[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissu...[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.展开更多
Natural antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. AMPs are multifunctional molecules that have an essential act...Natural antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. AMPs are multifunctional molecules that have an essential activity in infection and inflammation: they play an important role in the innate immune response, not only as antimicrobial agents, but also as immunomodulating molecules and as an important link between the innate and adaptive immune response. In this article, we will discuss the antimicrobial activity, together with the novel properties of some of these molecules as immune modulators on the innate and adaptive immune response.展开更多
OBJECTIVE Metrnl is a novel secreted protein with limited researches.In this study,we investigated metrnl tissue expression pattern in humans,and exploredthe possible role of its highest expression using animal models...OBJECTIVE Metrnl is a novel secreted protein with limited researches.In this study,we investigated metrnl tissue expression pattern in humans,and exploredthe possible role of its highest expression using animal models.METHODS We examined metrnl tissue expression pattern in a human tissue microarray containing 19types of tissues from 69 donors,and verified the highest expression in fresh human and mouse tissues.We then created an animal model of cell-specific knockout mice to study the role of metrnl.RESULTS Metrnl was the highest expressed in human gastrointestinal tract,and specifical y expressed in the intestinal epithelium.Consistently,Metrnl expression was also the highest expressed in mouse gastrointestinal tract among the detected tissues of 14 types.We developed intestinal epithelial cellspecific metrnl knockout mice with Vil in-Cre.In this animal model,metrnl levels displayed a statistically significant reduction in gut fluid,but not in blood serum.This cell specific deletion of metrnl did not affect body weight,food intake,blood glucose,colon length and histology,intestinal permeability,mucus production and mucin 2 expression under physiological conditions,but markedly reduced the expression of antimicrobial peptides,such as regenerating islet-derived 3 gamma and lactotransferrin.CONCLUSION Metrnl is rich in intestinal epithelial cells of humans and mice,mainly contributing to local gut metrnl level,and less affecting systemic circulating metrnl level.Metrnl plays a role in maintaining gut antimicrobial peptides.展开更多
Antimicrobial peptides(AMPs)are the natural antibiotics recognized for their broad-spectrum resistance to bacteria,fungi,viruses and parasites,and influencing the host immune responses.AMPs attributed to good biologic...Antimicrobial peptides(AMPs)are the natural antibiotics recognized for their broad-spectrum resistance to bacteria,fungi,viruses and parasites,and influencing the host immune responses.AMPs attributed to good biological effects have been used in various areas of human health,which are trying to completely replace antibiotics,owing to serious drug-resistance bacteria.However,limited bioactivity and potential biotoxicity of some AMPs was neglected,attributable to their hydrophobic structure with positive charges and nonspecific destruction of cell membranes.Various strategies have been used to design and biosynthetic optimized AMPs to improve their bioactivity,productivity,while lowering host toxicity and cost.Here,we focus on the progress made in understanding the AMPs,including biosynthesis(AMP-BioDesign 1.0 and 2.0),bioactivity(e.g.immune regulation and broad-spectrum or nonspecific actions against bacteria,viruses or parasites),and principle biotoxicity(e.g.hemolysis,acute toxicity and instability,ect).The application prospects of AMP for human health,clinical medicine(for novel drugs),tissue engineering and drug delivery system,respectively,are summarized in this review.Furthermore,future prospects and new strategy for the development of effective and low-toxic AMP formulations for human health are discussed.展开更多
BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae inf...BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae infections.Nanodefensin with a 14-carbon saturated fatty acid(ND-C14)is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of humanα-defensin 5(HD5)via an amide bond.However,it is unclear whether ND-C14 is effective against lung infections caused by S.pneumoniae.METHODS:In vitro,three groups were established,including the control group,and the HD5 and ND-C14 treatment groups.A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S.pneumoniae.The morphological changes of S.pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy.In vivo,mice were divided into sham,vehicle,and ND-C14 treatment groups.Mice in the sham group were treated with 25μL of phosphate-buffered saline(PBS).Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25μL of bacterial suspension with 2×108 CFU/mL(total bacterial count:5×10^(6) CFU),and then the mice were given 25μL PBS or intratracheally injected with 25μL of ND-C14(including 20μg or 50μg),respectively.Survival rates were evaluated in the vehicle and ND-C14 treatment groups.Bacterial burden in the blood and bronchoalveolar lavage fluid were counted.The lung histology of the mice was assessed.A propidium iodide uptake assay was used to clarify the destructive eff ect of ND-C14 against S.pneumoniae.RESULTS:Compared with HD5,ND-C14 had a better bactericidal eff ect against S.pneumoniae because of its stronger ability to destroy the membrane structure of S.pneumoniae in vitro.In vivo,ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S.pneumoniae.ND-C14 reduced bacterial burden and lung tissue injury.Moreover,ND-C14 had a membrane permeation eff ect on S.pneumoniae,and its destructive ability increased with increasing ND-C14 concentration.CONCLUSION:The ND-C14 may improve bactericidal eff ects on S.pneumoniae both in vitro and in vivo.展开更多
Background: Ticks are hematophagous parasites that transmit an extensive range of pathogens to their vertebrate hosts.Ticks can destroy invading microorganisms or alleviate infection via their rudimentary but orchestr...Background: Ticks are hematophagous parasites that transmit an extensive range of pathogens to their vertebrate hosts.Ticks can destroy invading microorganisms or alleviate infection via their rudimentary but orchestrated innate immune system.Antimicrobial peptides(AMPs)are important components of tick innate immunity.Among these humoral effector molecules,defensins are well-studied and widely identified in various species of Ixodidae(hard ticks)and Argasidae(soft ticks).This review was aimed at presenting the characterization of tick defensins from structure-based taxonomic status to antimicrobial function.Main text: All published papers written in English from 2001 to May 2022 were searched through PubMed and Web of Science databases with the combination of relevant terms on tick defensins.Reports on identification and characterization of tick defensins were included.Of the 329 entries retrieved,57 articles were finally eligible for our scoping review.Tick defensins mainly belong to the antibacterial ancient invertebrate-type defensins of the cis-defensins superfamily.They are generally small,cationic,and amphipathic,with six cysteine residues forming three intra-molecular disulfide bonds.Tick defensins primarily target membranes of a variety of pathogens,including Gram-positive and Gram-negative bacteria,fungi,viruses,and protozoa.Since tick defensins have a high degree of variability,we summarize their common biological properties and enumerate representative peptides.Along with the various and potent antimicrobial activities,the role of tick defensins in determining vector competence is discussed.Conclusions: Due to their broad-spectrum antimicrobial activities,tick defensins are considered novel candidates or targets for controlling infectious diseases.展开更多
Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constr...Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.展开更多
基金Supported by China Postdoctoral Science Foundation(20090451250)Youth Fund of Sichuan Provincial Department of Education(09zb054)Key Project of International Science and Technology Cooperation(2005DFA30720)
文摘[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.
文摘Natural antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. AMPs are multifunctional molecules that have an essential activity in infection and inflammation: they play an important role in the innate immune response, not only as antimicrobial agents, but also as immunomodulating molecules and as an important link between the innate and adaptive immune response. In this article, we will discuss the antimicrobial activity, together with the novel properties of some of these molecules as immune modulators on the innate and adaptive immune response.
基金The project supported by National Natural Science Foundation of China(81130061,81202572,81373414)
文摘OBJECTIVE Metrnl is a novel secreted protein with limited researches.In this study,we investigated metrnl tissue expression pattern in humans,and exploredthe possible role of its highest expression using animal models.METHODS We examined metrnl tissue expression pattern in a human tissue microarray containing 19types of tissues from 69 donors,and verified the highest expression in fresh human and mouse tissues.We then created an animal model of cell-specific knockout mice to study the role of metrnl.RESULTS Metrnl was the highest expressed in human gastrointestinal tract,and specifical y expressed in the intestinal epithelium.Consistently,Metrnl expression was also the highest expressed in mouse gastrointestinal tract among the detected tissues of 14 types.We developed intestinal epithelial cellspecific metrnl knockout mice with Vil in-Cre.In this animal model,metrnl levels displayed a statistically significant reduction in gut fluid,but not in blood serum.This cell specific deletion of metrnl did not affect body weight,food intake,blood glucose,colon length and histology,intestinal permeability,mucus production and mucin 2 expression under physiological conditions,but markedly reduced the expression of antimicrobial peptides,such as regenerating islet-derived 3 gamma and lactotransferrin.CONCLUSION Metrnl is rich in intestinal epithelial cells of humans and mice,mainly contributing to local gut metrnl level,and less affecting systemic circulating metrnl level.Metrnl plays a role in maintaining gut antimicrobial peptides.
基金supported by Grants from National Natural Science Foundation of China(Grant No.31900950).
文摘Antimicrobial peptides(AMPs)are the natural antibiotics recognized for their broad-spectrum resistance to bacteria,fungi,viruses and parasites,and influencing the host immune responses.AMPs attributed to good biological effects have been used in various areas of human health,which are trying to completely replace antibiotics,owing to serious drug-resistance bacteria.However,limited bioactivity and potential biotoxicity of some AMPs was neglected,attributable to their hydrophobic structure with positive charges and nonspecific destruction of cell membranes.Various strategies have been used to design and biosynthetic optimized AMPs to improve their bioactivity,productivity,while lowering host toxicity and cost.Here,we focus on the progress made in understanding the AMPs,including biosynthesis(AMP-BioDesign 1.0 and 2.0),bioactivity(e.g.immune regulation and broad-spectrum or nonspecific actions against bacteria,viruses or parasites),and principle biotoxicity(e.g.hemolysis,acute toxicity and instability,ect).The application prospects of AMP for human health,clinical medicine(for novel drugs),tissue engineering and drug delivery system,respectively,are summarized in this review.Furthermore,future prospects and new strategy for the development of effective and low-toxic AMP formulations for human health are discussed.
基金supported by the National Natural Science Foundation of China(82072148)Zhejiang Provincial Basic Public Welfare Research Program of Zhejiang Province(LGF21H150002)+1 种基金Zhejiang Medicine and Health Science and Technology Project(2022RC245&2023KY255)Ningbo Municipal Natural Science Foundation(2023J134).
文摘BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae infections.Nanodefensin with a 14-carbon saturated fatty acid(ND-C14)is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of humanα-defensin 5(HD5)via an amide bond.However,it is unclear whether ND-C14 is effective against lung infections caused by S.pneumoniae.METHODS:In vitro,three groups were established,including the control group,and the HD5 and ND-C14 treatment groups.A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S.pneumoniae.The morphological changes of S.pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy.In vivo,mice were divided into sham,vehicle,and ND-C14 treatment groups.Mice in the sham group were treated with 25μL of phosphate-buffered saline(PBS).Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25μL of bacterial suspension with 2×108 CFU/mL(total bacterial count:5×10^(6) CFU),and then the mice were given 25μL PBS or intratracheally injected with 25μL of ND-C14(including 20μg or 50μg),respectively.Survival rates were evaluated in the vehicle and ND-C14 treatment groups.Bacterial burden in the blood and bronchoalveolar lavage fluid were counted.The lung histology of the mice was assessed.A propidium iodide uptake assay was used to clarify the destructive eff ect of ND-C14 against S.pneumoniae.RESULTS:Compared with HD5,ND-C14 had a better bactericidal eff ect against S.pneumoniae because of its stronger ability to destroy the membrane structure of S.pneumoniae in vitro.In vivo,ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S.pneumoniae.ND-C14 reduced bacterial burden and lung tissue injury.Moreover,ND-C14 had a membrane permeation eff ect on S.pneumoniae,and its destructive ability increased with increasing ND-C14 concentration.CONCLUSION:The ND-C14 may improve bactericidal eff ects on S.pneumoniae both in vitro and in vivo.
基金This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions,National Natural Science Foundation of China(81971917,32170142 and 81271792)Jiangsu Natural Science Foundation(BK20211310)+2 种基金Open Project Fund from State Key Laboratory of Genetic Engineering,Fudan University(SKLGE1903)the Jiangsu Undergraduate Training Program for Innovation and Entrepreneurship,Soochow University(202010285133Y)the 23rd Undergraduates Extracurricular Academic Research Fund,Soochow University(KY20210120A).
文摘Background: Ticks are hematophagous parasites that transmit an extensive range of pathogens to their vertebrate hosts.Ticks can destroy invading microorganisms or alleviate infection via their rudimentary but orchestrated innate immune system.Antimicrobial peptides(AMPs)are important components of tick innate immunity.Among these humoral effector molecules,defensins are well-studied and widely identified in various species of Ixodidae(hard ticks)and Argasidae(soft ticks).This review was aimed at presenting the characterization of tick defensins from structure-based taxonomic status to antimicrobial function.Main text: All published papers written in English from 2001 to May 2022 were searched through PubMed and Web of Science databases with the combination of relevant terms on tick defensins.Reports on identification and characterization of tick defensins were included.Of the 329 entries retrieved,57 articles were finally eligible for our scoping review.Tick defensins mainly belong to the antibacterial ancient invertebrate-type defensins of the cis-defensins superfamily.They are generally small,cationic,and amphipathic,with six cysteine residues forming three intra-molecular disulfide bonds.Tick defensins primarily target membranes of a variety of pathogens,including Gram-positive and Gram-negative bacteria,fungi,viruses,and protozoa.Since tick defensins have a high degree of variability,we summarize their common biological properties and enumerate representative peptides.Along with the various and potent antimicrobial activities,the role of tick defensins in determining vector competence is discussed.Conclusions: Due to their broad-spectrum antimicrobial activities,tick defensins are considered novel candidates or targets for controlling infectious diseases.
基金sponsored by the National Natural Science Foundation of China,No.30970992
文摘Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.
