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
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
After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the ...After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.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
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
This review focuses on DNA vaccines, denoting the last two decades since the early substantiation of preclinical protection was published in Science in 1993 by Ulmer et al. In spite of being safely administered and ea...This review focuses on DNA vaccines, denoting the last two decades since the early substantiation of preclinical protection was published in Science in 1993 by Ulmer et al. In spite of being safely administered and easily engineered and manufactured DNA vaccine, it holds the future prospects of immunization by inducing potent cellular immune responses against infectious and non-infectious diseases. It is well documented that injection of DNA plasmid encoding a desired gene of interest can result in the subsequent expression of its products and lead to the induction of an immune response within a host. This is pertinent to prophylactic and therapeutic vaccination approach when the peculiar gene produces a protective epitope from a pathogen. The recent studies demonstrated by a number of research centers showed that these immune responses evoke protective immunity against several infectious diseases and cancers, which provides adequate support for the use of this approach. We attempt in this review to provide an informative and unbiased overview of the general principles and concept of DNA vaccines technology with a summary of a novel approach to the DNA vaccine, present investigations that describe the mechanism(s) of protective immunity provoked by DNA immunization and to highlight the advantages and disadvantages of DNA immunisation.展开更多
INTRODUCTIONEndogenesis infection plays an important role innosocomial infection.By studying progress ofbacteria translocation from intestinal tract,theconcept of gut-origin infection has been acceptedgradually.Becaus...INTRODUCTIONEndogenesis infection plays an important role innosocomial infection.By studying progress ofbacteria translocation from intestinal tract,theconcept of gut-origin infection has been acceptedgradually.Because of no ideal tracing method,there were some controversies.In order to solve theproblem,the PUC19 plasmid vector tracing展开更多
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.
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After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.
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.
基金Supported by the Department of Microbiology and Immunology,College of Medicine and Health Sciences,Sultan Qaboos University,Sultanate of Oman(Code Number:RP032015)
文摘This review focuses on DNA vaccines, denoting the last two decades since the early substantiation of preclinical protection was published in Science in 1993 by Ulmer et al. In spite of being safely administered and easily engineered and manufactured DNA vaccine, it holds the future prospects of immunization by inducing potent cellular immune responses against infectious and non-infectious diseases. It is well documented that injection of DNA plasmid encoding a desired gene of interest can result in the subsequent expression of its products and lead to the induction of an immune response within a host. This is pertinent to prophylactic and therapeutic vaccination approach when the peculiar gene produces a protective epitope from a pathogen. The recent studies demonstrated by a number of research centers showed that these immune responses evoke protective immunity against several infectious diseases and cancers, which provides adequate support for the use of this approach. We attempt in this review to provide an informative and unbiased overview of the general principles and concept of DNA vaccines technology with a summary of a novel approach to the DNA vaccine, present investigations that describe the mechanism(s) of protective immunity provoked by DNA immunization and to highlight the advantages and disadvantages of DNA immunisation.
基金the National Natural Science Foundation of China,No.39570042
文摘INTRODUCTIONEndogenesis infection plays an important role innosocomial infection.By studying progress ofbacteria translocation from intestinal tract,theconcept of gut-origin infection has been acceptedgradually.Because of no ideal tracing method,there were some controversies.In order to solve theproblem,the PUC19 plasmid vector tracing