Unveiling the protein-lipid interaction mechanism: How the sturgeon lipids diminish the surimi gel properties.

Sturgeon, with 4 times higher lipid content than silver carp (ubiquitously applied for surimi production in China), affects surimi gelling properties. However, how the flesh lipids affect gelling properties remains unclear. This study investigated how flesh lipids impact surimi gelling properties and elucidated the interaction mechanism between lipids and proteins. Results revealed yellow meat contains 7 times higher lipids than white meat. Stronger ionic protein-protein interactions were replaced by weaker hydrophobic forces and hydrogen bonds in protein-lipid interaction. Protein-lipid interaction zones encapsulated lipid particles, changing protein structure from α-helix to ß-sheet structure thereby gel structure becomes flexible and disordered, significantly diminishing surimi gel strength. Docking analysis validated fatty acid mainly binding at Ala577, Ile461, Arg231, Phe165, His665, and His663 of myosin. This study first reported the weakened surimi gelling properties from the perspective of free fatty acids and myosin interactions, offering a theoretical basis for sturgeon surimi production.

Atlantic salmon (Salmo salar) waste as a unique source of biofunctional protein hydrolysates: Emerging productions, promising applications, and challenges mitigation.

The Atlantic salmon is an extremely popular fish for its nutritional value and unique taste among several fish species. Researchers are focusing on the utilization of Atlantic salmon waste for generating protein hydrolysates rich in peptides and amino acids and investigating their health benefits. Several technological approaches, including enzymatic, chemical, and the recently developed subcritical water hydrolysis, are currently used for the production of Atlantic salmon waste protein hydrolysates. Hydrolyzing various wastes, e.g., heads, bones, skin, viscera, and trimmings, possessing antioxidant, blood pressure regulatory, antidiabetic, and anti-inflammatory properties, resulting in applications in human foods and nutraceuticals, animal farming, pharmaceuticals, cell culture, and cosmetics industries. Furthermore, future applications, constraints several challenges associated with industrial hydrolysate production, including sensory, safety, and economic constraints, which could be overcome by suggested techno processing measures. Further studies are recommended for developing large-scale, commercially viable production methods, focusing on eradicating sensory constraints and facilitating large-scale application.

Enhanced anti-inflammatory effect of fish myofibrillar protein by introducing pectin oligosaccharide and its molecular mechanisms.

This study investigated the efficacy of glycation with edible uronic acid-containing oligosaccharides via the Maillard reaction to enhance the anti-inflammatory effect of fish myofibrillar protein (Mf). Lyophilized Mf was reacted with pectin oligosaccharide (PO, half of the total protein weight) at 60 °C and 35 % relative humidity for up to 12 h to produce glycated Mf (Mf-PO). After pepsin and trypsin digestion, the anti-inflammatory effect was assessed by measuring the secretions of proinflammatory cytokines in LPS-stimulated RAW 264.7 macrophages, and the anti-inflammatory effect of Mf was enhanced by PO-glycation without marked lysine loss and browning. The effects on the expressions of genes related to the LPS-stimulated signaling pathway in macrophages were also examined. PO-glycation suppressed LPS-stimulated inflammation by suppressing expression of cd14 and enhancing suppressive effect of Mf on the TLR4-MyD88-dependent inflammatory signaling pathway. Therefore, as an edible reducing sugar, PO could be an effective bioindustrial material for developing anti-inflammatory Mf.

Effects of slurry ice treatment on the physicochemical changes and proteome of large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is susceptible to oxidative denaturation during storage. This work is to investigate the quality alterations by analyzing its physicochemical changes and proteomics throughout preservation under refrigeration, frozen, and slurry ice (SI) conditions. Results revealed that the freshness of large yellow croaker, as evaluated by indicators such as total volatile basic nitrogen, total viable count, and thiobarbituric acid reactive substances, was well maintained while stored in the SI group. Meanwhile, the water distribution in the muscle tissue of group SI exhibited slower fluctuations, thereby preserving the integrity of fish muscle cells. Based on label-free proteomic analysis, a considerable downregulation was observed in the mitogen-activated protein kinase (MAPK) signaling pathway, indicating that SI decelerated this metabolic pathway and effectively delayed the deterioration of muscle. Therefore, the application of SI provides potential for maintaining the quality stability of large yellow croaker.

Effect of temperature fluctuations during frozen storage on ice crystal distribution and quality of tilapia (Oreochromis mossambicus).

The study constructed a model of temperature fluctuation (TF, -20 °C âˆ¼ -10 °C) during frozen status to build a link between the tilapia fillets muscle of ice crystal morphology, moisture distribution, protein oxidation index and the edible quality. When TF treatment more than 3 times, the brightness, color and hardness of frozen tilapia fillets decreased significantly, and the cooking loss and thawing loss increased significantly. The free and unconjugated water in frozen fish fillets exceeded 97 % and did not change much after 9 times TF. The K and TVB-N values were within the safety standards (K < 60 %, TVB-N < 30 mg N/100 g). The ice crystals in the tissues were significantly increased. Protein carbonyls and Ca2+-ATPase were significantly reduced, and secondary structures were irregular. Network correlation analysis showed that ice crystal morphology was significantly correlated with the color, texture and protein oxidation index of frozen tilapia fillets. The results would provide theoretical approach for the transportation and sales of tilapia industrial enterprises.

Enhancement of the flavor and functional characteristics of cod protein isolate using an enzyme-microbe system.

Cod protein isolate (CPI), a by-product of the cod processing industry, represents a novel source of high value-added products. However, off-flavors in cod protein such as bitterness and fishy odor reduce its acceptability to consumers. Here, CPI was first debittered using aminopeptidase from Streptomyces canus (ScAPase) and then deodorized through probiotic fermentation. This is the first reported demonstration of complete removal of the bitterness of CPI using ScAPase. Subsequently, Syn3 and Syn4, as aromatic CPI (ACPI), were prepared from debittered CPI (DCPI) via fermentation with Lactobacillus acidophilus and Bifidobacterium longum, respectively. These products, DCPI and ACPI, were characterized by the absence of bitterness and fishy odor, along with a strong aromatic scent and high overall acceptability. Additionally, these products exhibited improved physicochemical properties, including enhanced oil-holding capacity, emulsifying activity, and resistance to digestion, compared to untreated CPI. However, significant differences were observed in their radical scavenging activities. The highest scavenging activity was detected in Syn3 against DPPH˙ (63.5%) and ˙OH (79.2%), in DCPI against O2- (32.0%), and in post-digestion Syn4 against ABTS˙+ (95.2%). Furthermore, after digestion treatment, these products significantly promoted the proliferation of probiotics. Notably post-digestion Syn4 showed the most substantial proliferation effect on Lactobacillus reuteri, Lactobacillus rhamnosus, and Bifidobacterium breve compared to other post-digestion samples. These results indicate that the treated CPI has the potential for applications in health food products.

Exploring the Physicochemical Characteristics of Marine Protein Hydrolysates and the Impact of In Vitro Gastrointestinal Digestion on Their Bioactivity.

Fish protein hydrolysates (FPHs) were obtained from different fish sources using a combination of microbial enzymes. The industrially produced FPHs from blue whiting (Micromesistius poutassou) and sprat (Sprattus sprattus) were compared to freeze-dried FPHs generated in-house from hake (Merluccius merluccius) and mackerel (Scomber scombrus) in terms of their physicochemical composition and functionality. Significant differences (p < 0.05) were observed in the protein, moisture, and ash contents of the FPHs, with the majority having high levels of protein (73.24-89.31%). Fractions that were more extensively hydrolysed exhibited a high solubility index (74.05-98.99%) at different pHs. Blue whiting protein hydrolysate-B (BWPH-B) had the highest foaming capacity at pH 4 (146.98 ± 4.28%) and foam stability over 5 min (90-100%) at pH 4, 6, and 8. The emulsifying capacity ranged from 61.11-108.90 m2/g, while emulsion stability was 37.82-76.99% at 0.5% (w/v) concentration. In terms of peptide bioactivity, sprat protein hydrolysate (SPH) had the strongest overall reducing power. The highest Cu2+ chelating activity was exhibited by hake protein hydrolysate (HPH) and mackerel protein hydrolysate (MPH), with IC50 values of 0.66 and 0.78 mg protein/mL, respectively, while blue whiting protein hydrolysate-A (BWPH-A) had the highest activity against Fe2+ (IC50 = 1.89 mg protein/mL). SPH scavenged DPPH and ABTS radicals best with IC50 values of 0.73 and 2.76 mg protein/mL, respectively. All FPHs displayed noteworthy scavenging activity against hydroxyl radicals, with IC50 values ranging from 0.48-3.46 mg protein/mL. SPH and MPH showed the highest scavenging potential against superoxide radicals with IC50 values of 1.75 and 2.53 mg protein/mL and against hydrogen peroxide with 2.22 and 3.66 mg protein/mL, respectively. While inhibition of α-glucosidase was not observed, the IC50 values against α-amylase ranged from 8.81-18.42 mg protein/mL, with SPH displaying the highest activity. The stability of FPHs following simulated gastrointestinal digestion (SGID) showed an irregular trend. Overall, the findings suggest that marine-derived protein hydrolysates may serve as good sources of natural nutraceuticals with antioxidant and antidiabetic properties.

Antioxidative, Glucose Management, and Muscle Protein Synthesis Properties of Fish Protein Hydrolysates and Peptides.

The marine environment is an excellent source for many physiologically active compounds due to its extensive biodiversity. Among these, fish proteins stand out for their unique qualities, making them valuable in a variety of applications due to their diverse compositional and functional properties. Utilizing fish and fish coproducts for the production of protein hydrolysates and bioactive peptides not only enhances their economic value but also reduces their potential environmental harm, if left unutilized. Fish protein hydrolysates (FPHs), known for their excellent nutritional value, favorable amino acid profiles, and beneficial biological activities, have generated significant interest for their potential health benefits. These hydrolysates contain bioactive peptides which are peptide sequences known for their beneficial physiological effects. These biologically active peptides play a role in metabolic regulation/modulation and are increasingly seen as promising ingredients in functional foods, nutraceuticals and pharmaceuticals, with potential to improve human health and prevent disease. This review aims to summarize the current in vitro, cell model (in situ) and in vivo research on the antioxidant, glycaemic management and muscle health enhancement properties of FPHs and their peptides.

Effect of protein oxidation on the structure and emulsifying properties of fish gelatin.

This study aimed to investigate the effect of oxidation on fish gelatin and its emulsifying properties. Fish gelatin was oxidized with varying concentrations of H2O2 (0-30 mM). Increased concentrations of the oxidant led to a decrease in amino acids in the gelatin, including glycine, lysine, and arginine. Additionally, the relative content of ordered secondary structure and triple helix fractions decreased. Zeta potential decreased, while particle size, surface hydrophobicity, and water contact angle increased. Regarding emulsifying behavior, oxidation promoted the adsorption of gelatin to the oil-water interface and reduced interfacial tension. With increased degrees of oxidation, the zeta potential and size of the emulsion droplets decreased. The oxidized gelatin exhibited better emulsifying activity but worse emulsifying stability. Based on these results, a mechanism for how oxidation affects the emulsifying properties of gelatin was proposed: the increase in gelatin's hydrophobicity and the decrease in triple helix structure induced by oxidation reduced the interfacial tension at the oil-water interface. This promoted protein adsorption at the oil-water interface, allowing the formation of smaller oil droplets and enhancing gelatin's emulsifying activity. However, the decrease in electrostatic repulsion between emulsion droplets and the decrease in solution viscosity increased the flocculation and aggregation of oil droplets, ultimately weakening the emulsifying stability of gelatin.

Functional and Mechanistic Dissection of Protein Glutaminase PG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar Proteins.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65 °C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Molecular Cloning, Prokaryotic Expression, and Immunological Characterization of ß-Enolase from Grass Carp (Ctenopharyngodon idella).

ß-Enolase is a cross-allergen commonly found in fungi, plants, and aquatic products. Although studies on the allergenicity of fish enolase have been reported in recent years, they are still limited to a few species of marine fish. Therefore, the detection of freshwater fish in the food industry requires more studies of the molecular characterization as well as the allergenicity of enolase. In this study, the nucleotide sequence of ß-enolase from grass carp was obtained by molecular cloning technology. Structural domain analysis showed that it contained the characteristic structural domains of the enolase superfamily, and homology analysis indicated that enolases are highly conserved evolutionarily. Recombinant ß-enolase was obtained by prokaryotic expression, and its allergenicity was assessed by ß-enolase-sensitized mice, which confirmed the ability of ß-enolase to trigger an allergic response and cause a rise in Th1 and Th2 immune responses in mice. These results suggest that ß-enolase could be used as a characterizing substance for the detection of fish allergens in the food industry as well as the preparation of drugs for allergy-related studies.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation.

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

Characterization and immune role of class B scavenger receptor member 1 in spotted sea bass (Lateolabrax maculatus).

Scavenger receptors (SRs) are integral to the innate immune system and function as pattern-recognition receptors that facilitate pathogen clearance and mediate anti-inflammatory responses. However, the role of SRs in the immune response of Lateolabrax maculatus against Aeromonas veronii is unclear. Here, we cloned scavenger receptor B1 from L. maculatus (LmSRB1) and performed bioinformatics analysis to study its potential functions. The open reading frame spans 1530 base pairs and encodes a 509-amino acid protein with a molecular mass of 57.44 kDa. Comparative analysis revealed high sequence conservation among fish species. Expression profiling revealed strong LmSRB1 transcription in various tissues, especially in head kidney and spleen. Following A. veronii exposure, LmSRB1 expression initially increased, peaking after 4-8 h, with a notable secondary peak at 72 h. Fluorescence in situ hybridization indicated that LmSRB1 mainly localized to the cytoplasm, and subcellular-localization studies confirmed LmSRB1 protein expression in the cytoplasm and cell membrane. Enzyme-linked immunosorbent assay data showed dose-dependent binding of LmSRB1 to A. veronii. Modulating LmSRB1 expression significantly altered the levels of IL-8, IL-1ß, TRAF6, and NIK. These results highlight the crucial role of LmSRB1 in L. maculatus's innate immune response to A. veronii and offer insights into improving the management of bacterial infections in aquaculture.

Immune response to Aeromonas hydrophila and molecular characterization of polymeric immunoglobulin receptor in juvenile Megalobrama amblycephala.

Polymeric immunoglobulin receptor (pIgR) is an important immune factor in the mucosal immune system of fish, which plays a key role in mediating the secretion and transport of immunoglobulin into mucus. In this study, the full-length cDNA sequence of Megalobrama amblycephala pIgR gene was firstly cloned and the immune response to Aeromonas hydrophila was detected. After being challenged by Aeromonas hydrophila at 3 d, significantly pathological features were observed in intestine, head kidney, spleen, liver and gill of Megalobrama amblycephala. The content of lysozyme (Lys) and the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) increased significantly at 1 d and reached the peak at 3 d, and the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in serum reached the peak at 5 d and 7 d after infection, respectively. The expression level of IL-1ß gene reached the peak at 3 d in intestine, 5 d in gill and spleen, 7 d in head kidney and liver of Megalobrama amblycephala after infected by Aeromonas hydrophila, respectively. The TNF-α gene expression reached the peak at 3 d in intestine and gill, 5 d in head kidney and spleen, 7 d in liver after infection, respectively. The experimental results showed that the infection of Aeromonas hydrophila caused the pathological changes of immune-related tissues and triggered the inflammation responses. The full-length cDNA sequence of Megalobrama amblycephala pIgR was 1828 bp, and its open reading frame (ORF) was 1023 bp, encoding 340 amino acids. The pIgR of Megalobrama amblycephala has a signal peptide sequence, followed by extracellular region, transmembrane region and intracellular region. The extracellular region includes two Ig-like domains (ILDs), and its tertiary structure is twisted "L". The phylogenetic tree was constructed using the adjacency method, and the pIgR genes of Megalobrama amblycephala and cyprinidae fish were clustered into a single branch. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pIgR gene in different tissues of Megalobrama amblycephala. The expression level of pIgR gene was the highest in liver, followed by intestine, head kidney, skin, middle kidney and spleen, lower in heart, gill and brain, and the lowest in muscle. After being infected by Aeromonas hydrophila, the expression level of Megalobrama amblycephala pIgR gene in intestine, head kidney, spleen, liver and gill showed a trend of increasing first and then decreasing within 28 d. The pIgR gene expression reached the peak in mucosal immune-related tissues (gill and intestine) was earlier than that in systemic immune-related tissues (head kidney and spleen), and the relative expression level of pIgR gene at peak in intestine (12.3 fold) was higher than that in head kidney (3.73 fold) and spleen (7.84 fold). These results suggested that Megalobrama amblycephala pIgR might play an important role in the mucosal immune system to against Aeromonas hydrophila infection.

Molecular structure and functional responses of IgM, IgT and IgD to Flavobacterium covae and Streptococcus iniae infection in Asian seabass (Lates calcarifer Bloch, 1790).

The Asian seabass (Lates calcarifer) faces significant disease threats, which are exacerbated by intensive farming practices and environmental changes. Therefore, understanding its immune system is crucial. The current study presents a comprehensive analysis of immune-related genes in Asian seabass peripheral blood leukocytes (PBLs) using Iso-seq technology, identifying 16 key pathways associated with 7857 immune-related genes, comprising 634 unique immune-related genes. The research marks the first comprehensive report on the entire immunoglobulin repertoire in Asian seabass, revealing specific characteristics of immunoglobulin heavy chain constant region transcripts, including IgM (Cµ, ighm), IgT (Cτ, ight), and IgD (Cδ, ighd). The study confirms the presence of membrane-bound form, ighmmb, ightmb, ighdmb of IgM, IgT and IgD and secreted form, ighmsc and ightsc of IgM and IgT, respectively, with similar structural patterns and conserved features in amino acids across immunoglobulin molecules, including cysteine residues crucial for structural integrity observed in other teleost species. In response to bacterial infections by Flavobacterium covae (formerly F. columnare genomovar II) and Streptococcus iniae, both secreted and membrane-bound forms of IgM (ighmmb and ighmsc) and IgT (ightmb and ightsc) show significant expression, indicating their roles in systemic and mucosal immunity. The expression of membrane-bound form IgD gene, ighdmb, predominantly exhibits targeted upregulation in PBLs, suggesting a regulatory role in B cell-mediated immunity. The findings underscore the dynamic and tissue-specific expression of immunoglobulin repertoires, ighmmb, ighmsc, ightmb, ightsc and ighdmb in Asian seabass, indicating a sophisticated immune response to bacterial pathogens. These findings have practical implications for fish aquaculture, and disease control strategies, serving as a valuable resource for advancing research in Asian seabass immunology.

Interleukin-17B in common carp (Cyprinus carpio L.): Molecular cloning and immune effects as immune adjuvant of Aeromonas veronii formalin-killed vaccine.

The interleukin-17 (IL-17) family of cytokines is critical for host defense responses and mediates different pro- or anti-inflammatory mediators through different signaling pathways. However, the function of the related family member, IL-17B, in teleosts is poorly understood. In the present study, an IL-17B homolog (CcIL-17B) in common carp (Cyprinus carpio) was identified, and sequence analysis showed that CcIL-17B had eight conserved cysteine residues, four of which could form two pairs of disulfide bonds, which in turn formed a ring structure composed of nine amino acids (aa). The deduced aa sequences of CcIL-17B shared 35.79-92.93 % identify with known homologs. The expression patterns were characterized in healthy and bacteria-infected carp. In healthy carp, IL-17B mRNA was highly expressed in the spleen, whereas Aeromonas veronii effectively induced CcIL-17B expression in the liver, head, kidney, gills, and intestine. The recombinant protein rCcIL-17B could regulate the expression levels of inflammatory cytokines (such as IL-1ß, IL-6, TNF-α, and IFN-γ) in primary cultured head kidney leukocytes in vitro. As an adjuvant for the formalin-killed A. veronii (FKA) vaccine, rCcIL-17B induced the production of specific antibodies more rapidly and effectively than Freund's complete adjuvant (FCA). The results of the challenge experiments showed that the relative percent survival (RPS) after vaccination with rCcIL-17B was 78.13 %. This percentage was significantly elevated compared to that observed in the alternative experimental groups (62.5 % and 37.5 %, respectively). Additionally, the bacterial loads in the spleen of the rCcIL-17B + FKA group were significantly lower than those in the control group from 12 h to 48 h after bacterial infection. Furthermore, histological analysis showed that the epithelial cells were largely intact, and the striated border structure was complete in the intestine of rCcIL-17B + FKA group. Collectively, our results demonstrate that CcIL-17B plays a crucial role in eliciting immune responses and evokes a higher RPS against A. veronii challenge compared to the traditional adjuvant FCA, indicating that rCcIL-17B is a promising vaccine adjuvant for controlling A. veronii infection.

A comprehensive study on the multifunctional properties of galectin-4 in red-lip mullet (Planiliza haematocheilus): Insights into molecular interactions, antimicrobial defense, and cell proliferation.

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish.

Interleukin-26 in channel catfish (Ictalurus punctatus): Molecular characterization and immune functional activity.

IL-26 is a crucial inflammatory cytokine that participates in defending host cells against infections. We initially cloned and identified the cDNA sequences of interleukin (IL)-26 in channel catfish (Ictalurus punctatus). The open reading frame (ORF) of IpIL-26 was 537 bp in length, encoding 178 amino acids (aa). Constitutive expression of IpIL-26 was observed in tested tissues, with the highest level found in the gill and spleen. To explore the function of IpIL-26 in channel catfish, different stimuli were used to act on both channel catfish and channel catfish kidney cells (CCK). The expression of IpIL-26 could be up-regulated by bacteria and viruses in multiple tissues. In vitro, recombinant IpIL-26 (rIpIL-26) could induce the expression levels of inflammatory cytokines such as TNF-α, IL-1ß, IL-6, IL-20, and IL-22 playing vital roles in defending the host against infections. Our results demonstrated that IpIL-26 might be an essential cytokine, significantly affecting the immune defense of channel catfish against pathogen infections.

Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides.

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 µM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Identification and screening of umami peptides from skipjack tuna (Katsuwonus pelamis) hydrolysates using EAD/CID based micro-UPLC-QTOF-MS and the molecular interaction with T1R1/T1R3 taste receptor.

In this study, the enzymatic hydrolysates of skipjack tuna, Katsuwonus pelamis, were purified by ultrafiltration and further identified through micro-ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (micro-UPLC-QTOF-MS). The potential umami peptides were identified using both conventional collision-induced dissociation (CID) and novel electron-activated dissociation (EAD) fragmentation techniques. Nine novel umami peptides with iUmami-SCM > 588 were screened. Sensory evaluation and electronic tongue analysis were performed to confirm the taste characteristics of the umami peptides, indicating that these umami peptides all exhibited varying degrees of umami taste. Molecular docking and molecular dynamics simulation were utilized to investigate the interaction with T1R1/T1R3 taste receptors. The docking results revealed that Asp234, Ser23, Glu231, and Ile237 appeared most frequently in all docking sites and formed stable complexes through hydrogen bonding and electrostatic interactions. Furthermore, molecular dynamics simulation allowed for a more comprehensive analysis of their interactions within a dynamic environment, providing a deeper understanding of the umami perception mechanism involving umami peptides and receptors.