5 μl of polymerase(instead of 18 PCR cycles and 1.0 μl of enzyme in the standard protocol) were sufficient for the reaction.
文摘This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of the drug resistant plasmids pFD10 and pFD11 with R144 as a control. The results obtained by this method are identical with that by the recombination method. However, the former is simple and more convenient. When the pFD10 plasmid coexisted with the F′lac pro plasmid, the fre-
基金This work was funded by the Medical and Health Science and Technology Program of Zhejiang Province(2021KY813)the National Natural Science Foundation of China(82174095)the National Natural Science Foundation of Zhejiang Province(LZ22H290001).
文摘Cancer-associated fibroblasts(CAFs)are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment,leading to the failure of triple-negative breast cancer(TNBC)immunotherapy.Therefore,depleting CAFs may enhance the effect of immunotherapy(such as PD-L1 antibody).Relaxin(RLN)has been demonstrated to significantly improve transforming growth factor-β(TGF-β)induced CAFs activation and tumor immunosuppressive microenvironment.However,the short half-life and systemic vasodilation of RLN limit its in vivo efficacy.Here,plasmid encoding relaxin(pRLN)to locally express RLN was delivered with a new positively charged polymer named polymeric metformin(PolyMet),which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before.In order to improve the stability of pRLN in vivo,this complex was further formed lipid poly-γ-glutamic acid(PGA)/PolyMetpRLN nanoparticle(LPPR).The particle size of LPPR was 205.5±2.9 nm,and the zeta potential was+55.4±1.6 mV.LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro.In vivo,it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment,which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration.Thus,LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse,and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody(aPD-L1).Altogether,this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.
基金This work was funded by the Foundation of Hubei Hongshan Laboratory,China(2022hszd014)the National Natural Science Foundation of China(31771752).
文摘Chloroplast is a discrete,highly structured,and semi-autonomous cellular organelle.The small genome of chloroplast makes it an up-and-coming platform for synthetic biology.As a special means of synthetic biology,chloroplast genetic engineering shows excellent potential in reconstructing various sophisticated metabolic pathways within the plants for specific purposes,such as improving crop photosynthetic capacity,enhancing plant stress resistance,and synthesizing new drugs and vaccines.However,many plant species exhibit limited efficiency or inability in chloroplast genetic transformation.Hence,new transformation technologies and tools are being constantly developed.In order to further expand and facilitate the application of chloroplast genetic engineering,this review summarizes the new technologies in chloroplast genetic transformation in recent years and discusses the choice of appropriate synthetic biological elements for the construction of efficient chloroplast transformation vectors.
基金supported in part by grants from the Laboratory of Lingnan Modern Agriculture Project (NT2021006)National Key Research and Development Program of China (2022YFD1800400)。
文摘Conjugative transfer of antibiotic resistance genes(ARGs)by plasmids is an important route for ARG dissemination.An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs,highlighting potential challenges for controlling this type of horizontal transfer.Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance.Although such inhibitors are rare,they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood.Here,we studied the effects of dihydroartemisinin(DHA),an artemisinin derivative used to treat malaria,on conjugation.DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene(mcr-1)by more than 160-fold in vitro in Escherichia coli,and more than two-fold(IncI2 plasmid)in vivo in a mouse model.It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla_(NDM-5)by more than twofold in vitro.Detection of intracellular adenosine triphosphate(ATP)and proton motive force(PMF),in combination with transcriptomic and metabolomic analyses,revealed that DHA impaired the function of the electron transport chain(ETC)by inhibiting the tricarboxylic acid(TCA)cycle pathway,thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer.Furthermore,expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure,indicating that the transfer apparatus for conjugation may be inhibited.Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.
基金the National Key Research and Development Program of China(2018YFE0192600)the Shanghai Agriculture Applied Technology Development Program,China(T20200104)+1 种基金the Fundamental Research Funds for the Central Universities,China(2020JB05)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-ZDRW202203).
文摘An extensively drug-resistant(XDR)Escherichia coli strain 258E was isolated from an anal swab sample of a chicken farm of Anhui province in China.Genomic analyses indicated that the strain 258E harbors an incompatibility group N(IncN)plasmid pEC258-3,which co-produces bla_(CTX-M-3),bla_(KPC-2),bla_(TEM-1B),qnrS1,aac(6')-Ib-cr,dfrA14,arr-3,and aac(6')-Ib3.Multiple genome arrangement analyses indicated that pEC258-3 is highly homologous with pCRKP-1-KPC discovered in Klebsiella pneumoniae from a patient.Furthermore,conjugation experiments proved that plasmid pEC258-3 can be transferred horizontally and may pose a significant potential threat in animals,community and hospital settings.
基金Supported by the National Key Research and Development Plan of China(2019YFC1905902,2019YFC1905900)Key Research and Development Plan in Shandong Province(2020CXGC010603,2021ZDSYS10,2022CXGC020206)+2 种基金"Open Competition Mechanism"Project of Qilu University of Technology(Shandong Academy of Sciences)(2022JBZ01-06)Taishan Industry Leading Talent Program(tscy20180103)National Natural Science Foundation of China(31801527)。
文摘Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.
文摘Objective:To compare the targeting effects of lactosarninated alginate(AlgNP)、polyethylene glycol - coated hydroxyapatite- poly- L- lysine nanoparticles (PLL- PCHNP)and relative nonlactosaminated ones load ed with exogenous gene on liver via peripheral intravenous route. Methods:Preparation of AlgNP based on control of gelification phenomenon of algiante by calcium ions and HA- PLLNP with collosol - gel method, both further modified with lactosaminated - poly- L - lysine synthesized by reductive lactosamination . We used pEGFPCl as the reporter gene to establish receptor- mediated and positive liver targeting nanoparticles- gene model. The potential of adsorbing DNA on nanoparticles was analysed by electrophoresis and spectrophotometer. Then different complexes were transferred into the rat's body by peripheral intravenous route and their targeting characteristics in liver were investigated by using radioisotope tracing assay. Results: PCHNP presented as needle - like particles with a diameter of 20nm by TEM and could be effectively combined with PLL. The diameter of AlgNP was 280nm. Agarpse gel electrophoresis showed both nanoparticles could effectively combine with DNA and the optimal proportion of PLLPCHNP and DNA was 30:1 (w/w); DNA mixed ratio of AlgPLL was 68.3 % by spectrophotometer. The radioactivities in liver for the two lactosaminated nanoparticles were higher than the nonlactosaminated ones. No statistic difference between AlgNP and AlgLacNP could be found . Conclusions: Lactosaminated naroparticles can target to liver more effectively by peripheral intravenous route than nonlactosaminated ones, which is closely concerned with the characteritics of the nanopartide complex.
