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Ferritin M is involved in the regulation of fish immunity. In this study, open reading frame (ORF) sequences of ferritin M from hybrid fish and its parental species were 534 bp. Tissue-specific analysis indicated that the highest level of ferritin M from red crucian carp was observed in kidney, while peaked expressions of ferritin M from white crucian carp and hybrid carp were observed in gill. Elevated levels of ferritin M from hybrid carp and its parental species were detected in immune-related tissues following Aeromonas hydrophila infection or in cultured fish cell lines after lipopolysaccharide (LPS) challenge. Ferritin M overexpression could attenuate NF-κB and TNFα promoter activity in their respective fish cells. Purified ferritin M fusion proteins elicited in vitro binding activity to A. hydrophila and Edwardsiella tarda, lowered bacterial dissemination to tissues and alleviated inflammatory response. Furthermore, treatment with ferritin M fusion proteins could mitigate bacteria-induced liver damage and rescue antioxidant activity. These results suggested that ferritin M in hybrid fish showed a similar immune defense against bacteria infection in comparison with those of its parental species., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1β (IL-1β) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1β secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila challenge could promote the erythrocyte hemolysis, increase free hemoglobin (FHB) level and generate malondialdehyde (MDA) production in plasma but decrease the levels of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (ALP) and lysozyme (LZM) of red crucian carp (RCC, 2 N = 100) and triploid hybrid fish (3 N fish, 3 N = 150) following A. hydrophila challenge. Elevated expression levels of heat shock protein 90 alpha (HSP90α), matrix metalloproteinase 9 (MMP-9), free fatty acid receptor 3 (FFAR3), paraoxonase 2 (PON2) and cytosolic phospholipase A2 (cPLA2) were observed in A. hydrophila-infected fish. In addition, A. hydrophila challenge could significantly increase expressions of cortisol, leucine, isoleucine, glutamate and polyunsaturated fatty acids (PUFAs) in RCC and 3 N, while glycolysis and tricarboxylic acid cycle appeared to be inactive. We identified differential fatty acid derivatives and their metabolic networks as crucial biomarkers from metabolic profiles of different ploidy cyprinid fish subjected to A. hydrophila infection. These results highlighted the comparative metabolic strategy of different ploidy cyprinid fish against bacterial infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Complement 1 inhibitor (C1INH) serving as a multifunctional factor can participate in the regulation of complement cascades and attenuate the activation of various proteases. In this study, we obtained EcC1INH cDNA and the tissue-specific analysis indicate that the highest expression level of EcC1INH mRNA was detected in liver. Moreover, Vibrio alginolyticus challenge can significantly increase EcC1INH mRNA expression in liver and kidney. N-terminal domain of EcC1INH could decrease LPS binding activity to cell surface, while loss of positively charged residues (PCRs) Arg21, His22, Lys50, Arg61 in N-terminal domain of EcC1INH can significantly reduce its interaction with LPS. Furthermore, LPS injection experiment indicated that the binding of EcC1INH N-terminal domain to LPS can antagonize LPS-induced inflammatory signaling pathway and attenuate the production of proinflammatory cytokines in vivo, indicating that EcC1INH was involved in negative regulation of inflammatory response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The tumor suppressor protein p53 plays a critical role in cell cycle, apoptosis and DNA repair. In this study, the full-length pufferfish p53 (Pf-p53) was obtained, containing an open reading frame of 1095 bp, a 5'UTR of 157 bp and a 3'UTR of 285 bp with a poly (A) tail. The Pf-p53 encoded a polypeptide of 364 amino acids with a theoretical isoelectric point of 8.03 and predicted molecular weight of 40.6 kDa. Pf-p53 was ubiquitously expressed in various tissues with a high-level expression in kidney, liver and gill. Vibrio alginolyticus challenge could induce ROS production and disrupt Ca 2+ homeostasis, subsequently leading to the induction of DNA damage and apoptosis, while the Vibrio alginolyticus-induced oxidative stress can also increase the non-specific immunity. The pufferfish challenged with Vibrio alginolyticus showed a sharp increase of Pf-p53 transcript in liver. Subcellular localization analysis revealed that Pf-p53 was primarily localized in nucleus. Furthermore, overexpression of Pf-p53 in Hela cells could inhibit cell proliferation and the transcriptional activities of the NF-ĸB promoter. Taken together, our results indicated that Pf-p53 may play an important role in the immune response to Vibrio alginolyticus challenge., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The α2-macroglobulin receptor (α2MR) is a major domain of complement component 3b, which may play an important role in regulating the downstream complement system in teleosts. In order to characterize the domain thoroughly larger than currently available quantities are required. Thus, in this study the Epinephelus coioides α2MR (Ec-α2MR) was expressed and secreted by the methylotrophic yeast Pichia pastoris with variations in pH and induction time to identify optimal production conditions. At pH 5.5 with 48h induction 13mg of Ec-α2MR (ca. 90% purity) was obtained from 500ml of culture. The Ec-α2MR protein product was validated by MALDI-TOF MS sequence analysis, and both Western blotting and ELISAs demonstrated that it possessed the expected activity, binding to C3b or C3b homolog antibodies, and thus can be used for future studies of the interactions and functions of complement proteins in teleosts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The effect of Ec-FABP10 (Epinephelus coiodes-FABP10) on growth performance, enzyme activity, respiratory burst, MDA level, ATP content, immune-related gene expression of juvenile orange-spotted grouper (E. coioides). The commercial diet supplemented with FABP10 protein was feed to orange-spotted grouper for six weeks. No significant difference was observed in the specific growth rates, while the survival rate in the FABP10 additive group was significantly higher. After the feeding trial, the groupers were exposed to acute low temperature challenge. The decreased level of respiratory burst activity was observed in the FABP10 additive group after the exposure to the acute low temperature stress, while the blood cell count increased significantly at 15 °C and a significant increase of ATP content was observed at 10 °C. Higher enzymatic activities of CAT and SOD were observed at 20 °C and 15 °C, respectively. Meanwhile, the lower level of MDA was observed after the exposure to acute low temperature challenge by comparing with the controls. Further transcript expression analyses of FABP10, SOD2, GPX4, HSPA4 and LIPC in liver by quantitative real-time PCR demonstrated that the up-regulated transcript expression of FABP10, SOD2, HSPA4 and LIPC was observed in FABP10 additive group at 15 °C, while the transcript expression of GPX4 increased significantly at 20 °C. Western blotting analysis confirmed that FABP10 protein expression strongly increased at 15 ± 0.5 °C in FABP10 additive group. These results showed that FABP10 additive diet could moderate the metabolic and immune abilities mainly via ROS pathway in the orange-spotted grouper., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)