Molecular characterization and immune response of suppressor of cytokine signaling 5b from redlip mullet (Planiliza haematocheilus): Disclosing its anti-viral potential and effect on cell proliferation.

The suppressor of cytokine signaling (SOCS) proteins family comprising eight proteins (SOCS1-7 and cytokine-inducible SH2-containing (CIS)) are classical feedback inhibitors of cytokine signaling. Although the biological role of CIS and SOCS1-3 have been extensively studied, the biological functions of SOCS4-7 remain unclear. Here, we elucidated the molecular characteristics, expression profile, immune response, anti-viral potential, and effect on cell proliferation of Phsocs5b, a member of the SOCS protein family from redlip mullet (Planiliza haematocheilus); phsocs5b comprised 1695 nucleotides. It was 564 amino acids long with a molecular weight of 62.3 kDa and a theoretical isoelectric point of 8.95. Like SOCS4-7 proteins, Phsocs5b comprised an SH2 domain, SOCS box domain, and a long N-terminal. SH2 domain is highly identical to its orthologs in other vertebrates. Phsocs5b, highly expressed in the brain tissue, was localized in the cytoplasm. Temporal changes in phsocs5b expression were observed following immune stimulation with polyinosinic: polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. In FHM cells, Phsocs5b overexpression suppressed the viral hemorrhagic septicemia virus (VHSV) infection and epidermal growth factor receptor (egfr) expression but increased the mRNA levels of pi3k, akt, pro-inflammatory cytokines (il1ß and il8), and anti-viral genes (isg15 and ifn). Overall, our findings suggest that Phsocs5b attenuates VHSV infection, either by hindering the cell entry via degradation of Egfr, enhancing pro-inflammatory cytokines and anti-viral factor production, or both. The results also indicated that Phsocs5b could directly activate Pi3k/Akt pathway by itself, thus enhancing the proliferation and migration of cells. Taken together, Phsocs5b may be considered a potential therapeutic target to enhance immune responses while positively regulating the proliferation and migration of cells.

Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection.

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin-/- compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin-/- fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin-/- fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.

Molecular characterization and expression analysis of B-cell lymphoma-2 protein in Amphiprion clarkii and its role in virus infections.

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.

Molecular delineation, expression profiling, immune response, and anti-apoptotic function of a novel clusterin homolog from big-belly seahorse (Hippocampus abdominalis).

Clusterin (CLU) is a glycoprotein that contains α- and ß-chains. CLU exerts multifunctional activities and plays a role in different cell signaling pathways that are associated with various diseases such as proteotoxic and oxidative stress, as well as cell death and survival. However, its role in marine teleost fish remains unclear. Therefore, the present study was carried out to characterize and investigate the immune responses and anti-apoptotic effects of CLU of the big-belly seahorse (Hippocampus abdominalis) (HaCLU) on oxidative stress-induced cell death. The HaCLU open reading frame was 1389 bp long and encoded a protein with 462 amino acids, a molecular weight of 51.28 kDa and an isoelectric point of 5.41. In-silico results demonstrated that HaCLU has a signal peptide in the 1-29 amino acid region, while the α- and ß-chains were in the 34-227 and 228-455 amino acid regions, respectively. Multiple sequence alignment clarified the low homology of the α-chain with other orthologs. The highest HaCLU mRNA expression level was observed in the liver, followed by the heart, spleen, and brain tissues of healthy big-belly seahorses. Further, HaCLU mRNA expression level was elevated in the liver in response to different stimuli, including lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae. HaCLU potentiates cell viability and weakens chromatin condensation in the nucleus of FHM cells following H2O2-induced oxidative stress and subsequent cell death. HaCLU overexpression resulted in a reduced Bax/Bcl-2 mRNA expression ratio. This study revealed the role of HaCLU in immune regulation against pathogenic infections and its anti-apoptotic effects on oxidative stress-induced cell death.

Preparation and characterization of succinyl chitosan and succinyl chitosan nanoparticle film: In vitro and in vivo evaluation of wound healing activity.

Development of novel wound dressing materials having the ability to prevent bacterial infections and capable of accelerating the tissue regeneration process is utmost important, since the wounds in patients can cause severe health issues. In the present work, we synthesized novel N-succinyl chitosan nanoparticles (N-SuC NPs) films and tested their antimicrobial, cytotoxicity, and in vitro and in vivo wound healing activity. N-SuC NPs were synthesized by ionic gelation method, and subsequently N-SuC NPs films were prepared by solution casting method using synthesized N-SuC NPs. The prepared N-SuC NPs films showed significant antimicrobial activity against Escherichia coli and Staphylococcus aureus with a minimum inhibitory concentration of 6 mg/mL and <8 mg/mL, respectively. The biocompatibility and the in vitro wound healing activity of N-SuC NPs films were analyzed using human dermal fibroblast (HDF) cells. In vivo cutaneous wound healing of the N-SuC NPs film was investigated using the Wister rat model, and the studies showed that the N-SuC NPs film significantly accelerated the wound healing process by inducing more blood vessels formation and tissue granulation. The experimental results showed that synthesized N-SuC NPs film had excellent antimicrobial, cytotoxicity and wound healing activity, indicating that it could be used in biomedical applications.

Succinyl chitosan gold nanocomposite: Preparation, characterization, in vitro and in vivo anticandidal activity.

Herein, we have successfully synthesized a novel N-Succinyl chitosan/gold nanocomposite (N-SuC/Au NC) using N-SuC and gold(III) chloride, and investigated the biocompatibility and antifungal activity. The synthesized N-SuC/Au NC was characterized by UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscope, and inductively coupled plasma atomic emission spectroscopy. The N-SuC/Au NC exhibited a strong inhibition effect towards pathogenic Candida albicans. Morphological analysis revealed the destruction of C. albicans cell membrane due to N-SuC/Au NC treatment. The in vitro and in vivo toxicity of N-SuC/Au NC was analyzed with HEK293T mammalian cells and zebrafish larvae, respectively. The synthesized N-SuC/Au NC demonstrated no cytotoxicity towards HEK293T cells up to 1200 µg/mL concentration. The survival rate of the zebrafish larvae at 120 hpf, was found as 100% up to 1200 µg/mL of N-SuC/Au NC exposure. The in vivo studies further confirmed the inhibitory effects of N-SuC/Au NC on the formation of C. albicans hyphae in infected zebrafish muscle tissue.