An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases.

Influence of Material Removal Strategy on Machining Deformation of Aluminum Plates with Asymmetric Residual Stresses.

In this paper, the effects of material removal strategies and initial stress states on the machining deformation of aluminum alloy plates were investigated through a combination of finite element simulation and experiments. We developed different machining strategies described by Tm+Bn, which removal m mm materials form top and n mm materials from the bottom of the plate. The results demonstrate that the maximum deformation of structural components with the T10+B0 machining strategy could reach 1.94 mm, whereas with the T3+B7 machining strategy was only 0.065 mm, decreasing by more than 95%. The asymmetric initial stress state had a significant impact on the machining deformation of the thick plate. The machined deformation of thick plates increased with the increase in the initial stress state. The concavity of the thick plates changed with the T3+B7 machining strategy due to the asymmetry of the stress level. The deformation of frame parts was smaller when the frame opening was facing the high-stress level surface during machining than when it was facing the low-stress level. Moreover, the modeling results for the stress state and machining deformation were accurate and in good accordance with the experimental findings.

Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia.

Immunoglobulins (Igs) are important effector molecules that mediate humoral immunity. A typical Ig consists of two heavy and two light chains. In teleosts, three Ig heavy chain isotypes (Igµ, Igδ and Igτ) and three Ig light chain isotypes (Igκ, Igλ and Igσ) have been identified. Compared to the heavy chains, teleost Ig light chains have been poorly studied due to the lack of antibodies. In this study, a mouse anti-Nile tilapia Igλ monoclonal antibody (mAb) was prepared, which could specifically recognize Igλ in serum and Igλ+ B cells in tissues. Further, the composition of IgM+ and Igλ+ B cell subsets was analyzed using this antibody and a mouse anti-tilapia IgM heavy chain mAb. The ratio of IgM+Igλ+ B cells to total IgM+ B cells in head kidney and peripheral blood was about 30%, while that in spleen was about 50%; the ratio of IgM-Igλ+ B cells to total Igλ+ B cells in head kidney and peripheral blood was about 45%, while that in spleen was about 25%. The IgM-Igλ+ B cells was speculated to be IgT+ B cells. Finally, we detected an increase in the level of specific antibodies against the surface antigen-Sip of Streptococcus agalactiae in serum after S. agalactiae infection, indicating that mouse anti-tilapia Igλ mAb can be used to detect the antibody level after immunization of Nile tilapia, which lays a foundation for the evaluation of immunization effect of tilapia vaccine.

Presence of two RIG-I-like receptors, MDA5 and LGP2, and their dsRNA binding capacity in a perciform fish, the snakehead Channa argus.

Fish retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are critical RNA sensors in cytoplasm and are involved in antiviral innate immunity. However, some species of fish lack RIG-I gene, and the function of RLR members in RIG-I-absent fish is poorly understood. In the present study, MDA5, LGP2 and MAVS genes were identified in commercially important snakehead Channa argus. But, RIG-I gene was not found in this fish, and a systematic analysis of RLRs in available genome database of fish indicated the absence of RIG-I in the Acanthomorphata, Clupeiformes and Polypteriformes, suggesting that loss events of RIG-I gene may have occurred independently three times in the evolutionary history of fish. The MDA5, LGP2 and MAVS in snakehead have conserved protein domains and genomic location based on sequence, phylogenetic and syntenic analyses. These genes are constitutively expressed in healthy fish and can be induced by polyinosinic and polycytidylic acid (poly(I:C)) stimulation in vitro. It is further revealed that the snakehead MDA5 and LGP2 have binding capacity with dsRNA, such as poly(I:C), and MDA5 can interact with MAVS, implying the antiviral function of MDA5 in the RIG-I-absent fish.

Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production.

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.

MOF Nanosheet-Based Mixed Matrix Membranes with Metal-Organic Coordination Interfacial Interaction for Gas Separation.

In the mixed matrix membrane (MMM), the interface between the filler and the polymer matrix will directly affect the gas separation performance of the membranes. Reasonable interfacial design in MMMs is thus important and necessary. In this work, metal-organic coordination interaction is used to construct the interface in metal-organic framework (MOF) nanosheet-based polyimide MMMs where ultrathin Co-benzenedicarboxylate MOF nanosheets (CBMNs) with a thickness less than 5 nm and a lateral size more than 5 µm are synthesized as fillers and a carboxyl-functionalized polyimide (6FDA-durene-DABA) is used as a polymer matrix. Because of the high aspect ratio (>1000) of CBMNs, abundant metal-organic coordination bonds are formed between Co2+ in CBMNs and the -COOH group in 6FDA-durene-DABA. As a result, the 6FDA-durene-DABA/CBMN MMMs exhibit improved separation performance for the CO2/CH4 and H2/CH4 gas pairs with H2/CH4 and CO2/CH4 selectivities up to 42.0 ± 4.0 and 33.6 ± 3.0, respectively. The enhanced interfacial interaction leads to the comprehensive separation performance of CO2/CH4 and H2/CH4 gas pairs approaching or surpassing the 2008 Robeson upper bound. In addition, the CO2 plasticization pressure of the MMMs is significantly enhanced up to ∼20 bar, which is 2 times that of the pure 6FDA-durene-DABA membrane. When separating a mixed gas of CO2/CH4, the selectivity of CO2/CH4 remains stable at around 23 and the CO2 permeability keeps around 400 barrer during the long-term test.

Hamp Type-1 Promotes Antimicrobial Defense via Direct Microbial Killing and Regulating Iron Metabolism in Grass Carp (Ctenopharyngodon idella).

Hepcidin is an antimicrobial peptide and regulator of iron homeostasis which has two isoforms in most fishes and some mammals. Previous studies have reported that the two hepcidin isoforms have different roles. Hamp type-1 plays a regulatory role in iron metabolism and hamp type-2 mostly performs an antimicrobial role. In this study, we found that Ctenopharyngodon idella (C. idella) have only one hepcidin isoform (hamp type-1), which showed both broad-spectrum antibacterial and iron regulatory functions. C. idella hepcidin mature peptide (hepcidin-25) and truncated peptide (hepcidin-20) exhibited bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner in part through membrane rupture and binding to bacterial genomic DNA. The data from challenge tests demonstrated that the administration of hepcidin-25 significantly reduced mortality rates of C. idella by A. hydrophila infection, probably due to direct bactericidal activities of the peptide and a reduction of iron content in the fish serum. In addition, a comparison between hepcidin-20 and -25 suggests that the N terminal 5 amino acids play a critical role in reducing iron content in fish serum. Our findings revealed an important role of hamp type-1 in maintaining iron homeostasis and fighting against bacterial infections, suggesting the hepcidin has implications for the prevention and control of bacterial infection in aquaculture.

Ca2+ plays an antiviral role by increasing p53 expression to achieve protection against spring viraemia of carp virus infection.

Calcium (Ca) is a messenger that regulates a multitude of physiological processes, but its functions in antiviral progress remain undefined. In this study, we found that Ca2+ enhances fish survival to defend against spring viraemia of carp virus (SVCV) infection by reversing the instability of p53 mediated by the viral protein. First, Ca2+ significantly protected cells and fish against SVCV infection by inducing early apoptosis. Additionally, p53 expression, which was inhibited by SVCV N protein, was upregulated by Ca2+ treatment. Then, the mechanism underlying the reduction of K63-linked p53 ubiquitination by SVCV N protein via the K358 site was completely prevented by Ca2+. These findings reveal the role of Ca2+ in lower vertebrates in the antiviral response, which is connected to and corresponds with viral immune evasion, providing a solution to fish diseases caused by pathogens.

Human inflammatory dendritic cells in malignant pleural effusions induce Th1 cell differentiation.

Dendritic cells are crucial for the initiation and regulation of immune responses against cancer and pathogens. DCs are heterogeneous and highly specialized antigen-presenting cells. Human DCs comprise several subsets with different phenotypes and functional properties. In the steady state, human DC subsets have been well studied. However, the components of DC subsets and their immune functions during the inflamed setting are poorly understood. We identified and characterized DC subsets in the malignant pleural effusions of NSCLC patients. We analyzed the capacity of these DC subsets to induce T-cell differentiation. We observed the presence of inflammatory DCs (infDCs) and macrophages in the malignant pleural effusions of NSCLC patients, as identified by the CD11C+HLA-DR+CD16-BDCA1+ and CD11C+HLA-DR+CD16+BDCA1- phenotypes, respectively. InfDCs represented approximately 1% of the total light-density cells in the pleural effusion and were characterized by the expression of CD206, CD14, CD11b, and CD1α, which were absent on blood DCs. InfDCs also expressed CD80, although at a low level. As infDCs did not express CD40, CD83 and CD275, they remained functionally immature. We found that TLR agonists promoted the maturation of infDCs. Compared with macrophages, infDCs had a weaker capacity to phagocytose necrotic tumor cell lysates. However, only infDCs induced autologous memory CD4+ T-cell differentiation into Th1 cells. For the first time, we found that infDCs were present in the malignant pleural effusions of NSCLC patients. We conclude that infDCs represent a distinct human DC subset and induce Th1 cell differentiation in the presence of TLR agonists.

Membrane IgM+ plasma cells in grass carp (Ctenopharyngodon idella): Insights into the conserved evolution of IgM+ plasma cells in vertebrates.

Teleost fish are the most primitive bony vertebrates that contain B cells; thus, comparative analysis of teleost naïve/mature B cells and plasma cells can provide helpful evidence for understanding the evolution paradigms of these two B-cell subpopulations in vertebrates. In this study, we developed monoclonal antibody against grass carp IgM and identified two different IgM+ cell subsets: IgM+ lymphocytes (Lym), resembling naïve/mature B cells, and IgM+ myeloid cells (Mye), resembling plasma cells. Like plasma cells in mammals, the size of IgM+ Mye is significantly larger than that of IgM+ Lym, as revealed by flow cytometric analysis and transmission electron microscopy. The IgM+ Mye were further verified as plasma cells because they showed gene expression patterns similar with those of human plasma cells and a great capacity to secrete IgM. Like mammalian IgM+ and IgA+ plasma cells, not IgG+ plasma cells, grass carp IgM+ Mye also expressed membrane immunoglobulins, a feature conserved in IgM+ plasma cells in vertebrates. Furthermore, recombinant CD40L or IL-21 alone could induce the plasma cell generation and IgM secretion, while the combination of CD40L and IL-21 had greater effect on IgM secretion, but not on plasma cell generation. This study fills an important gap in the knowledge of plasma cells in teleost fish and provides critical insights into the conserved evolution of IgM+ plasma cells in vertebrates.

Infectious hematopoietic necrosis virus N protein suppresses fish IFN1 production by targeting the MITA.

Interferon (IFN) is a vital antiviral factor in host in the early stages after the viral invasion. Meanwhile, viruses have to survive by taking advantage of the cellular machinery and complete their replication. As a result, viruses evolved several immune escape mechanisms to inhibit host IFN expression. However, the mechanisms used to escape the host's IFN system are still unclear for infectious hematopoietic necrosis virus (IHNV). In this study, we report that the N protein of IHNV inhibits IFN1 production in rainbow trout by degrading the MITA. Firstly, the upregulation of IFN1 promoter activity stimulated by poly I:C was suppressed by IHNV infection. Consistent with this result, the overexpression of the N protein of IHNV blocked the IFN1 transcription that was activated by poly I:C and MITA. Secondly, MITA was remarkably decreased by the overexpression of N protein at the protein level. Further analysis demonstrated that the N protein targeted MITA and promoted the ubiquitination of MITA. Taken together, these data suggested that the production of rainbow trout IFN1 could be suppressed by the N protein of IHNV via degrading MITA.

Spring viraemia of carp virus modulates p53 expression using two distinct mechanisms.

p53, which regulates cell-cycle arrest and apoptosis, is a crucial target for viruses to release cells from cell-cycle checkpoints or to protect cells from apoptosis for their own benefit. Viral evasion mechanisms of aquatic viruses remain mysterious. Here, we report the spring viremia of carp virus (SVCV) degrading and stabilizing p53 in the ubiquitin-proteasome pathway by the N and P proteins, respectively. Early in an SVCV infection, significant induction was observed in the S phase and p53 was decreased in the protein level. Further experiments demonstrated that p53 interacted with SVCV N protein and was degraded by suppressing the K63-linked ubiquitination. However, the increase of p53 was observed late in the infection and experiments suggested that p53 was bound to SVCV P protein and stabilized by enhancing the K63-linked ubiquitination. Finally, lysine residue 358 was the key site for p53 K63-linked ubiquitination by the N and P proteins. Thus, our findings suggest that fish p53 is modulated by SVCV N and P protein in two distinct mechanisms, which uncovers the strategy for the subversion of p53-mediated host innate immune responses by aquatic viruses.

Zebrafish NDRG1a Negatively Regulates IFN Induction by Promoting the Degradation of IRF7.

Viral infection activates the transcription factor IFN regulatory factor 7 (IRF7), which plays a critical role in the induction of IFNs and innate antiviral immune response. How virus-induced IFN signaling is controlled in fish is not fully understood. In this study, we demonstrate that N-myc downstream-regulated gene 1a (NDRG1a) in zebrafish plays a role as a negative regulator for virus-triggered IFN induction. First, the activation of the IFN promoter stimulated by the polyinosinic-polycytidylic acid or spring viremia of carp virus was decreased by the overexpression of NDRG1a. Second, NDRG1a interacted with IRF7 and blocked the IFN transcription activated by IRF7. Furthermore, NDRG1a was phosphorylated by TANK-binding kinase 1 (TBK1) and promoted the K48-linked ubiquitination and degradation of IRF7. Finally, the overexpression of NDRG1a blunted the transcription of several IFN-stimulated genes, resulting in the host cells becoming susceptible to spring viremia of carp virus infection. Our findings suggest that fish NDRG1a negatively regulates the cellular antiviral response by targeting IRF7 for ubiquitination and degradation, providing insights into the novel role of NDRG1a on the innate antiviral immune response in fish.

Dietary gossypol reduced intestinal immunity and aggravated inflammation in on-growing grass carp (Ctenopharyngodon idella).

The present study explored the effects of dietary gossypol on the gut health of on-growing grass carp. The fish were fed six diets containing different levels of free gossypol (0, 121.38, 243.94, 363.89, 759.93 and 1162.06 mg/kg diet) from gossypol-acetic acid for 60 days and then challenged with Aeromonas hydrophila for 14 days. The results showed that dietary gossypol (1) could aggravate enteritis and damage the structure of intestinal epithelial cells, (2) decreased the lysozyme (LZ) and Acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and it down-regulated the Hepcidin (rather than distal intestine (DI)), immunoglobulin Z (IgZ), liver-expressed antimicrobial peptide (LEAP)-2B, Mucin2 and ß-defensin-1 mRNA levels in the proximal intestine (PI), mid intestine (MI) and DI, (3) up-regulated intestinal pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), interleukin 1ß (IL-1ß), IL-6 (only in PI), IL-8 and IL-12p35 mRNA levels partly related to nuclear factor kappa B (NF-κB) signalling, and (4) down-regulated the mRNA levels of anti-inflammatory cytokines such as transforming growth factor (TGF)-ß1, TGF-ß2, interleukin 4/13A (IL-4/13A) (except IL-4/13B), IL-10 and IL-11 partly relating to target of rapamycin (TOR) signalling in the intestines of on-growing grass carp. Moreover, the dietary gossypol had no impact on the LEAP-2A, IL-12P40, IL-17D, IL-10, NF-κBp52, IKKα and eIF4E-binding proteins 2 (4E-BP2) mRNA levels in the intestines. Finally, based on the intestinal histopathological results, enteritis morbidity, LZ activity and IgM content, the safe dose of gossypol in the diets for on-growing grass carp should be less than 103.42 mg/kg diet.

Characterization of grass carp CD40 and CD154 genes and the association between their polymorphisms and resistance to grass carp reovirus.

In bony fish, CD40 and CD154 are two very important costimulatory molecules involved in T and B cell cooperation in thymus-dependent antibody production. In the current study, we identified the cDNAs of CD40 and CD154 and analyzed their genomic structures in grass carp. Quantitative real-time PCR indicated that the CD40 and CD154 were mainly expressed in immune organs. After challenge with grass carp reovirus (GCRV), these two genes were up-regulated at 72 h in head kidney and spleen. Moreover, seven and five single nucleotide polymorphisms (SNPs) were identified in the CD40 and CD154 respectively. Statistical analysis indicated that three SNPs in the coding region of the CD40 were significantly associated with the resistance of grass carp against GCRV. These results indicated that CD40 and CD154 play important roles in the responses to GCRV in grass carp. The SNP markers in the CD40 associated with the resistance to GCRV may facilitate the disease-resistant breeding of grass carp.

Deoxynivalenol decreased the growth performance and impaired intestinal physical barrier in juvenile grass carp (Ctenopharyngodon idella).

Deoxynivalenol (DON) is one of the most common mycotoxin contaminants of animal feed worldwide and brings significant threats to the animal production. However, studies concerning the effect of DON on fish intestine are scarce. This study explored the effects of DON on intestinal physical barrier in juvenile grass carp (Ctenopharyngodon idella). A total of 1440 juvenile grass carp (12.17 ±â€¯0.01 g) were fed six diets containing graded levels of DON (27, 318, 636, 922, 1243 and 1515 µg/kg diet) for 60 days. This study for the first time documented that DON caused body malformation in fish, and histopathological lesions, oxidative damage, declining antioxidant capacity, cell apoptosis and destruction of tight junctions in the intestine of fish. The results indicated that compared with control group (27 µg/kg diet), DON: (1) increased the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) content, and up-regulated the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1: Keap1a but not Keap1b), whereas decreased glutathione (GSH) content and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes (except GSTR in MI) and NF-E2-related factor 2 (Nrf2), as well as the protein levels of Nrf2 in fish intestine. (2) up-regulated cysteinyl aspartic acid-protease (caspase) -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl2-associated X protein (Bax), Fas ligand (FasL) and c-Jun N-terminal protein kinase (JNK) mRNA levels, whereas down-regulated B-cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in fish intestine. (3) down-regulated the mRNA levels of ZO-1, ZO-2b, occludin, claudin-c, -f, -7a, -7b, -11 (except claudin-b and claudin-3c), whereas up-regulated the mRNA levels of claudin-12, -15a (not -15b) and myosin light chain kinase (MLCK) in fish intestine. All above data indicated that DON caused the oxidative damage, apoptosis and the destruction of tight junctions via Nrf2, JNK and MLCK signaling in the intestine of fish, respectively. Finally, based on PWG, FE, PC and MDA, the safe dose of DON for grass carp were all estimated to be 318 µg/kg diet.

Deficiency of dietary pyridoxine disturbed the intestinal physical barrier function of young grass carp (Ctenopharyngodon idella).

The aim of this study was to assess the effects of dietary pyridoxine (PN) deficiency on intestinal antioxidant capacity, cell apoptosis and intercellular tight junction in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (231.85 ±â€¯0.63 g) were fed six diets containing graded levels of PN (0.12-7.48 mg/kg diet) for 10 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results showed that compared with the optimal PN level, PN deficiency (1) increased the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC), decreased the activities and mRNA levels of antioxidant enzymes such as copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) (P < .05); (2) up-regulated the mRNA levels of cysteinyl aspartic acid-protease-3 (caspase-3), caspase-7, caspase-8, caspase-9, Bcl-2 associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1) and Fas ligand (FasL), and down-regulated the mRNA levels of inhibitor of apoptosis proteins (IAP), B-cell lymphoma protein-2 (Bcl-2) and myeloid cell leukaemia-1 (Mcl-1) (P < .05); (3) down-regulated the mRNA levels of ZO-1, occludin [only in middle intestine (MI)], claudin-b, claudin-c, claudin-f, claudin-3c, claudin-7a, claudin-7b and claudin-11, and up-regulated the mRNA levels of claudin-12 and claudin-15a (P < .05), which might be partly linked to Kelch-like-ECH-associated protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2), p38 mitogen-activated protein kinase (p38MAPK) and myosin light chain kinase (MLCK) signalling in the intestines of fish. However, the activities and mRNA levels of MnSOD, the mRNA levels of Keap1b, c-Jun N-terminal protein kinase (JNK) and claudin-15b in three intestinal segments, and the mRNA levels of occludin in the proximal intestine (PI) and distal intestine (DI) were not affected by graded levels of PN. These data indicate that PN deficiency could disturb the intestinal physical barrier function of fish. Additionally, based on the quadratic regression analysis for MDA content and GST activity, the dietary PN requirements for young grass carp were estimated as 4.85 and 5.02 mg/kg diet, respectively.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling.

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ±â€¯0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKß, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.

The improved growth performance and enhanced immune function by DL-methionyl-DL-methionine are associated with NF-κB and TOR signalling in intestine of juvenile grass carp (Ctenopharyngodon idella).

The present study investigated the effects of dietary DL-methionyl-DL-methionine (Met-Met) on growth performance, intestinal immune function and the underlying signalling molecules in juvenile grass carp (Ctenopharyngodon idella). Fish were fed one DL-methionine (DL-Met) group (2.50 g/kg diet) and six graded levels of Met-Met groups (0, 0.79, 1.44, 1.84, 2.22 and 2.85 g/kg diet) for 10 weeks, and then challenged with Aeromonas hydrophila for 14 days. Results indicated that the optimal Met-Met supplementation: (1) increased fish growth performance, intestinal lysozyme (LZ) and acid phosphatase (ACP) activities, complement (C3 and C4) and immunoglobulin M (IgM) contents, up-regulated hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, ß-defensin-1 and Mucin2 mRNA levels; (2) down-regulated tumour necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), interleukin 1ß (IL-1ß), IL-8 [only in the distal intestine (DI)], IL-12p35, IL-12p40 and IL-15 (not IL-17D) mRNA levels partially related to the down-regulation of IκB kinase ß (IKKß) and IKKγ (rather than IKKα), nuclear factor kappa B (NF-κB) p65 and c-Rel (rather than NF-κB p52) mRNA levels and the up-regulation of inhibitor of κBα (IκBα) mRNA levels; (3) up-regulated IL-4/13A, IL-4/13B, IL-6, IL-10, IL-11 and transforming growth factor (TGF)-ß1 (not TGF-ß2) mRNA levels partially associated with the target of rapamycin (TOR) signalling pathway [TOR/ribosomal protein S6 kinases 1 (S6K1), eIF4E-binding proteins (4E-BP)] in three intestinal segments of juvenile grass carp. These results suggest that Met-Met supplementation improves growth and intestinal immune function in fish. Furthermore, according to a positive effect, the optimal Met-Met supplementation was superior to the optimal DL-Met supplementation at improving the growth performance and enhancing the intestinal immune function in fish. Finally, based on percent weight gain (PWG), protection against enteritis morbidity and immune index (LZ activity), the optimal Met-Met supplementation for juvenile grass carp was estimated as 1.61, 1.64 and 1.68 g/kg diet, respectively, as the basal diet contains 8.03 g/kg total sulfur amino acids (TSAA) (4.26 g methionine/kg and 3.77 g cysteine/kg).

Sodium butyrate enhanced physical barrier function referring to Nrf2, JNK and MLCK signaling pathways in the intestine of young grass carp (Ctenopharyngodon idella).

This study evaluated the effect of dietary sodium butyrate (SB) supplementation on the intestinal physical barrier function of young grass carp (Ctenopharyngodon idella). The fish were fed one powdery sodium butyrate (PSB) diet (1000.0 mg kg-1 diet) and five graded levels of microencapsulated sodium butyrate (MSB) diets: 0.0 (control), 500.0, 1000.0, 1500.0 and 2000.0 mg kg-1 diet for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila to explore the effect of SB supplementation on intestinal physical barrier function and the potential mechanisms in fish. The results showed that optimal SB supplementation: (1) down-regulated the cysteine-aspartic protease-2 (caspase-2), caspase-3 (rather than PI), caspase-7, caspase-8 (rather than PI), caspase-9, fatty acid synthetase ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell lymphoma 2 associated X protein (Bax) and c-Jun Nterminal protein kinase (JNK) mRNA levels, up-regulated the B-cell lymphoma protein-2 (Bcl-2) (rather than PI), inhibitor of apoptosis proteins (IAP) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in the intestine (P < 0.05), inhibited the intestinal cell apoptosis, maintained the intestine cell structure integrity; (2) increased NF-E2-related factor 2 (Nrf2) mRNA levels and nucleus protein levels, and down-regulated kelch-like-ECH-associated protein (Keap1b) (rather than Keap1a) mRNA levels in the intestine, up-regulated copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase 1a (GPx1a), GPx1b, GPx4a, GPx4b, glutathione S-transferases R (GSTR), GSTP1, GSTP2, GSTO1, GSTO2 and glutathione reductase (GR) mRNA levels in the intestine, increased the corresponding antioxidant enzymes activity (P < 0.05), thus enhancing the ability of scavenging free radicals and decreasing the reactive oxygen species (ROS) content, decreasing the lipid and protein peroxidation, as well as alleviating oxidative damage; (3) down-regulated the molecule myosin light-chain kinase (MLCK) mRNA levels in the intestine, and up-regulated the occludin, zonula occludens-1 (ZO-1), ZO-2, claudin-b, claudin-c, claudin-f, claudin-3c (rather than PI), claudin-7a, claudin-7b and claudin-11 mRNA levels, down-regulated claudin-12, claudin-15a and claudin-15b mRNA levels (P < 0.05), thus maintaining the structural integrity between cells. This study suggests that SB supplementation could improve fish intestinal physical barrier function. Furthermore, according to the positive effect, MSB was superior to PSB on improving intestinal physical barrier function of fish. Finally, based on protein carbonyl content in the PI, the optimal SB supplementation (MSB as SB source) for young grass carp was estimated to be 338.8 mg kg-1 diet.