Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

In vitro analysis of the expression of inflammasome, antiviral, and immune genes in an Oreochromis niloticus liver cell line following stimulation with bacterial ligands and infection with tilapia lake virus.

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1ß and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 106 TCID50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1ß showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent.

Teleost Hox code defines regional identities competent for the formation of dorsal and anal fins.

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.

Salinity-responsive histone PTMs identified in the gills and gonads of Mozambique tilapia (Oreochromis mossambicus).

BACKGROUND: Histone post-translational modifications (PTMs) are epigenetic marks that can be induced by environmental stress and elicit heritable patterns of gene expression. To investigate this process in an ecological context, we characterized the influence of salinity stress on histone PTMs within the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus). A total of 221 histone PTMs were quantified in each tissue sample and compared between freshwater-adapted fish exposed to salinity treatments that varied in intensity and duration. RESULTS: Four salinity-responsive histone PTMs were identified in this study. When freshwater-adapted fish were exposed to seawater for two hours, the relative abundance of H1K16ub significantly increased in the gills. Long-term salinity stress elicited changes in both the gills and testes. When freshwater-adapted fish were exposed to a pulse of severe salinity stress, where salinity gradually increased from freshwater to a maximum of 82.5 g/kg, the relative abundance of H1S1ac significantly decreased in the gills. Under the same conditions, the relative abundance of both H3K14ac and H3K18ub decreased significantly in the testes of Mozambique tilapia. CONCLUSIONS: This study demonstrates that salinity stress can alter histone PTMs in the gills and gonads of Mozambique tilapia, which, respectively, signify a potential for histone PTMs to be involved in salinity acclimation and adaptation in euryhaline fishes. These results thereby add to a growing body of evidence that epigenetic mechanisms may be involved in such processes.

The anti-inflammatory role of zDHHC23 through the promotion of macrophage M2 polarization and macrophage necroptosis in large yellow croaker (Larimichthys crocea).

Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (Larimichthys crocea), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 LczDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all LczDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, LczDHHC23 exhibited the most pronounced upregulation following Pseudomonas plecoglossicida (P. plecoglossicida) infection of macrophage/monocyte cells (MO/MΦ). Silencing LczDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, LczDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon LczDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by P. plecoglossicida infection. These findings highlight the non-PAT immunomodulatory function of LczDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting LczDHHC23 as a potential therapeutic target for immune modulation in aquatic species.

Magnetic orientation in juvenile Atlantic herring (Clupea harengus) could involve cryptochrome 4 as a potential magnetoreceptor.

The Earth's magnetic field can provide reliable directional information, allowing migrating animals to orient themselves using a magnetic compass or estimate their position relative to a target using map-based orientation. Here we show for the first time that young, inexperienced herring (Clupea harengus, Ch) have a magnetic compass when they migrate hundreds of kilometres to their feeding grounds. In birds, such as the European robin (Erithacus rubecula), radical pair-based magnetoreception involving cryptochrome 4 (ErCRY4) was demonstrated; the molecular basis of magnetoreception in fish is still elusive. We show that cry4 expression in the eye of herring is upregulated during the migratory season, but not before, indicating a possible use for migration. The amino acid structure of herring ChCRY4 shows four tryptophans and a flavin adenine dinucleotide-binding site, a prerequisite for a magnetic receptor. Using homology modelling, we successfully reconstructed ChCRY4 of herring, DrCRY4 of zebrafish (Danio rerio) and StCRY4 of brown trout (Salmo trutta) and showed that ChCRY4, DrCRY4 and ErCRY4a, but not StCRY4, exhibit very comparable dynamic behaviour. The electron transfer could take place in ChCRY4 in a similar way to ErCRY4a. The combined behavioural, transcriptomic and simulation experiments provide evidence that CRY4 could act as a magnetoreceptor in Atlantic herring.

The Tilapia Cyst Tissue Enclosing the Proliferating Myxobolus bejeranoi Parasite Exhibits Cornified Structure and Immune Barrier Function.

Myxozoa, a unique group of obligate endoparasites within the phylum Cnidaria, can cause emerging diseases in wild and cultured fish populations. Recently, the myxozoan Myxobolus bejeranoi has been identified as a prevalent pathogen infecting the gills of cultured hybrid tilapia, leading to systemic immune suppression and considerable mortality. Here, we employed a proteomic approach to examine the impact of M. bejeranoi infection on fish gills, focusing on the structure of the granulomata, or cyst, formed around the proliferating parasite to prevent its spread to surrounding tissue. Enrichment analysis showed increased immune response and oxidative stress in infected gill tissue, most markedly in the cyst's wall. The intense immune reaction included a consortium of endopeptidase inhibitors, potentially combating the myxozoan arsenal of secreted proteases. Analysis of the cyst's proteome and histology staining indicated that keratin intermediate filaments contribute to its structural rigidity. Moreover, we uncovered skin-specific proteins, including a grainyhead-like transcription factor and a teleost-specific S100 calcium-binding protein that may play a role in epithelial morphogenesis and cysts formation. These findings deepen our understanding of the proteomic elements that grant the cyst its distinctive nature at the critical interface between the fish host and myxozoan parasite.

Genome-Wide Identification, Molecular Characterization, and Expression Analysis of the HSP70 and HSP90 Gene Families in Thamnaconus septentrionalis.

Heat shock proteins (HSPs) are a class of highly conserved proteins that play an important role in biological responses to various environmental stresses. The mariculture of Thamnaconus septentrionalis, a burgeoning aquaculture species in China, frequently encounters stressors such as extreme temperatures, salinity variations, and elevated ammonia levels. However, systematic identification and analysis of the HSP70 and HSP90 gene families in T. septentrionalis remain unexplored. This study conducted the first genome-wide identification of 12 HSP70 and 4 HSP90 genes in T. septentrionalis, followed by a comprehensive analysis including phylogenetics, gene structure, conserved domains, chromosomal localization, and expression profiling. Expression analysis from RNA-seq data across various tissues and developmental stages revealed predominant expression in muscle, spleen, and liver, with the highest expression found during the tailbud stage, followed by the gastrula, neurula, and juvenile stages. Under abiotic stress, most HSP70 and HSP90 genes were upregulated in response to high temperature, high salinity, and low salinity, notably hspa5 during thermal stress, hspa14 in high salinity, and hsp90ab1 under low salinity conditions. Ammonia stress led to a predominance of downregulated HSP genes in the liver, particularly hspa2, while upregulation was observed in the gills, especially for hsp90b1. Quantitative real-time PCR analysis corroborated the expression levels under environmental stresses, validating their involvement in stress responses. This investigation provides insights into the molecular mechanisms of HSP70 and HSP90 in T. septentrionalis under stress, offering valuable information for future functional studies of HSPs in teleost evolution, optimizing aquaculture techniques, and developing stress-resistant strains.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Integrative Metabolomics, Enzymatic Activity, and Gene Expression Analysis Provide Insights into the Metabolic Profile Differences between the Slow-Twitch Muscle and Fast-Twitch Muscle of Pseudocaranx dentex.

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

The Expansion of Sirtuin Gene Family in Gilthead Sea Bream (Sparus aurata)-Phylogenetic, Syntenic, and Functional Insights across the Vertebrate/Fish Lineage.

The Sirtuin (SIRT1-7) family comprises seven evolutionary-conserved enzymes that couple cellular NAD availability with health, nutrition and welfare status in vertebrates. This study re-annotated the sirt3/5 branch in the gilthead sea bream, revealing three paralogues of sirt3 (sirt3.1a/sirt3.1b/sirt3.2) and two of sirt5 (sirt5a/sirt5b) in this Perciform fish. The phylogeny and synteny analyses unveiled that the Sirt3.1/Sirt3.2 dichotomy was retained in teleosts and aquatic-living Sarcopterygian after early vertebrate 2R whole genome duplication (WGD). Additionally, only certain percomorphaceae and gilthead sea bream showed a conserved tandem-duplicated synteny block involving the mammalian-clustered sirt3.1 gene (psmd13-sirt3.1a/b-drd4-cdhr5-ctsd). Conversely, the expansion of the Sirt5 branch was shaped by the teleost-specific 3R WGD. As extensively reviewed in the literature, human-orthologues (sirt3.1/sirt5a) showed a high, conserved expression in skeletal muscle that increased as development advanced. However, recent sirt3.2 and sirt5b suffered an overall muscle transcriptional silencing across life, as well as an enhanced expression on immune-relevant tissues and gills. These findings fill gaps in the ontogeny and differentiation of Sirt genes in the environmentally adaptable gilthead sea bream, becoming a good starting point to advance towards a full understanding of its neo-functionalization. The mechanisms originating from these new paralogs also open new perspectives in the study of cellular energy sensing processes in vertebrates.

Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

IL-20 is produced by CD3γδ T cells and induced in the mucosal tissues of grass carp during infection with Aeromonas hydrophila.

Interleukin (IL) 20 is a multifunctional cytokine and plays a vital role in regulating autoimmune diseases, inflammation, and immune responses. IL-20 homologs have been described in fish. However, due to the lack of antibodies, cellular sources and immunological functions of fish IL-20 in response to infections have not been fully characterized. In this study, a monoclonal antibody (mAb) was generated against the recombinant grass carp (Ctenopharyngodon idella) IL-20 protein and characterized by immunoblotting, immunofluorescent microscopy and flow cytometry. It was shown that the IL-20 mAb specifically recognized recombinant IL-20 proteins expressed in the E. coli cells and HEK293 cells. Using confocal microscopy, the IL-20+ cells were identified in the head kidney, gills and intestine of grass carp, and induced after infection with Aeromonas hydrophila. Moreover, the IL-20 protein was found to be secreted mainly by CD3γδ T cells which were located predominantly in the gill filaments and intestinal mucosa. Taken together, our results suggest that IL-20 producing T cells are required for the mucosal immunity against bacterial infection in fish.

Selection of texture-associated biomarkers in chilled and iced grass carp (Ctenopharyngodon idella) fillets via DIA-based proteomics.

Consumers care about the texture of fresh fish flesh, but a rapid quantitative analytical method for this has not been properly established. In this study, texture-associated biomarkers were selected by DIA-based proteomics for possible future application. Results indicated a significant decline in texture and moisture characteristics with extended storage under chilled and iced conditions, and flesh quality was categorized into three intervals. A total of 8 texture-associated biomarkers were identified in the chilled storage group, and 3 distinct ones in the iced storage group. Biomarkers were further refined based on their expression levels. Isobutyryl-CoA dehydrogenase, mitochondrial and [Phosphatase 2A protein]-leucine-carboxy methyltransferase were identified as effective texture-associated biomarkers for chilled fish, and Staphylococcal nuclease domain-containing protein 1 for iced fish. This study provided suitable proteins as indicators of fresh fish flesh texture, which could help establish a rapid and convenient texture testing method in future studies.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate.

Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.

Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

Japanese medaka (Oryzias latipes) Nectin4 plays an important role against red spotted grouper nervous necrosis virus infection.

Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4△4 (+4 bp) and OlNectin4△7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.