Identification, functional characterization and immune response profiles of interleukin-10 in Nibea albiflora.

Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5'-UTR (untranslated region) of 350 bp, a 3'-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost.

A Na+ channel receptor of FMRFamide in the cephalopod Sepiella japonica: Identification, characterisation, and expression profiling during different stages of gonadal development.

FMRFamide, a member of the neuropeptide family, is involved in numerous physiological processes. FMRFamide-activated sodium channels (FaNaCs) are a family of non-voltage-gated, amiloride-sensitive, Na+-selective channels triggered by the neuropeptide FMRFamide. In the present study, the full-length cDNA of the FaNaC receptor of Sepiella japonica (SjFaNaC) was cloned. The cDNA of SjFaNaC was 3004 bp long with an open reading frame (ORF) of 1812 bp, encoding 603 amino acid residues with no signal peptide at the N-terminus. Sequence analysis indicated that SjFaNaC shared a high identity with other cephalopods FaNaCs and formed a sister clade with bivalves. The protein structure was predicted using SWISS-MODEL with AcFaNaC as the template. Quantitative real-time PCR (qRT-PCR) revealed that SjFaNaC transcripts were highly expressed in both female and male reproductive organs, as well as in the optic lobe and brain of the central nervous system (CNS). Results of in situ hybridisation (ISH) showed that SjFaNaC mRNA was mainly distributed in the medulla and deep retina of the optic lobe and in both the supraesophageal and subesophageal masses of the brain. Subcellular localisation indicated that the SjFaNaC protein was localised intracellularly and on the cell surface of HEK293T cells. In summary, these findings may lay the foundation for future exploration of the functions of SjFaNaC in cephalopods.

Chemical Characterization of Honeysuckle Polyphenols and Their Alleviating Function on Ultraviolet B-Damaged HaCaT Cells by Modulating the Nrf2/NF-κB Signaling Pathways.

Scientific evidence attests that the epidermis receives excessive ultraviolet B (UVB) radiation, triggering the generation of substantial quantities of reactive oxygen species (ROS), which disrupted the delicate equilibrium of oxidation-reduction, leading to oxidative stress and inflammation. The historical use of honeysuckle polyphenols (HPs) has garnered our attention due to their efficacy in inhibiting oxidative damage. In this study, HPs were prepared from honeysuckle flowers employing an ultrasonic-assisted extraction method and quantitatively analyzed by a LC-MS/MS, and the mechanisms underlying HPs' antioxidative and anti-inflammatory effects on a UVB-irradiated HaCaT cell model were systematically investigated. The results showed that HPs had a significant cellular repair effect on UVB-irradiated HaCaT cells (p < 0.001). The mechanism of action indicated that HPs could allow Nrf2 to enter the nucleus by regulating the dissociation of Nrf2 from Keap1, which further increases the activity of downstream proteases (SOD and CAT), increases ROS scavenging, and reduces the intracellular malondialdehyde (MDA) level. In addition, HPs could down-regulate Toll-like receptor 4 (TLR4) and inhibit NF-κB (P65) dissociating from IκBα, resulting in a decrease in NF-κB (P65) entry into the nucleus and a decrease in inflammatory factors (TNF-α, IL-6, and IL-1ß). In addition, four key compounds in HPs, including chlorogenic acid, quercetin, isorhamnetin, and luteolin, were selected to verify the mechanism of HPs repairing UVB damage using molecular docking techniques. The experiment suggested that four key active compounds could effectively occupy the Kelch homologue (Kelch) structural domain of Keap1, competitively bind with Nrf2, and facilitate the promotion of Nrf2 binding, ultimately enhancing the translocation of Nrf2 into the nucleus. In addition, four key active compounds could effectively interact with NF-κB (P65) through hydrogen bonding, van der Waals forces, and electrostatic forces to inhibit its entry into the nucleus. In summary, HPs can effectively repair the damage of HaCaT cells by UVB radiation and can be used to develop health and cosmetic products for the treatment of UV radiation-induced diseases.

Molecular Characterization, Expression and In Situ Hybridization Analysis of a Pedal Peptide/Orcokinin-type Neuropeptide in Cuttlefish Sepiella japonica.

BACKGROUND: Neuropeptide pedal peptide (PP) and orcokinin (OK), which are structurally related active peptides, have been widely discovered in invertebrates and constitute the PP/OK neuropeptide family. They have complex structures and play myriad roles in physiological processes. To date, there have been no related reports of PP/OK-type neuropeptide in cephalopods, which possess a highly differentiated multi-lobular brain. METHODS: Rapid Amplification of cDNA Ends (RACE) was employed to obtain the open reading frame (ORF) of PP/OK-type neuropeptide in Sepiella japonica (termed as Sj-PP/OK). Various software were used for sequence analysis. Semi-quantitative PCR was applied to analyze the tissue distribution profile, quantitative real-time PCR (qRT-PCR) was used to study spatio-temporal expression throughout the entire growth and development period, and in situ hybridization (ISH) was employed to observe the tissue location of Sj-PP/OK. RESULTS: in the present study, we identified the ORF of Sj-PP/OK. The putative precursor of Sj-PP/ OK encodes 22 mature peptides, of which only tridecapeptides could undergo post-translationally amidated at C-terminus. Each of these tridecapeptides possesses the most conserved and frequent N-terminus Asp-Ser-Ile (DSI). Sequence analysis revealed that Sj-PP/OK shared comparatively low identity with other invertebrates PP or OK. The tissue distribution profile showed differences in the expression level of Sj-PP/OK between male and female. qRT-PCR data demonstrated that Sj-PP/OK was widely distributed in various tissues, with its expression level increasing continuously in the brain, optic lobe, liver, and nidamental gland throughout the entire growth and development stages until gonad maturation. ISH detected that Sj-PP/OK positive signals existed in almost all regions of the optic lobe except the plexiform zone, the outer edge of all functional lobes in the brain, epithelial cells and the outer membrane layer of the accessory nidamental gland. These findings suggest that Sj-PP/OK might play a role in the regulation of reproduction, such as vitellogenin synthesis, restoration, and ova encapsulation. CONCLUSION: The study indicated that Sj-PP/OK may be involved in the neuroendocrine regulation in cephalopods, providing primary theoretical basis for further studies of its regulation role in reproduction.

Identification, functional characterization and expression pattern of interferon-gamma (IFN-γ) and interferon-gamma receptor 1 (IFNGR1) in Nibea albiflora.

Interferon-gamma (IFN-γ) is an inflammatory cytokine that plays a crucial role in regulating both innate and cell-mediated immune responses by binding to a receptor complex made up of IFNGR1 and IFNGR2. In this study, the complete cDNA of IFN-γ and IFNGR1 from Nibea albiflora were cloned and functionally characterized (named NaIFN-γ and NaIFNGR1), whose complete cDNA sequences were 1593 bp and 2792 bp, encoding 201 and 399 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that the concluded amino acids sequences of NaIFN-γ and NaIFNGR1 shared high identity with their teleost orthologues including the IFN-γ signature and nuclear localization signal (NLS) motif in NaIFN-γ and FN Ⅲ domain in NaIFNGR1. Real-time PCR showed that NaIFN-γ and NaIFNGR1 constitutively expressed in all tested tissues, such as the head-kidney, spleen, liver, kidney, gill, muscle, blood, and intestine with the highest expression of NaIFN-γ and NaIFNGR1 appearing in the liver and gill, respectively. After experiencing stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus (V. alginolyticus) or Vibrio parahaemolyticus (V. parahaemolyticus), NaIFN-γ and NaIFNGR1 mRNA were up-regulated with the time-dependent model. Due to the presence of a nuclear localization signal (NLS), the subcellular localization revealed that NaIFN-γ dispersed throughout the cytoplasm and nucleus. NaIFNGR1, as a member of Cytokine receptor family B, was primarily expressed on the cell membrane. When NaIFN-γ and NaIFNGR1 were co-transfected, their fluorescence signals overlapped on the membrane of HEK 293T cells indicating the potential interaction between IFN-γ and IFNGR1. The GST-pull-down results further showed that NaIFN-γ could directly interact with the extracellular region of NaIFNGR1, further confirming the affinity between IFN-γ and IFNGR1. Taken together, the results firstly demonstrated that the NaIFN-γ ligand-receptor system existed in N.albiflora and played a pivotal part in N.albiflora's immune response against pathogenic bacterial infections, which contributed to the better understanding of the role of IFN-γ in the immunomodulatory mechanisms of teleost.

Transcriptomic analysis of large yellow croaker (Larimichthys crocea) reveals the suppression of the inflammatory response from Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is the most productive marine fish in China. Cryptocaryon irritans is an extremely destructive parasite that causes great economic losses in large yellow croaker aquaculture industry. Therefore, it is very necessary to study the immune response of large yellow croaker in response to C. irritans infection. In this study, the transcriptomic profiles of large yellow croaker were sequenced and analyzed in the brain and head kidney at 72 h after C. irritans infection. Cytokines and chemokines related terms were significantly enriched based on the GO enrichment of down-regulated differentially expressed genes (DEGs) from the head kidney. Meanwhile, cytokine-cytokine receptor interaction was significantly enriched based on the KEGG enrichment of up-regulated DEGs from the brain and down-regulated DEGs from the head kidney, respectively. Moreover, the majority of inflammation-related DEGs were significantly up-regulated in the brain, but distinctly down-regulated in the head kidney. These results showed that the brain and head kidney might play different roles against C. irritans infection, and the inflammatory response of large yellow croaker may be restrained during C. irritans infection. Taken together, the transcriptomic analyses will be helpful to more comprehensively understand the immune mechanism of teleost against C. irritans infection, and provide a theoretical basis for the prevention and treatment of Cryptosporidiosis.

Novel Ca-Chelating Peptides from Protein Hydrolysate of Antarctic Krill (Euphausia superba): Preparation, Characterization, and Calcium Absorption Efficiency in Caco-2 Cell Monolayer Model.

Antarctic krill (Euphausia superba) is the world's largest resource of animal proteins and is thought to be a high-quality resource for future marine healthy foods and functional products. Therefore, Antarctic krill was degreased and separately hydrolyzed using flavourzyme, pepsin, papain, and alcalase. Protein hydrolysate (AKH) of Antarctic krill prepared by trypsin showed the highest Ca-chelating rate under the optimized chelating conditions: a pH of 8.0, reaction time of 50 min, temperature of 50 °C, and material/calcium ratio of 1:15. Subsequently, fourteen Ca-chelating peptides were isolated from APK by ultrafiltration and a series of chromatographic methods and identified as AK, EAR, AEA, VERG, VAS, GPK, SP, GPKG, APRGH, GVPG, LEPGP, LEKGA, FPPGR, and GEPG with molecular weights of 217.27, 374.40, 289.29, 459.50, 275.30, 300.36, 202.21, 357.41, 536.59, 328.37, 511.58, 516.60, 572.66, and 358.35 Da, respectively. Among fourteen Ca-chelating peptides, VERG presented the highest Ca-chelating ability. Ultraviolet spectrum (UV), Fourier Transform Infrared (FTIR), and scanning electron microscope (SEM) analysis indicated that the VERG-Ca chelate had a dense granular structure because the N-H, C=O and -COOH groups of VERG combined with Ca2+. Moreover, the VERG-Ca chelate is stable in gastrointestinal digestion and can significantly improve Ca transport in Caco-2 cell monolayer experiments, but phytate could significantly reduce the absorption of Ca derived from the VERG-Ca chelate. Therefore, Ca-chelating peptides from protein hydrolysate of Antarctic krill possess the potential to serve as a Ca supplement in developing healthy foods.

Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from the Collagens of Monkfish (Lophius litulon) Swim Bladders: Isolation, Characterization, Molecular Docking Analysis and Activity Evaluation.

The objective of this study was to isolate and characterize collagen and angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) peptides from the swim bladders of monkfish (Lophius litulon). Therefore, acid-soluble collagen (ASC-M) and pepsin-soluble collagen (PSC-M) with yields of 4.27 ± 0.22% and 9.54 ± 0.51%, respectively, were extracted from monkfish swim bladders using acid and enzyme methods. The ASC-M and PSC-M contained Gly (325.2 and 314.9 residues/1000 residues, respectively) as the major amino acid, but they had low imino acid content (192.5 and 188.6 residues/1000 residues, respectively) in comparison with collagen from calf skins (CSC) (216.6 residues/1000 residues). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and ultraviolet (UV) absorption spectrums of ASC-M and PSC-M illustrated that they were mainly composed of type I collagen. Subsequently, three ACEi peptides were isolated from a PSC-M hydrolysate prepared via a double-enzyme system (alcalase + neutrase) and identified as SEGPK (MHP6), FDGPY (MHP7) and SPGPW (MHP9), with molecular weights of 516.5, 597.6 and 542.6 Da, respectively. SEGPK, FDGPY and SPGPW displayed remarkable anti-ACE activity, with IC50 values of 0.63, 0.94 and 0.71 mg/mL, respectively. Additionally, a molecular docking assay demonstrated that the affinities of SEGPK, FDGPY and SPGPW with ACE were -7.3, -10.9 and -9.4 kcal/mol, respectively. The remarkable ACEi activity of SEGPK, FDGPY and SPGPW was due to their connection with the active pockets and/or sites of ACE via hydrogen bonding, hydrophobic interaction and electrostatic force. Moreover, SEGPK, FDGPY and SPGPW could protect HUVECs by controlling levels of nitric oxide (NO) and endothelin-1 (ET-1). Therefore, this work provides an effective means for the preparation of collagens and novel ACEi peptides from monkfish swim bladders, and the prepared ACEi peptides, including SEGPK, FDGPY and SPGPW, could serve as natural functional components in the development of health care products to control hypertension.

Molecular characterization and expression of twenty interleukin-17 transcripts in the common Chinese cuttlefish (Sepiella japonica) in response to Vibrio harveyi infection.

The common Chinese cuttlefish (Sepiella japonica) is an essential species for stock enhancement by releasing juveniles in the East China Sea now. S. japonica is susceptible to bacterial diseases during parental breeding. In vertebrates, Interleukin-17 (IL-17) cytokine family plays critical roles in both acute and chronic inflammatory responses. In Cephalopoda, few studies have been reported on IL-17 genes so far. In this study, twenty IL-17 transcripts obtained from S. japonica were divided into eight groups (designated as Sj_IL-17-1 to Sj_IL-17-8). Multiple alignment analysis showed that IL-17s in S. japonica and human both contained four ß-folds (ß1-ß4), except for Sj_IL-17-6 with two ß-folds (ß1 and ß2), and the third and fourth ß-folds of Sj_IL-17-5 and Sj_IL-17-8 were longer than those of other Sj_IL-17. Protein structure and conserved motifs analysis demonstrated that Sj_IL-17-5 and Sj_IL-17-6 displayed different protein structure with respect to other six Sj_IL-17 proteins. The homology and phylogenetic analysis of amino acids showed that Sj_IL-17-5, Sj_IL-17-6 and Sj_IL-17-8 had low homology with the other five Sj_IL-17s. Eight Sj_IL-17 mRNAs were ubiquitously expressed in ten examined tissues, with dominant expression in the hemolymph. qRT-PCR data showed that the mRNA expression levels of Sj_IL-17-2, Sj_IL-17-3, Sj_IL-17-6, and Sj_IL-17-8 were significantly up-regulated in infected cuttlefishes, and Sj_IL-17-2, Sj_IL-17-6, Sj_IL-17-7, and Sj_IL-17-8 mRNAs Awere significantly up-regulated after bath infection of Vibrio harveyi, suggesting that certain Sj_IL-17s were involved in the immune response of S. japonica against V. harveyi infection. These results implied that Sj_IL-17s were likely to have distinct functional diversification. This study aims to understand the involvement of Sj_IL-17 genes in immune responses of cuttlefish against bacterial infections.

Antioxidant Peptides from Monkfish Swim Bladders: Ameliorating NAFLD In Vitro by Suppressing Lipid Accumulation and Oxidative Stress via Regulating AMPK/Nrf2 Pathway.

In this study, we investigate the ameliorating functions of QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) from monkfish swim bladders on an FFA-induced NAFLD model of HepG2 cells. The lipid-lowering mechanisms revealed that these five oligopeptides can up-regulate the expression of phospho-AMP-activated protein kinase (p-AMPK) proteins to inhibit the expression of the sterol regulatory element binding protein-1c (SREBP-1c) proteins on increasing lipid synthesis and up-regulating the expression of the PPAP-α and CPT-1 proteins on promoting the ß-oxidation of fatty acids. Moreover, QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) can significantly inhibit reactive oxygen species' (ROS) production, promote the activities of intracellular antioxidases (superoxide dismutase, SOD; glutathione peroxidase, GSH-PX; and catalase, CAT) and bring down the content of malondialdehyde (MDA) derived from lipid peroxidation. Further investigations revealed that the regulation of these five oligopeptides on oxidative stress was achieved through activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to raise the expression levels of the heme oxygenase 1 (HO-1) protein and downstream antioxidant proteases. Therefore, QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) could serve as candidate ingredients to develop functional products for treating NAFLD.

Marine Bioactive Peptides-Structure, Function and Application.

Marine organisms live in harsh marine habitats, causing them to have significantly different and more diverse proteins than those of terrestrial organisms [...].

Eighteen Novel Bioactive Peptides from Monkfish (Lophius litulon) Swim Bladders: Production, Identification, Antioxidant Activity, and Stability.

In the study, papain was chosen from five proteases to hydrolyze proteins of monkfish swim bladders for effectively utilizing monkfish (Lophius litulon) processing byproducts, and the hydrolysis conditions of papain were optimized as hydrolysis temperature of 65 °C, pH 7.5, enzyme dose 2.5% and time 5 h using single-factor and orthogonal experiments. Eighteen peptides were purified from the swim bladder hydrolysate of monkfish by ultrafiltration and gel permeation chromatography methods and identified as YDYD, QDYD, AGPAS, GPGPHGPSGP, GPK, HRE, GRW, ARW, GPTE, DDGGK, IGPAS, AKPAT, YPAGP, DPT, FPGPT, GPGPT, GPT and DPAGP, respectively. Among eighteen peptides, GRW and ARW showed significant DPPH· scavenging activities with EC50 values of 1.053 ± 0.003 and 0.773 ± 0.003 mg/mL, respectively; YDYD, QDYD, GRW, ARW and YPAGP revealed significantly HO· scavenging activities with EC50 values of 0.150 ± 0.060, 0.177 ± 0.035, 0.201 ± 0.013, 0.183 ± 0.0016 and 0.190 ± 0.010 mg/mL, respectively; YDYD, QDYD, ARW, DDGGK and YPAGP have significantly O2-· scavenging capability with EC50 values of 0.126 ± 0.0005, 0.112 ± 0.0028, 0.127 ± 0.0002, 0.128 ± 0.0018 and 0.107 ± 0.0002 mg/mL, respectively; and YDYD, QDYD and YPAGP showed strong ABTS+· scavenging ability with EC50 values of 3.197 ± 0.036, 2.337 ± 0.016 and 3.839 ± 0.102 mg/mL, respectively. YDYD, ARW and DDGGK displayed the remarkable ability of lipid peroxidation inhibition and Ferric-reducing antioxidant properties. Moreover, YDYD and ARW can protect Plasmid DNA and HepG2 cells against H2O2-induced oxidative stress. Furthermore, eighteen isolated peptides had high stability under temperatures ranging from 25-100 °C; YDYD, QDYD, GRW and ARW were more sensitive to alkali treatment, but DDGGK and YPAGP were more sensitive to acid treatment; and YDYD showed strong stability treated with simulated GI digestion. Therefore, the prepared antioxidant peptides, especially YDYD, QDYD, GRW, ARW, DDGGK and YPAGP from monkfish swim bladders could serve as functional components applied in health-promoting products because of their high-antioxidant functions.

Bioactive Peptides from Skipjack Tuna Cardiac Arterial Bulbs (II): Protective Function on UVB-Irradiated HaCaT Cells through Antioxidant and Anti-Apoptotic Mechanisms.

The aim of this study was to investigate the protective function and mechanism of TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) from skipjack tuna cardiac arterial bulbs on skin photoaging using UVB-irradiated HaCaT cell model. The present results indicated that TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) had significant cytoprotective effect on UVB-irradiated HaCaT cells (p < 0.001). Hoechst 33342 staining showed that apoptosis of UV-irradiated HaCaT cells could be significantly reduced by the treatment of TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM); JC-1 staining showed that TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) could protect HaCaT cells from apoptosis by restoring mitochondrial membrane potential (MMP); Furthermore, TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) could significantly down-regulate the ratio of Bax/Bcl-2 and reduce the expression level of the apoptosis-executing protein Caspase-3 by decreasing the expression of protein Caspase-8 and Caspase-9 (p < 0.05). The action mechanism indicated that TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) could up-regulate the expression levels of Nrf2, NQO1 and HO-1 (p < 0.05), which further increased the activity of downstream proteases (SOD, CAT and GSH-Px), and scavenged reactive oxygen species (ROS) and decreased the intracellular levels of malondialdehyde (MDA). In addition, molecular docking indicated that TCP3 (PKK) and TCP6 (YEGGD) could competitively inhibit the Nrf2 binding site because they can occupy the connection site of Nrf2 by binding to the Kelch domain of Keap1 protein. TCP9 (GPGLM) was inferred to be non-competitive inhibition because it could not bind to the active site of the Kelch domain of Keap1 protein. In summary, the antioxidant peptides TCP3 (PKK), TCP6 (YEGGD) and TCP9 (GPGLM) from cardiac arterial bulbs of skipjack tuna can effectively protect HaCaT cells from UVB-irradiated damage and can be used in the development of healthy and cosmetic products to treat diseases caused by UV radiation.

Comprehensive Analysis of Whole-Transcriptome Profiles in Response to Acute Hypersaline Challenge in Chinese Razor Clam Sinonovacula constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Molecular cloning, expression analysis and immune-related functional identification of tumor necrosis factor alpha (TNFα) in Sepiella japonica under bacteria stress.

Tumor necrosis factor α (TNFα), a cytokine mainly secreted by active macrophages and monocytes, causes hemorrhagic necrosis of tumor tissues, kills tumor cells, regulates inflammatory responses, and plays a crucial role in innate immunity. In this study, TNFα of Sepiella japonica (named as SjTNFα) was acquired, whose full-length cDNA was 1206 bp (GenBank accession no. ON357428), containing a 5' UTR of 185 bp, a 3' UTR of 137 bp and an open reading frame (ORF) of 1002bp to encode a putative peptide of 333 amino acids for constructing the transmembrane domain and the cytoplasmic TNF domain. Its predicted pI was 8.69 and the theoretical molecular weight was 44.72 KDa. Multiple sequence alignment and phylogenetic analysis showed that SjTNFα had the highest homology to Octopus sinensis, they fell into a unified branch and further clustered with other animals. Real-time PCR indicated that SjTNFα was widely expressed in all subject tissues, including spleen, pancreas, gill, heart, brain, optic lobe, liver and intestine, and exhibited the highest in the liver and the lowest in the brain. The relative expression of SjTNFα varied at the developmental period of juvenile stage, pre-spawning and oviposition in the squid, with the highest in the liver at the juvenile stage and oviposition, and in the optic lobe of pre-spawning. After being infected with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of SjTNFα in liver and gill were both upregulated with time, and the highest expression appeared at 24 h and 8 h in liver for different infection, and at 4 h in gill consistently. Cell localization showed that SjTNFα distributed on membrane of HEK293 cells because it was a type II soluble transmembrane protein. When HEK293 cells were stimulated with LPS of different concentrations, the NF-κB pathway was activated in the nucleus and the corresponding mRNA was transferred through the intracellular signal transduction pathway, resulting in the synthesis and release of TNFα, which made the expression of SjTNFα was up-regulated obviously. These findings showed that SjTNFα might play an essential role in the defense of S. japonica against bacteria challenge, which contributed to the understanding of the intrinsic immune signaling pathway of Cephalopoda and the further study of host-pathogen interactions.

Characterization of a novel Toll-like receptor 13 homologue from a marine fish Nibea albiflora, revealing its immunologic function as PRRs.

Congenital immunity mediated by Toll-like receptor (TLR) family is the first line of defense for disease-resistant immunity of fish and plays a vital role as a bridge between innate immunity and acquired immunity. As a less known member of the TLR family TLR13 can participate in the immune and inflammatory reactions of the body for recognizing the conserved sequence of 23S rRNA in bacteria and induce immune response. In this study, the full-length cDNA of TLR13 from Nibea albiflora (named as NaTLR13) was cloned and was functionally characterized. It was 4210bp (GenBank accession no. MT701899) including an open reading frame (ORF) of 2886bp to encode 962 amino acids with molecular weight of 110.37 kDa and the theoretical isoelectric point of 9.08. There were several conservative structures in NaTLR13 such as 15 leucine-rich repeat sequences (LRRs), a Toll-IL-1 receptor domain (TIR), an LRR-CT terminal domain, two LRR-TYP structures and two transmembrane domains. The multiple sequence alignment and phylogenetic analysis manifested that NaTLR13 had high similarity with Larimichthys crocea and Collichthys lucidus (88.79% and 87.02%, respectively) and they fell into the same branch. The Real-time PCR showed that NaTLR13 was expressed in all selected tissues, with the highest in the spleen, followed by the liver, kidney, gill, heart and muscle. After being challenged by Vibrio alginolyticus, Vibrio parahaemolyticus or Poly (I:C), the expression of NaTLR13 increased firstly, then decreased and finally stabilized with time for its immune defense function. Subcellular localization analysis revealed that NaTLR13 was unevenly distributed in the cytoplasm with green fluorescence and MyD88 was evenly spread in the cytoplasm with red signals. When NaTLR13 and MyD88 were co-transfected, they obviously overlapped and displayed orange-yellow color, which showed that the homologous TLR13 might interact with MyD88 for NFκB signaling pathway transmission. The functional domains of NaTLR13 (named NaTLR13-TIR and NaTLR13-LRR) were expressed in E.coli BL21 (DE3) and purified by Ni-NAT Superflow Resin conforming to the expected molecular weights, and the recombinant proteins could bind to three Vibrios (V.alginolyticus, V.parahaemolyticus and Vibrio harveyi), indicating that NaTLR13 could be bounden to bacteria through its functional domain. These results suggested that NaTLR13 might play an important role in the defense of N.albiflora against bacteria or viral infection and the data would provide some information for further understanding the regulatory mechanism of the innate immune system in fish.

Bioactive Peptides from Skipjack Tuna Cardiac Arterial Bulbs: Preparation, Identification, Antioxidant Activity, and Stability against Thermal, pH, and Simulated Gastrointestinal Digestion Treatments.

Cardiac arterial bulbs of Skipjack tuna (Katsuwonus pelamis) are rich in elastin, and its hydrolysates are high quality raw materials for daily cosmetics. In order to effectively utilizing Skipjack tuna processing byproducts-cardiac arterial bulbs and to prepare peptides with high antioxidant activity, pepsin was selected from six proteases for hydrolyzing proteins, and the best hydrolysis conditions of pepsin were optimized. Using ultrafiltration and chromatographic methods, eleven antioxidant peptides were purified from protein hydrolysate of tuna cardiac arterial bulbs. Four tripeptides (QGD, PKK, GPQ and GLN) were identified as well as seven pentapeptides (GEQSN, GEEGD, YEGGD, GEGER, GEGQR, GPGLM and GDRGD). Three out of them, namely the tripeptide PKK and the pentapeptides YEGGD and GPGLM exhibited the highest radical scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and superoxide anion assays. They also showed to protect plasmid DNA and HepG2 cells against H2O2-induced oxidative stress. Furthermore, they exhibited high stability under temperature ranged from 20-100 °C, pH values ranged from 3-11, and they simulated gastrointestinal digestion for 240 min. These results suggest that the prepared eleven antioxidant peptides from cardiac arterial bulbs, especially the three peptides PKK, YEGGD, and GPGLM, could serve as promising candidates in health-promoting products due to their high antioxidant activity and their stability.

Novel angiotensin-converting enzyme inhibitory peptides from tuna byproducts-milts: Preparation, characterization, molecular docking study, and antioxidant function on H2O2-damaged human umbilical vein endothelial cells.

To prepare peptides with high angiotensin-converting enzyme (ACE) inhibitory (ACEi) activity, Alcalase was screened from five proteases and employed to prepare protein hydrolysate (TMH) of skipjack tuna (Katsuwonus pelamis) milts. Subsequently, 10 novel ACEi peptides were isolated from the high-ACEi activity TMH and identified as Tyr-Asp-Asp (YDD), Thr-Arg-Glu (TRE), Arg-Asp-Tyr (RDY), Thr-Glu-Arg-Met (TERM), Asp-Arg-Arg-Tyr-Gly (DRRYG), Ile-Cys-Tyr (ICY), Leu-Ser-Phe-Arg (LSFR), Gly-Val-Arg-Phe (GVRF), Lys-Leu-Tyr-Ala-Leu-Phe (KLYALF), and Ile-Tyr-Ser-Pro (IYSP) with molecular weights of 411.35, 404.41, 452.45, 535.60, 665.69, 397.48, 521.61, 477.55, 753.91, and 478.53 Da, respectively. Among them, the IC50 values of ICY, LSFR, and IYSP on ACE were 0.48, 0.59, and 0.76 mg/mL, respectively. The significant ACEi activity of ICY, LSFR, and IYSP with affinities of -7.0, -8.5, and -8.3 kcal/mol mainly attributed to effectively combining with the ACEi active sites through hydrogen bonding, electrostatic force, and hydrophobic interaction. Moreover, ICY, LSFR, and IYSP could positively influence the production of nitric oxide (NO) and endothelin-1 (ET-1) secretion in human umbilical vein endothelial cells (HUVECs) and weaken the adverse impact of norepinephrine (NE) on the production of NO and ET-1. In addition, ICY, LSFR, and IYSP could provide significant protection to HUVECs against H2O2 damage by increasing antioxidase levels to decrease the contents of reactive oxide species and malondialdehyde. Therefore, the ACEi peptides of ICY, LSFR, and IYSP are beneficial functional molecules for healthy foods against hypertension and cardiovascular diseases.

Sequence and functional features of a novel scavenger receptor homolog, SCARA5 from Yellow drum (Nibea albiflora).

As an important member in SR-As, member 5 (SCARA5) can swallow apoptotic cells and foreign bodies, and participate multiple signaling pathways to inhibit tumor occurrence, development growth and metastasis. To explore its immune function, SCARA5 was identified from the yellow drum (Nibea albiflora) according to its transcriptome data, and its full-length cDNA was 6968 bp (named as NaSCARA5, GenBank accession no: MW070211) encoding 497 amino acids with a calculated molecular weight of 55.12 kDa, which had the typical motifs of SR family, such as transmembrane helix region, coil region, Pfam collagens region and SR region. BLASTp and the phylogenetic relationship analysis illustrated that the sequences shared high similarity with known SCARA5 of teleosts. Quantitative real time RT-PCR analysis showed that NaSCARA5 was expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the spleen (24.42-fold compared with that in heart). After being infected with Polyinosinic:polycytidylic acid (PolyI:C), Vibrio alginolyticus and Vibrio parahaemolyticus, NaSCARA5 mRNA were up-regulated with time dependent mode in spleen, which suggested that NaSCARA5 might play an important role in the immune process of fish. The extracellular domain of NaSCARA5 was successfully expressed in BL21 (DE3), and yielded the target protein of the expected size with many active sites for their conferring protein-protein interaction functions. After being purified by Ni-NAT Superflow resin and renatured, it was found to bind all the tested bacteria (V.parahaemolyticus,V.alginolyticus and Vibrio harveyi). The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into epithelioma papulosum cyprini (EPC) cells, and it was mainly expressed on the cell membrane indicating that NaSCARA5 was a typical transmembrane protein. The aforementioned results indicated that NaSCARA5 played a significant role in the defense against pathogenic bacteria infection as PRRs, which may provide some further understandings of the regulatory mechanisms in the fish innate immune system for SR family.

Seventeen novel angiotensin converting enzyme (ACE) inhibitory peptides from the protein hydrolysate of Mytilus edulis: isolation, identification, molecular docking study, and protective function on HUVECs.

In the study, seventeen angiotensin converting enzyme (ACE) inhibitory peptides were isolated from the protein hydrolysate of blue mussel (Mytilus edulis) and identified as MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK, and FKWH, respectively. Among them, IK, YEGDP, WF, and SWISS showed the strongest ACE inhibitory activity with IC50 values of 0.77 ± 0.020, 0.19 ± 0.010, 0.40 ± 0.015, and 0.32 ± 0.017 mg mL-1, respectively. Molecular docking study indicated that IK, YEGDP, WF, and SWISS exhibited better inhibitory activity attributed to its effective interaction with the active site of ACE by hydrogen bonding, electrostatic force and hydrophobic interaction. Furthermore, IK, YEGDP and WF perform an important protective function on human umbilical vein endothelial cells (HUVECs) by increasing nitric oxide (NO) content, decreasing endothelin-1 (ET-1) secretion, and antagonizing the adverse impact of norepinephrine on the secretion of NO and ET-1. In addition, YEGDP and WF could provide protection to HUVECs against H2O2 damage by increasing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and NO levels to decrease the contents of reactive oxygen species (ROS) and malondialdehyde. Therefore, seventeen ACE inhibitory peptides, especially YEGDP and WF, might be used as natural ingredients for the development of products with antihypertensive functions.