Evolution, expression, and characterisation of liver-expressed antimicrobial peptide genes in ancient chondrostean sturgeons.

Liver-expressed antimicrobial peptide 2 (leap-2) is an evolutionarily ancient molecule that acts as the key component in vertebrate innate immunity against invading pathogens. Leap-2 has been identified and characterised in several teleosts, but not yet in chondrosteans. Herein, the complete coding sequences of leap-2b and leap-2c were identified from expressed sequence tags (ESTs) isolated from Dabry's sturgeon (Acipenser dabryanus) and Chinese sturgeon (A. sinensis), designated as adleap-2b, adleap-2c, asleap-2b, and asleap-2c, respectively. Adleap-2b and adleap-2c sequences share 98% and 100% sequence identity with asleap-2b, and asleap-2c, respectively. Sequence alignment revealed that all four genes contain four cysteine residues, conserved in all fish leap-2 homologs, that form two disulfide bonds. Comparative analysis of the exon-intron structure revealed a three exon/two intron structure for that leap-2 genes in animals, but intron 1 is much longer in sturgeons than in other species. The adleap-2c gene was expressed mainly in the liver of Dabry's sturgeon, and transcription of adleap-2c was significantly up-regulated (p < 0.05) in the liver and midkidney in response to Aeromonas hydrophila challenge. These results suggest adleap-2c may contribute to the defence against pathogenic bacterial invasion. The findings further our understanding of the function of adleap-2c and the molecular mechanism of innate immunity in chondrosteans.

Characterization and expression analysis of g- and c-type lysozymes in Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Expression pattern analysis of IRF4 and its related genes revealed the functional differentiation of IRF4 paralogues in teleost.

In mammals, interferon regulatory factor 4 (IRF4) plays an important role in the process of development and differentiation of B cells, T cells and dendritic cells. It can regulate immune pathway through IRF5, MyD88, IL21, PGC1α, and NOD2. In the present study, we investigated the expression pattern of IRF4 paralogues and these related genes for the first time in teleosts. The results showed that these genes were all expressed predominantly in known immune tissues while IRF5 was also relatively highly expressed in muscle. IRF4b, IL21, MyD88, IRF5 and NOD2 showed maternal expression in the oocyte and the higher expression of IRF4a, Mx and PGC1α before hatching might be involved in the embryonic innate defense system. Zebrafish embryonic fibroblast (ZF4) cells were infected with GCRV and SVCV. During GCRV infection, the expression of Mx was significantly up-regulated from 3 h to 24 h, reaching the highest level at 12 h (101.5-fold over the controls, P < 0.001). And the expression of IRF4a was significantly up-regulated from 3 h to 48 h, reaching the highest level at 12 h (13.75-fold over the controls, P < 0.001). While the expression of IRF4b was only slightly up-regulated at 12 h and 24 h (3.39-fold, 1.93-fold) above control levels, respectively. Whereas the expression of Mx was significantly up-regulated during SVCV infection from 1 h to 48 h, reaching the highest level at 24 h (11.49-fold over the controls, P < 0.001). IRF4a transcripts were significantly up-regulated from 6 h to 24 h, reaching the highest level at 24 h (41-fold over the controls, P < 0.01). IRF4b only showed a slightly up-regulation by SVCV at 24 h (3.2-fold over the controls, P < 0.01). IRF4a and IRF4b displayed a distinct tissue expression pattern, embryonic stages expression and inducible expression in vivo and in vitro, suggesting that IRF4 paralogues might play different roles in immune system.

Bioinformatics and expression analysis of finTRIM genes in grass carp, Ctenopharyngodon idella.

The tripartite motifs (TRIMs) constitute a large family of proteins containing a Really Interesting New Gene (RING) domain, a B-box domain and coiled-coil region followed by different C-terminal domains. TRIM proteins play multiple roles in various cellular processes, including cell growth, differentiation, apoptosis and antiviral immunity. Fish novel large multigene TRIM genes (finTRIM/ftr) appear only in teleosts and play a vital role in antiviral responses. Phylogenetic analysis revealed the existence of different subsets of novel fish TRIM 14 genes (finTRIM14/ftr14), ftr51, ftr67, ftr72, ftr82, ftr83, and ftr99 in grass carp (Ctenopharyngodon idella), suggesting lineage-specific diversification events. Therefore, the number of finTRIM genes varies greatly among species. The ftr genes in grass carp, which are closely related to zebrafish and possess various evolutionary branches, have evolved faster than human TRIMs. The predicted protein domains were almost identical RING zinc finger domains, with the exception of ftr72, the B-box domain (excluding ftr67, ftr82, ftr83), and the B30.2 domain, which evolved under positive selection (with the exception of ftr67, and ftr72). The genes were predominantly expressed in the spleen, gill and head kidney. These findings indicate that the ftr genes in grass carp are involved diverse cellular processes, including innate immune responses.

Analysis of the expression patterns of the novel large multigene TRIM gene family (finTRIM) in zebrafish.

Tripartite motif (TRIM) proteins are receiving increased research interest because of their roles in a wide range of cellular biological processes in innate immunity. In zebrafish (Danio rerio), the functions of the finTRIM (ftr) family are unclear. In the present study, we investigated the expression pattern of ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 in zebrafish for the first time. The results showed that ftr12, ftr67, and ftr84 are maternally expressed in the oocyte and highly expressed at the early stage (0-4 hpf) of embryo (P < 0.05), suggesting their involvement in the embryonic innate defense system. The ftr82 gene was highly expressed at 8 hpf (P < 0.05), which implied that the embryos could synthesize their own immunity-related mRNAs. However, ftr51 and ftr83 were highest at 8 hpf (2.33 and 51.53 relative to ß-actin respectively) and might mediate embryonic development. The expression levels of ftr12, ftr51, and ftr67 were highest in the gill, intestines, and liver, respectively. Ftr82, ftr83, and ftr84 were predominantly expressed in the kidney, suggesting that these finTRIMs might play roles in both immunity and non-immunity-related tissue compartments. Zebrafish embryonic fibroblast (ZF4) cells were infected with Grass carp reovirus (GCRV) and Spring viremia of carp virus (SVCV). During GCRV infection, the expression of ftr12 was significantly upregulated from 12 h to 24 h; and ftr51 and ftr67 increased from 3 h to 12 h. The expressions of ftr82, ftr83, and ftr84 were only upregulated at 12 h, 12 h, and 24 h, respectively. All of these genes were significantly downregulated at 48 h (P < 0.05). Challenge with SVCV upregulated the expressions of ftr12 and ftr51 at 12 h and 48 h (P < 0.05), respectively, and ftr67 reached its highest expression level at 3 h. ftr82 showed only a slight upregulation at 6 h and 48 h, and ftr83 and ftr84 were consecutively increased, reaching their highest levels at 12 h (P < 0.05). Meanwhile, ftr67 and ftr83 were significantly downregulated at 48 h (P < 0.05). Our research demonstrated that ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 probably have important roles in innate immune responses and in non-immunity-related tissues.

Dietary exposure to sulfamethazine alters fish intestinal homeostasis and promotes resistance gene transfer.

The present study was undertaken to explore the effects of sulfamethazine (SMZ) dietary exposure on the enrichment of the intestine microbial structure, and antibiotic resistance gene (ARGs) transmission in marine medaka, with respect to antibiotic dose, duration, and sex. In male fish, a dietary exposure of 10 µg/L SMZ led to a heightened SMZ enrichment in the intestine, whereas metabolite (N-SMZ) levels were elevated at a higher exposure concentration (100 µg/L). Conversely, female fish exhibited stable levels of accumulation and metabolic rates across the exposure period. The composition of intestinal microorganisms revealed that exposure duration exerted a greater impact on the abundance and diversity of gut microbes, and microbial responses to SMZ varied across exposure time points. The expansion of Bacteroidetes and Ruegeria likely stimulated SMZ metabolism and contributed to the more balanced level of SMZ and N-SMZ observed in females. In males, short-term SMZ stress resulted in a disruption of intestinal homeostasis, while the rise in the abundance of the Fusobacteria and Propionigeniuma suggested a potential enhancement in intestinal anti-inflammatory capacity over time. Overall, female medaka exhibited greater adaptability to SMZ, and males appear to experience prolonged effects due to SMZ. A total of 11 ARGs and 5 mobile genetic elements (MGEs) were identified. Ruegeria is the main carrier of two types of MGEs (IS1247, ISSm2-Xanthob), and may serve as an indicator of ARG transmission. Therefore, it is rational to consider some fish breeding areas in natural waters as potential "reservoirs" of antibiotic resistance. This research will provide a valuable reference for the transmission of drug resistance along the food chain.

Metagenomic evidence for increasing antibiotic resistance in progeny upon parental antibiotic exposure as the cost of hormesis.

Antibiotics are widely consumed in the intensive mariculture industry. A better understanding of the effect of antibiotics on intergenerational antibiotic resistance in organisms is urgent since intergenerational transmission is crucial for the spread of antibiotic resistance genes (ARGs) in the environment. Herein, marine medaka (Oryzias melastigma) chronically exposed to low doses of sulfamethazine (SMZ) hormetically affected the progeny, characterized by increased richness and diversity of fecal microbiota and intestinal barrier-related gene up-regulation. Progeny immunity was modulated and caused by genetic factors due to the absence of significant SMZ accumulation in F1 embryos. In addition, some of the top genera in the progeny were positively correlated with immune diseases, while the expression of some immune-related genes, such as TNFα, IL1R2, and TLR3 changed significantly. This further indicated that the host selection caused by changes in progeny immunity was probably the primary determinant of progeny intestinal microbial colonization. Metagenomic analysis revealed that Proteobacteria represented the primary carriers of ARGs, while parental SMZ exposure facilitated the distribution and enrichment of multiple ARGs involved in the antibiotic inactivation in the progeny by promoting the diversity of Gammaproteobacteria and Bacteroidetes, further illustrating that antibiotic selection pressure persisted even if the offspring were not exposed. Therefore, SMZ induced hormesis in the progeny at the expense of increasing antibiotic resistance. Collectively, these findings provide a comprehensive overview of the intergenerational effect of antibiotics and serve as a reminder that the ARG transmission induced by the intergenerational impact of antibiotics on organisms should not be ignored.

Identification and characterization of three CXC chemokines in Asian swamp eel (Monopterus albus) uncovers a third CXCL11_like group in fish.

Chemokines direct cell migration in development and immune defense, and bridge between innate and adaptive immune responses. The chemokine gene family has been rapidly evolving and has undergone species/lineage-specific expansion. Mammals possess inflammatory CXC chemokines CXCL1-8/15 and CXCL9-11 sub-groups, and homeostatic CXCL12-14, 16-17. Orthologues of mammalian CXCL12-14, three chemokines related to CXCL1-8/15 (CXCL8_L1-3), two chemokines related to CXC9-11 (CXCL11_L1-2), and five fish-specific chemokines (CXCL_F1-5) have been described in teleosts. In this study, we reported three novel CXC chemokines in Asian swamp eel Monopterus albus, a commercially important freshwater fish species in China. Two of them belong to the fish-specific CXCL_F2 group, named CXCL_F2a/b, that share 89.5% amino acid identity. The other (CXCL11_L3) belongs to a third CXCL11_L related to the mammalian CXCL9-11 subfamily found only in percomorph fish species, and is the only CXCL9-11 related molecules in this lineage. Mammalian CXCL9-11 attract Th1 cells, and block the migration of Th2 cells in an immune response. This study suggests that all major lineages of teleosts have a CXCL9-11 related chemokine that will aid future functional investigation of CXCL11_L in fish. Cxcl_f2a is highly expressed constitutively in the skin of swamp eels that may attract immune cells to protect the skin in the absence of scales. Cxcl11_l3 and cxcl_f2b are highly expressed in immune tissues/organs and are up-regulated by the viral mimic poly I:C, but not bacterial infection in vivo, suggesting their role in anti-viral defense. The two cxcl_f2 paralogues are differentially expressed and modulated, indicating sub- and/or neo-functionalization.

Omega-3 polyunsaturated fatty acids affect lipopolysaccharide-induced maturation of dendritic cells through mitogen-activated protein kinases p38.

OBJECTIVE: The omega-3 polyunsaturated fatty acids (PUFAs) play a key role as immune response modulators and suppressors of immunologic functions, such as lymphocyte proliferation, cytokine production, and cell surface molecular expression in T lymphocytes, monocytes, and natural killer cells. However, little is known about the effect of omega-3 PUFAs on dendritic cells (DCs). We studied the effect of omega-3 PUFAs on DCs and the related intracellular signal transduction pathway. METHODS: Dendritic cells were generated from human peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factors and interleukin (IL)-4 and treated with eicosapentaenoic acid (EPA), docosahexanoic acid (DHA), and stearic acid for 24 h. Lipopolysaccharide (LPS) was used for maturation of the DCs. The expressions of CD40, CD80, CD86, and human leukocyte antigen-DR (HLA-DR) were analyzed by flow cytometry; production of IL-12 and tumor necrosis factor-alpha were detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The proliferative ability of allogeneic T cells stimulated by DCs was evaluated by tritiated thymidine ((3)H-TdR). Western blot analysis of p38 mitogen-activated protein kinase was conducted. RESULTS: The omega-3 PUFAs reduced expression levels of costimulatory molecules CD80 and CD86 and major histocompatibility complex HLA-DR. IL-12 and tumor necrosis factor-alpha levels decreased significantly in the EPA and DHA groups. EPA and DHA also significantly reduced the proliferative ability of allogeneic T cells stimulated by DCs. The omega-3 PUFAs significantly inhibited LPS-induced p38 phosphorylation. CONCLUSION: The omega-3 PUFAs may inhibit LPS-induced DC maturation and upregulate cytokine production. Impaired p38 mitogen-activated protein kinase activity is a potential critical intracellular signaling transduction mechanism.

First Study on Interferon Regulatory Factor in Sturgeon: Expression Pattern of Interferon Regulatory Factor in Dabry's Sturgeon Acipenser dabryanus.

Interferon regulatory factors (IRFs) are crucial regulators in initiating the host innate immune response against pathogen invasions. Dabry's sturgeon (Acipenser dabryanus) is particularly a valuable fish species found in the Yangtze River, China for which there is scarce immunological data. In the present study, we investigated the expression profile of sturgeon IRF genes. All adIRFs were composed of 8 exons and 7 introns, except adIRF1, which possessed 9 exons interrupted by 8 introns. Moreover, the predicted protein sequence has a DNA-binding domain (DBD) sharing high identity with spotted gar (Lepisosteus oculatus). Regarding the expression patterns, 5 adIRF genes were found to be constitutively expressed in all tissues examined, and were significantly higher in lymphoid organs (eg, blood, kidney, intestine, and spleen). Following Aeromonas hydrophila infection, the expression of adIRF1 and adIRF3 were upregulated in the spleen and caudal kidney, while both the adIRF5 and adIRF8 genes were downregulated in caudal kidney. In addition, adIRF4 was significantly upregulated at 3 h postinfection by A. hydrophila in the spleen and caudal kidney. These results suggest that adIRFs are related to the immune response to bacterial infection, which will help clarify the function of these IRFs and provide a fundamental basis for protecting the Dabry's sturgeon.

[Effects of Pollen Typhae total flavone on glucose and lipid metabolism in 3T3-L1 adipocytes].

OBJECTIVE: To observe the effects of Pollen Typhae total flavone (PTF) on glucose and lipid metabolism in 3T3-L1 adipocytes. METHODS: The content of glucose which disappeared from the culture medium after incubation with drugs for 24 hours was determined as glucose consumption of the cells. The activity of cells was detected by XTT method. The transport of glucose was observed by (3)H-glucose uptake method. The efflux of free fatty acid (FFA) from adipocytes was observed by the concentration of FFA in the culture medium. RESULTS: The glucose concentration in culture medium was significantly decreased with a concentration-dependent effect, when PTF concentrations were from 0.025 g/L to 0.4 g/L. The toxic effect on cells appeared while PTF concentration was 0.4 g/L, and the MTT value decreased. PTF also significantly increased glucose transportation in the 3T3-L1 adipocytes as rosiglitazone (ROS) did. At the same time, FFA concentration in culture medium was significantly decreased as compared to the normal control group, while ROS-treated group did not show any difference. CONCLUSION: PTF can increase insulin sensitivity by increasing glucose transportation and consumption in the 3T3-L1 adipocytes as well as decreasing the FFA efflux from the cells.