Molecular characterization, immune responses, and functional aspects of atypical prototype galectin from redlip mullet (Liza haematocheila) as a pattern recognition receptor in host immune defense system.

Galectins are a family of lectins that are widely distributed ß-galactoside-binding proteins identified in diverse organisms. Galectin family have appeared as pattern recognition receptors (PRRs) responsible for initiating and controlling the innate immunity. The present study aimed to study the binding ability and potential role in PRRs of galectin-related protein B-like (LhGal B-like) from redlip mullet (Liza haematocheila) involved in the host immune responses. We constructed a cDNA library of redlip mullet and identified the LhGal B-like sequence. By sequence analysis and multiple sequence alignment, we revealed that LhGal B-like contains a conserved carbohydrate recognition domain (CRD) and consists of 135 amino acids with a predicted molecular weight of 16.07 kDa. In addition, pairwise comparison results showed that LhGal B-like shares higher sequence identity (82.2-95.2%) and similarity (89-95.9%) with fish species than those (34.1-37.8% and 57.2-58.1%, respectively) with other species. The phylogenetic tree showed that LhGal B-like clustered into the fish group and was evolutionally related to Mastacembelus armatus. The tissue distribution results revealed that LhGal B-like was expressed ubiquitously in all the tested tissues, where it was highly expressed in the brain, followed by gills and muscle. The immune modulated expression of LhGal B-like was observed by injecting lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). According to the results, in the gills, the mRNA expression of LhGal B-like was significantly upregulated upon LPS treatment after 48 h and upon poly I:C treatment after 48 and 72 h. In addition, the result showed significant upregulations upon LPS and poly I:C treatment after 24 h. However, significant downregulation was also shown in the earlier phase after injection of poly I:C and L. garvieae in gills. Further, the binding affinity of recombinant LhGal B-like (rLhGal B-like) was evaluated using carbohydrate, pathogen-associated molecular patterns (PAMP) and bacterial binding assays. The rLhGal B-like could bind all the examined carbohydrates but had a higher affinity to α-lactose. PAMPs and bacterial binding experiments verified a wide range of PAMP molecules and bacterial strains that rLhGal B-like could bind to. Moreover, we examined the agglutination activity of rLhGal B-like, and the result showed that it could aggregate all the gram-positive and gram-negative bacteria. Taken together, our findings reveal the functional aspects of LhGal B-like as a PRR and the potential involvement of LhGal B-like in the innate immunity of redlip mullet.

Identification of reactive oxygen species modulator 1 (Romo 1) from black rockfish (Sebastes schlegelii) and deciphering its molecular characteristics, immune responses, oxidative stress modulation, and wound healing properties.

Reactive oxygen species modulator 1 (Romo1) is a mitochondrial inner membrane protein that induces mitochondrial reactive oxygen species (ROS) generation. In this study, we identified the Romo1 homolog from the black rockfish (Sebastes schlegelii), named it as SsRomo1, and characterized it at the molecular as well as functional levels. An open reading frame consisting of 240 bp was identified in the SsRomo1 complementary DNA (cDNA) sequence that encodes a 79 amino acid-long polypeptide with a molecular weight of 8,293 Da and a theoretical isoelectric point (pI) of 9.89. The in silico analysis revealed the characteristic features of SsRomo1, namely the presence of a transmembrane domain and the lack of a signal peptide. Homology analysis revealed that SsRomo1 exhibits the highest sequence identity with its fish counterparts (>93%) and shares a similar percentage of sequence identity with mammals (>92%). Additionally, it is closely clustered together with the fish clade in the constructed phylogenetic tree. The subcellular localization analysis confirmed its mitochondrial localization within the fathead minnow (FHM) cells. Under normal physiological conditions, the SsRomo1 mRNA is highly expressed in the rockfish ovary, followed by the blood and testis, indicating the abundance of mitochondria in these tissues. Furthermore, the significant upregulation of SsRomo1 in cells treated with lipopolysachharide (LPS), polyinosinic:polycytidylic acid, and Streptococcus iniae suggest that the increased ROS production is induced by SsRomo1 to eliminate pathogens during infections. Incidentally, we believe that this study is the first to determine the involvement of SsRomo1 in LPS-mediated nitric oxide (NO) production in RAW267.4 cells, based on their higher NO production as compared to that in the control. Moreover, overexpression of SsRomo1 enhanced the wound healing ability of FHM cells, indicating its high invasion and migration properties. We also determined the hydrogen peroxide-mediated cell viability of SsRomo1-overexpressed FHM cells and observed a significant reduction in viability, which is possibly due to increased ROS production. Collectively, our observations suggest that SsRomo1 plays an important role in oxidative stress modulation upon immune stimulation and in maintenance of tissue homeostasis in black rockfish.

Parasitic leiomyoma in the trocar site after laparoscopic myomectomy: A case report.

BACKGROUND: Laparoscopic myomectomy is increasingly used for resecting gynecological tumors. Leiomyomas require morcellation for retrieval from the peritoneal cavity. However, morcellated fragments may implant on the peritoneal cavity during retrieval. These fragments may receive a new blood supply from an adjacent structure and develop into parasitic leiomyomas. Parasitic leiomyomas can occur spontaneously or iatrogenically; however, trocar-site implantation is an iatrogenic complication of laparoscopic uterine surgery. We describe a parasitic leiomyoma in the trocar-site after laparoscopic myomectomy with power morcellation. CASE SUMMARY: A 50-year-old woman presented with a palpable abdominal mass without significant medical history. The patient had no related symptoms, such as abdominal pain. Computed tomography findings revealed a well-defined contrast-enhancing mass measuring 2.2 cm, and located on the trocar site of the left abdominal wall. She had undergone laparoscopic removal of uterine fibroids with power morcellation six years ago. The differential diagnosis included endometriosis and neurogenic tumors, such as neurofibroma. The radiologic diagnosis was a desmoid tumor, and surgical excision of the mass on the abdominal wall was successfully performed. The patient recovered from the surgery without complications. Histopathological examination revealed that the specimen resected from the trocar site was a uterine leiomyoma. CONCLUSION: Clinicians should consider the risks and benefits of laparoscopic vs laparotomic myomectomy for gynecological tumors. Considerable caution must be exercised for morcellation to avoid excessive tissue fragmentation.

Molecular characterization, expression, and functional analysis of cystatin B in the big-belly seahorse (Hippocampus abdominalis).

Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses.

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.

Modulation of fish immune response by interferon regulatory factor 4 in redlip mullet (Liza haematocheilus): Delineation through expression profiling, antiviral assay, and macrophage polarization analysis.

Interferon regulatory factor 4 (IRF4) is a crucial member of IRF family, which acts as an imperative transcription factor in the development and maturation of multiple lineages of blood cells and also plays a pivotal role in host defense against microbial infections. In the present study, we aimed to investigate the detailed structural and functional aspects of a redlip mullet IRF4 homolog (LhIRF4). The LhIRF4 open reading frame consists of 1347 base pairs encoding 449 amino acids, with the DNA-binding domain sharing significant homology with that of other vertebrate IRF4 homologs. The highest transcription levels of LhIRF4 were observed in the mullet intestine and spleen under normal physiological conditions. Furthermore, a time-dependent upregulation of LhIRF4 transcription was observed in the spleen and head kidney tissues upon pathogenic challenges. When overexpressed in mullet cells, LhIRF4 was localized to the nucleus and significantly stimulated the transcription of several host antiviral genes. Moreover, the overexpression of LhIRF4 strongly inhibited the replication of viral hemorrhagic septicemia virus (VHSV) in vitro. The function of LhIRF4 in regulation of macrophage M2 polarization has also been evidently demonstrated in RAW 264.7 cells. Taken together, our findings indicate the profound role of LhIRF4 in modulating immune responses against microbial infections in redlip mullet.

Cytosolic ß-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of ß-catenin homolog from redlip mullet (Planiliza haematocheila).

ß-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of ß-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish ß-catenin by identifying and characterizing the ß-catenin homolog (PhCatß) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatß consists of 2352 bp, which encodes a putative ß-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that ß-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatß and other fish ß-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatß in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatß transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatß stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatß resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatß could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatß plays a role in macrophage activation and antiviral immune response in redlip mullet.

Innovative digital tools for new trends in teaching and assessment methods in medical and dental education.

With the goal of providing optimal care to patients, student-centered active learning and the development of clinical competency have become vital components of the education of future physicians capable of sustainably coping with future challenges. However, the shape of future medicine is dramatically changing based on advances in information and communication technology, and the current classroom model seems to have difficulties in fully preparing students for the future of medicine. New trends in teaching and assessment methods include computer-aided instruction, virtual patients, augmented reality, human patient simulations, and virtual reality for the assessment of students' competency. The digital technologies introduced in medical and dental education include Google Forms to collect students' answers, YouTube livestreaming, google art & culture (an online art museum), and choose-your-own-adventure as a story-telling technique. Innovations in digital technology will lead the way toward a revolution in medical and dental education, allowing learning to be individualized, interactive, and efficient.

Expression profiling, immune functions, and molecular characteristics of the tetraspanin molecule CD63 from Amphiprion clarkii.

CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response.

Immune responses, subcellular localization, and antiviral activity of interferon-induced protein 35 (IFP35) in rock bream (Oplegnathus fasciatus).

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-myc-interactor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

The possible role of catalase in innate immunity and diminution of cellular oxidative stress: Insights into its molecular characteristics, antioxidant activity, DNA protection, and transcriptional regulation in response to immune stimuli in yellowtail clownfish (Amphiprion clarkii).

Catalase, a key enzyme in the antioxidant defense grid of organisms, scavenges free radicals to curtail their harmful effects on the host, supporting proper immune function. Herein, we report the identification and characterization of a catalase homolog from Amphiprion clarkii (ClCat), followed by its functional characterization. An open reading frame was identified in the cDNA sequence of ClCat at 1581 bp, which encodes a protein of 527 amino acids (aa) with a molecular mass of 60 kDa. In silico analyses of ClCat revealed characteristic features of the catalase family and a lack of a signal peptide. Multiple sequence alignment of ClCat indicated the conservation of functionally important residues among its homologs. According to phylogenetic analysis, ClCat was of vertebrate origin, positioned within the teleost clade. During native conditions, ClCat mRNA was highly expressed in blood, followed by the liver and kidney. Moreover, significant changes in ClCat transcription were observed after stimulation with LPS, poly I:C, and Vibrio harveyi, in a time-dependent manner. Recombinant ClCat (rClCat) was characterized, and its peroxidase activity was determined. Furthermore, the optimum temperature and pH for rClCat were determined to be 30-40 °C and pH 7, respectively. Oxidative stress tolerance and chromatin condensation assays indicated enhanced cell survival and reduced apoptosis, resulting from reactive oxygen species scavenging by rClCat. The DNA-protective function of rClCat was further confirmed via a metal-catalyzed oxidation assay. Taken together, our findings propose that rClCat plays an essential role in maintaining cellular oxidative homeostasis and host immune protection.

Molecular and functional characterization of a mitochondrial glutathione reductase homolog from redlip mullet (Liza haematocheila): Disclosing its antioxidant properties in the fish immune response mechanism.

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione (GSH), with the catalytic activity of NADPH. Most animals and aquatic organisms, including fish, possess high levels of this enzyme system to neutralize oxidative stress in cells. The current study was conducted to broaden our knowledge of GSHR in fish by identifying a mitochondrial isoform of this enzyme (LhGSHRm) in redlip mullet, Liza haematocheila, and clarifying its structure and function. The complete open reading frame of LhGSHRm consists of 1527 base pairs, encoding 508 amino acids, with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of important amino acids in this fish. Phylogenetic analysis demonstrated the closest evolutionary relationship between LhGSHRm and other fish GSHRm counterparts. In tissue distribution analysis, the highest mRNA expression of LhGSHRm was observed in the gill tissue under normal physiological conditions. Following pathogenic challenges, the LhGSHRm transcription level was upregulated in a time-dependent manner in the gill and liver tissues, which may modulate the immune reaction against pathogens. rLhGSHRm showed considerable glutathione reductase activity in an enzyme assay. Further, the biological activity of rLhGSHRm in balancing cellular oxidative stress was observed in both disk diffusion and DPPH assays. Collectively, these results support that LhGSHRm has profound effects on modulating the immune reaction in fish to sustain precise redox homeostasis.

Identification and characterization of cystatin B from black rockfish, Sebastes schlegelii, indicating its potent immunological importance.

Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine (8GG9) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5'-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3'-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts.

Laparoscopic Gastrectomy Using Instruments with a Minimal Diameter for Early Gastric Cancer: A Feasible Alternative to Conventional Laparoscopic Gastrectomy for Experienced Surgeons.

Background: The application of laparoscopic surgery using instruments that are 3 mm or less in diameter for patients with early gastric cancer (EGC) has not yet been established. We aimed to evaluate the feasibility and safety of laparoscopic gastrectomy using instruments with minimal diameter. Methods: We retrospectively analyzed 41 patients who underwent laparoscopic subtotal gastrectomy with D1-positive lymph node dissection for EGC. Among them, 17 patients underwent laparoscopic gastrectomy using instruments with a minimal diameter (experimental group), while 24 patients underwent conventional laparoscopic gastrectomy (control group). In the experimental group, we used two 3-mm trocars, one 5-mm trocar, and the GelPOINT® Advanced Access Platform. We compared operative outcomes between the two groups and assessed the learning curve of laparoscopic gastrectomy using instruments with minimal diameter. Results: The operative outcomes were similar between the two groups. The preoperative-to-postoperative day 2 ratio of neutrophil count in the experimental group was significantly lower than in the control group (2.07 versus 2.65; P = .038). Morbidity was not observed in the experimental group and 3 patients experienced complications in the control group, although it was not significantly different (P = .252). The operation time according to the accumulation of cases was stable without any significant change in the experimental group. Conclusions: Laparoscopic gastrectomy using instruments with minimal diameter is technically feasible and safe for EGC and could also be a good alternative to conventional laparoscopic gastrectomy to minimize the impact of surgical invasiveness when performed by experienced surgeons.

Cloning and functional characterization of rockfish peroxiredoxin 4 homolog with its innate immune responses.

The fourth member of the typical 2-cysteine peroxiredoxin (Prx4) is a well-known antioxidant enzyme, which reduces different peroxides in their catalytic process. The present study reports the identification of the rockfish Sebastes schlegelii Prx4 (SsPrx4) at a genomic level, as well as the characterization of its structural and functional features. SsPrx4 harbors a complete ORF of 786 bp encoding a polypeptide (29 kDa) of 262 amino acids (aa) with an isoelectric point of 6.2. Thioredoxin 2 domain was prominent in the SsPrx4 sequence, which has a signal peptide (31 bp) at the N-terminus. Hence, the SsPrx4 may be functionally active in the cytoplasm of rockfish cells. Moreover, two VCP motifs and three catalytic triad residues (112T, 115C, 191R) were identified in the SsPrx4 protein sequence. A peroxidatic cysteine (115CP) and resolving cysteines (236CR) were detected at the VCP motifs. The rockfish Prx4 genome consists of seven exons, which are similar to the architecture of other Prx4 orthologs. The deduced amino acid sequence of SsPrx4 shares a relatively high amino acid sequence identity (91.6%) and close evolutionary relationship with Miichthys miiuy and Stegastes partitus Prx4. The potential for scavenging extracellular H2O2 was evidenced by the purified recombinant SsPrx4 protein (rSsPrx4) in vitro system. Moreover, rSsPrx4 may protect the plasmid DNA in a metal-catalyzed oxidation system and catalyze the reduction of an insulin disulfide bond. Quantitative real-time PCR revealed that SsPrx4 mRNA was ubiquitously expressed in fourteen different tissues, with the highest expression observed in the liver followed by the ovary, and kidney tissues. Transcriptional modulations were observed in liver and spleen tissues of rockfish after injecting them with bacterial stimuli, including Streptococcus iniae, LPS, and a viral mimic of poly I:C. Together, the results suggest that SsPrx4 may play an important role in both the antioxidant and innate immune defense of black rockfish. These findings provide structural and functional insights into the SsPrx4 of the teleost.

A manganese superoxide dismutase (MnSOD) from red lip mullet, Liza haematocheila: Evaluation of molecular structure, immune response, and antioxidant function.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6 h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25 °C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N'-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet.

Characterization of four C1q/TNF-related proteins (CTRPs) from red-lip mullet (Liza haematocheila) and their transcriptional modulation in response to bacterial and pathogen-associated molecular pattern stimuli.

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses.

Identification of thioredoxin domain-containing protein 17 from big-belly seahorse Hippocampus abdominalis: Molecular insights, immune responses, and functional characterization.

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses.

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, Sebastes schlegelii.

C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from Sebastes schlegelii was identified and designated as SsC1inh. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, SsC1inh mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish.