Metabolite features and oxidative response in kidney of red crucian carp (Carassius auratus red var) after Aeromonas hydrophila challenge.

Aeromonas hydrophila can threaten the survival of freshwater fish. In this study, A. hydrophila challenge could induce tissue damage, promote antioxidant imbalance as well as alter the transcript levels of oxidative stress indicators, apoptotic genes and metabolic enzyme genes in kidney of red crucian carp (RCC). Metabolomics analysis revealed that A. hydrophila challenge had a profound effect on amino acid metabolism and lipid metabolism. In addition, we further identified dipeptides, fatty acid derivatives, cortisol, choline and tetrahydrocortisone as crucial biomarkers in kidney of RCC subjected to A. hydrophila infection. These results highlighted the importance of metabolic strategy against bacterial infection.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals.

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.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

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-1ß (IL-1ß) 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-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Comparative analysis of erythrocyte hemolysis, plasma parameters and metabolic features in red crucian carp (Carassius auratus red var) and triploid hybrid fish following Aeromonas hydrophila challenge.

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.

Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection.

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents.

A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var.

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.

Chimeric ferritin H in hybrid crucian carp exhibits a similar down-regulation in lipopolysaccharide-induced NF-κB inflammatory signal in comparison with Carassius cuvieri and Carassius auratus red var.

Ferritin H can participate in the regulation of teleostean immunity. ORF sequences of RCC/WCC/WR-ferritin H were 609 bp, while WR-ferritin H gene possessed chimeric fragments or offspring-specific mutations. In order to elucidate regulation of immune-related signal transduction, three fibroblast-like cell lines derived from caudal fin of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) were characterized and designated as RCCFCs, WCCFCs and WRFCs. A sharp increase of ferritin H mRNA was observed in RCCFCs, WCCFCs and WRFCs following lipopolysaccharide (LPS) challenge. Overexpression of RCC/WCC/WR-ferritin H can decrease MyD88-IRAK4 signal and antagonize NF-κB, TNFα promoter activity in RCCFCs, WCCFCs and WRFCs, respectively. These results indicated that ferritin H in hybrid offspring harbors highly-conserved domains with a close sequence similarity to those of its parents, playing a regulatory role in inflammatory signals.

Application of N-methyl-D-aspartate receptor nanopore in screening ligand molecules.

N-methyl-D-aspartate receptors (NMDARs) are crucial for excitatory synaptic transmission in the central nervous system. To study NMDARs more accurately and conveniently, we developed a stable NMDAR nanopore in a planar lipid bilayer. Pharmacological properties were validated using the allosteric modulator Ro 25-6981 and antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). The cyanotoxin ß-N-methylamino-L-alanine (BMAA) found in fresh water systems is suspected to be associated with the development of neurodegenerative diseases. Therefore, BMAA and its two isomers L-2, 4-Diaminobutyric acid dihydrochloride (DAB) and N-(2-aminoethyl) glycine (AEG) and an endogenous excitotoxin, quinolinic acid (QA), were studied using the NMDAR nanopores to assess their effects on NMDAR modulation. We demonstrated that the NMDAR nanopore could reliably detect its ligand molecules at the single-channel level. The study also demonstrated the practicability of NMDAR nanopores, and results were validated using two-electrode voltage-clamp (TEVC) recording. Compared with TEVC recording, the NMDAR nanopores conducted ion channel gating at the single-channel level without being affected by other proteins on the cell membrane. The highly sensitive and accurate NMDAR nanopore technique thus has a unique advantage in screening NMDAR ligand molecules that could be associated with neurodegenerative disease.