Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signal...Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule(PaNOD2) from ayu(Plecoglossus altivelis).Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-κB signaling and Western blotting analysis detected the phosphorylation of three MAP kinases(p-38, Erk1/2, and JNK1/2).Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide(MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of Pa NOD2 with the ligand. Overexpression of PaNOD2 activated the NF-κB signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-α and IL-1β in HEK293 T cells and ayu head kidney-derived monocytes/macrophages(MO/MΦ).Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.展开更多
With the rapid development of intensive farming,the aquaculture industry uses a great many antibiotics for the prevention and treatment of bacterial diseases.Despite their therapeutic functions,the overuse and accumul...With the rapid development of intensive farming,the aquaculture industry uses a great many antibiotics for the prevention and treatment of bacterial diseases.Despite their therapeutic functions,the overuse and accumulation of antibiotics also pose a threat to aquaculture organisms.In the present study,ayu(Plecoglossus altivelis)was used as a fish model to study the impacts of ciprofloxacin(CIP)overuse on intestinal homeostasis and immune response during subsequent Pseudomonas plecoglossicida infection.Based on 16S rRNA gene amplification and Illumina sequencing,we found that CIP pre-exposure caused significant variation in intestinal microbiota,including increased species richness,altered microbiota composition and interaction networks,and increased metabolic dysfunction.Furthermore,immunohistochemical analysis indicated that CIP pre-exposure resulted in severe mucosal layer damage,goblet cell reduction,and epithelial cell necrosis of the intestinal barrier in infected ayu.Quantitative real-time polymerase chain reaction(qRT-PCR)showed that disruption of intestinal homeostasis impaired systemic antiinfection immune responses in the intestine,gill,spleen,and head kidney,while inhibiting IL-1β,TNF-α,and IL-10 expression and promoting TGF-βexpression.Our findings indicated that CIP administration can directly affect intestinal microbiota composition and intestinal integrity in ayu fish.This perturbation of intestinal homeostasis is likely responsible for the lower survival rate of hosts following subsequent infection as the capacity to mount an effective immune response is compromised.This study also provides preliminary clues for understanding the effects of antibiotic overuse on higher vertebrates through trophic transfer.展开更多
The Dmrt family of genes are involved in sex differentiation in different species of invertebrates, and some vertebrates including human. In this study, we cloned the full-length eDNA of ayu （Plecoglossus altivelis）...The Dmrt family of genes are involved in sex differentiation in different species of invertebrates, and some vertebrates including human. In this study, we cloned the full-length eDNA of ayu （Plecoglossus altivelis） Draft1 and DmrtA2. Sequence and phylogenetic tree analyses showed ayu Dmrtl showed highest similarity to that of Oncorhynchus mykiss while ayu DmrtA2 is most similar to that of Oryzias latipes. Fluorescence-based quantitative reverse transcription PCR （qRT-PCR） revealed the Draft1 was predominantly expressed in the testis. At the larval stages, Dmrtl mRNA expression level was highest during 52-64 days post hatching （dph） and at the gastrula stage during embryonic development. DmrtA2, meanwhile, was specifically expressed in the ovary and was highly expressed in the female brain tissue, but not male brain tissue. During the larval stages, DmrtA2 expression remained high before day 34, and then fluctuated while generally decreasing. During embryonic development, DmrtA2 expression increased gradually and peaked at the hatching stage. Our data suggest that ayu Dmrtl might participate in the differentiation and maintenance of testis while DmrtA2 may play a role in ovary-differentiation and mature-ovary maintenance. DmrtA2 might also participate in brain development.展开更多
基金This work was supported by a Research Startup Grant from Chongqing University of Medical Sciences (No. B9001-0228418046), the Natural Sciences Foundation of Chongqing (No. 2005BB5035), Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
基金supported by the National Natural Science Foundation of China(31772876,31702374)Natural Science Foundation of Zhejiang Province(LZ18C190001,LQ17C190001)+1 种基金Zhejiang Xinmiao Talents Program(2017R405023)K.C.Wong Magna Fund in Ningbo University
文摘Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule(PaNOD2) from ayu(Plecoglossus altivelis).Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-κB signaling and Western blotting analysis detected the phosphorylation of three MAP kinases(p-38, Erk1/2, and JNK1/2).Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide(MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of Pa NOD2 with the ligand. Overexpression of PaNOD2 activated the NF-κB signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-α and IL-1β in HEK293 T cells and ayu head kidney-derived monocytes/macrophages(MO/MΦ).Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.
基金supported by the Key Research and Development Project of Zhejiang Province(2021C02062)National Natural Science Foundation of China(32173004,31972821)+2 种基金Natural Science Foundation of Zhejiang Province(LZ18C190001)Natural Science Foundation of Ningbo City(202003N4011)Research Project of State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products(2010DS700124-ZZ2008)。
文摘With the rapid development of intensive farming,the aquaculture industry uses a great many antibiotics for the prevention and treatment of bacterial diseases.Despite their therapeutic functions,the overuse and accumulation of antibiotics also pose a threat to aquaculture organisms.In the present study,ayu(Plecoglossus altivelis)was used as a fish model to study the impacts of ciprofloxacin(CIP)overuse on intestinal homeostasis and immune response during subsequent Pseudomonas plecoglossicida infection.Based on 16S rRNA gene amplification and Illumina sequencing,we found that CIP pre-exposure caused significant variation in intestinal microbiota,including increased species richness,altered microbiota composition and interaction networks,and increased metabolic dysfunction.Furthermore,immunohistochemical analysis indicated that CIP pre-exposure resulted in severe mucosal layer damage,goblet cell reduction,and epithelial cell necrosis of the intestinal barrier in infected ayu.Quantitative real-time polymerase chain reaction(qRT-PCR)showed that disruption of intestinal homeostasis impaired systemic antiinfection immune responses in the intestine,gill,spleen,and head kidney,while inhibiting IL-1β,TNF-α,and IL-10 expression and promoting TGF-βexpression.Our findings indicated that CIP administration can directly affect intestinal microbiota composition and intestinal integrity in ayu fish.This perturbation of intestinal homeostasis is likely responsible for the lower survival rate of hosts following subsequent infection as the capacity to mount an effective immune response is compromised.This study also provides preliminary clues for understanding the effects of antibiotic overuse on higher vertebrates through trophic transfer.
基金Special Preliminary Study of 973(2008CB117015)Changjiang Scholars and Innovative Research Team Projects(IRT0734)+1 种基金Priority Themes of Major Science and Technology in Zhejiang Province(2009C12077)the K.C.Wong Magna Fund of Ningbo University
文摘The Dmrt family of genes are involved in sex differentiation in different species of invertebrates, and some vertebrates including human. In this study, we cloned the full-length eDNA of ayu （Plecoglossus altivelis） Draft1 and DmrtA2. Sequence and phylogenetic tree analyses showed ayu Dmrtl showed highest similarity to that of Oncorhynchus mykiss while ayu DmrtA2 is most similar to that of Oryzias latipes. Fluorescence-based quantitative reverse transcription PCR （qRT-PCR） revealed the Draft1 was predominantly expressed in the testis. At the larval stages, Dmrtl mRNA expression level was highest during 52-64 days post hatching （dph） and at the gastrula stage during embryonic development. DmrtA2, meanwhile, was specifically expressed in the ovary and was highly expressed in the female brain tissue, but not male brain tissue. During the larval stages, DmrtA2 expression remained high before day 34, and then fluctuated while generally decreasing. During embryonic development, DmrtA2 expression increased gradually and peaked at the hatching stage. Our data suggest that ayu Dmrtl might participate in the differentiation and maintenance of testis while DmrtA2 may play a role in ovary-differentiation and mature-ovary maintenance. DmrtA2 might also participate in brain development.
