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Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×10 5 copies/μL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period., Competing Interests: Declaration of Competing Interest The author declares no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

The structure and process of the graphene/Si heterojunction near-infrared photodetector were optimized to enhance the operating speed limit. The introduction of a well-designed structure improved the rise time from 12.6 μs to 115 ns, albeit at the expense of the responsivity, which decreased from 1.25 A W -1 to 0.56 A W -1 . Similarly, the falling time was improved from 38 μs to 288 ns with a sacrifice in responsivity from 1.25 A W -1 to 0.29 A W -1 , achieved through the introduction of Ge-induced defect-recombination centers within the well. Through a judicious well design and the introduction of recombination defect centers, the minimum pulse width could be improved from 50.6 μs to 435 ns, facilitating 2 MHz operation. This represents more than 100 times increase compared to previously reported graphene and graphene/Si hybrid photodetectors., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS., Results: The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7 th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ)., Conclusion: Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture., (© 2024. The Author(s).)

Aquaculture, a crucial sector of the global food industry, faces a myriad of issues due to parasitic invasions. One such parasite, Microcotyle sebastis , which afflicts Korean rockfish in South Korea, has a significant economic impact. The impending danger of resistance to traditional anthelmintics necessitates the exploration of new antiparasitic candidates. Although the efficacy of salinomycin against aquatic parasites such as ciliates and sporozoans is known, its influence on monogeneans has yet to be studied. Therefore, this study investigated the efficacy and safety of salinomycin for the treatment of M. sebastis infections, presenting the first exploration of salinomycin's therapeutic potential against monogeneans. In vitro examinations revealed a minimum effective concentration of salinomycin of 5 mg/kg, which led to necrosis of the haptor upon dislodging from the gill filaments. The one-time oral administration of the drug at concentrations of 5 mg/kg and 10 mg/kg showed a significant dose-dependent reduction in parasite counts, with no apparent behavioral side effects in Korean rockfish. Biochemical analyses monitored the liver, heart, and kidney enzymes, specifically aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatine kinase-myocardial band (CK-MB). At both 20 °C and 13 °C, no significant differences were observed in the levels of AST and ALT. However, at 20 °C, alterations in BUN levels were evident on Day 14, a deviation not observed at 13 °C. The CK-MB analysis revealed elevated enzyme levels at both temperatures when compared to the control group, reflecting the similar changes observed in terrestrial animals administered salinomycin. The biochemical data suggest that the oral administration of salinomycin is potentially more favorable at 13 °C than at 20 °C. Although our findings warrant further comprehensive studies, including on the long-term and potential effects on nontarget species and water quality, they also suggest that salinomycin could be considered as an alternative or adjunctive treatment if resistance to the currently used praziquantel against M. sebastis is confirmed.

The high virulence of the white spot syndrome virus (WSSV) in acute infections and the lack of effective treatments underscore the necessity for a rapid, accurate, and efficient diagnostic process to control white spot disease. The analytical sensitivity of diagnostic assays (polymerase chain reactions; PCRs and rapid diagnostic kit) at different severity grades of WSSV infection were determined using the 95% limit of detection (LOD 95% ). The LOD 95% of nested, real-time, and one-step PCRs and rapid diagnostic kit were 0.70, 4.67, 1.19, and 79,434.65 viral genome copies/reactions, respectively. From the intramuscular challenge tests conducted under different dose and temperature conditions, the WSSV severity grades of time-course collected whiteleg shrimp ( Litopenaeus vannamei ) and dead or live shrimps were determined based on the viral loads of the pleopod. By applying the WSSV severity grades, a total of 92 shrimps were classified as G0. Furthermore, 92, 66, 199, and 79 shrimps were classified as G1, G2, G3, and G4, respectively. Additionally, 222 shrimps were classified as negative as WSSV was not confirmed in the nested PCR assay. Diagnostic sensitivity (DSe) and specificity (DSp) values of molecular diagnostic assays were compared with those of nested PCR in artificial WSSV-infected shrimp to evaluate diagnostic performance. The real-time and one-step PCRs exhibited a DSe >92.4% for G0 grade (approximately 10 1 -10 2 copies/mg), indicating WSSV detection at low copy numbers. The rapid diagnostic kit presented a DSe >92.4% for G2 grade (approximately 10 4 -10 5 copies/mg), suggesting the detection of WSSV-infected shrimp with clinical signs during the endemic period. These results suggest that the strategy of presumptive diagnosis using one-step PCR and rapid diagnostic kit at different seasonal periods followed by confirmatory diagnosis using real-time PCR assay could aid in controlling WSD with rapidity, accuracy, and cost-effectiveness., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

Parasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused on Microcotyle sebastis infections in Korean rockfish ( Sebastes schlegelii ) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl against M. sebastis infections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility of M. sebastis to levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug's efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity of M. sebastis to levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl against M. sebastis in Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.

White spot syndrome virus (WSSV) is the most problematic pathogen in crustaceans. In this study, we investigated the horizontal transmission model of WSSV based on the correlation between the disease severity grade and viral shedding rate and determined the minimum infective dose of WSSV via the waterborne route. Intramuscular injection challenges at different doses and water temperatures revealed that the thresholds of viral shedding and mortality were G1 (3.1 × 10 3 copies/mg) and G2 (8.5 × 10 4 copies/mg), respectively. Furthermore, a positive linear correlation was observed between viral copies of pleopods and viral shedding rate (y = 0.7076x + 1.414; p < 0.001). Minimum infective doses of WSSV were determined via an immersion challenge. Infection was observed within 1, 3, and 7 d in 10 5 -, 10 3 -, and 10 1 copies/mL of seawater, respectively. In the cohabitation challenge, infection was observed within six days with viral loads of 10 1 to 10 2 copies/mL of seawater, which further increased in the recipient group. Our results indicate a positive correlation between disease severity grade and viral shedding rate of infected shrimp and suggest that the waterborne transmission of WSSV depends on the viral load and exposure period.

Red sea bream iridovirus (RSIV) causes significant economic losses in the aquaculture industry. We analyzed the pathogenicity of RSIV in flathead grey mullets ( Mugil cephalus ), the correlation of histopathological lesions, and interspecies horizontal transmission, through immersion infection and cohabitation challenges. Flathead grey mullets, which were challenged by immersion infection, exhibited mortality at 14 and 24 days after RSIV exposure. Viral shedding in seawater peaked 2-3 days before or after the observed mortality. Specific lesions of RSIV were observed in the spleen and kidney, and the correlation between histopathological grade and viral load was the highest in the spleen. In a cohabitation challenge, flathead grey mullets were the donors, and healthy rock bream, red sea bream, and flathead grey mullets were the recipients. Viral shedding in seawater was the highest in flathead grey mullet and rock bream at 25 °C, with 10 6.0 RSIV copies L/g at 14 dpi. No mortality was observed in any group challenged at 15 °C, and no RSIV was detected in seawater after 30 dpi. The virus shed from RSIV-infected flathead grey mullets caused horizontal transmission through seawater. These findings suggest that rapid decision-making is warranted when managing disease in fish farms.

This study aimed to investigate the effects of different injection sites, including dorsal, cheek, and pectoral fin muscles, on the pharmacological properties of amoxicillin (AMOX) in olive flounder ( Paralichthys olivaceus ) after a single intramuscular (IM) injection of 40 mg/kg. The AMOX concentration was measured using high-performance liquid chromatography-tandem mass spectrometry, followed by a non-compartmental model analysis. The peak serum concentrations (C max ) achieved 3 h after dorsal, cheek, and pectoral fin IM injections were 202.79, 203.96, and 229.59 μg/mL, respectively. The area under the concentration-time curve (AUC) was 1697.23, 2006.71, and 1846.61 µg/mL·h, respectively. The terminal half-life (t 1/2 λ Z ) was prolonged for cheek and pectoral fin IM injections (10.12 and 10.33 h, respectively) compared to dorsal IM injection (8.89 h). In the pharmacokinetic-pharmacodynamic analysis, a higher T > minimum inhibitory concentration (MIC) and AUC/MIC values were observed after AMOX was injected into the cheek and pectoral fin muscles compared to the dorsal muscle. Muscle residue depletion was below the maximum residue level from day 7 after IM injection at all three sites. These findings suggest that the cheek and pectoral fin sites provide advantages regarding systemic drug exposure and prolonged action compared with the dorsal site.

Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream ( Oplegnathus fasciatus ) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream ( Pagrus major ), and flathead grey mullet ( Mugil cephalus ) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture.

Precise regulation of kinases and phosphatases is crucial for human metabolic homeostasis. This study aimed to investigate the roles and molecular mechanisms of protein tyrosine phosphatase type IVA1 (PTP4A1) in regulating hepatosteatosis and glucose homeostasis. Method: Ptp4a1 -/- mice, adeno-associated virus encoding Ptp4a1 under liver-specific promoter, adenovirus encoding Fgf21 , and primary hepatocytes were used to evaluate PTP4A1-mediated regulation in the hepatosteatosis and glucose homeostasis. Glucose tolerance test, insulin tolerance test, 2-deoxyglucose uptake assay, and hyperinsulinemic-euglycemic clamp were performed to estimate glucose homeostasis in mice. The staining, including oil red O, hematoxylin & eosin, and BODIPY, and biochemical analysis for hepatic triglycerides were performed to assess hepatic lipids. Luciferase reporter assays, immunoprecipitation, immunoblots, quantitative real-time polymerase chain reaction, and immunohistochemistry staining were conducted to explore the underlying mechanism. Results: Here, we found that deficiency of PTP4A1 aggravated glucose homeostasis and hepatosteatosis in mice fed a high-fat (HF) diet. Increased lipid accumulation in hepatocytes of Ptp4a1 -/- mice reduced the level of glucose transporter 2 on the plasma membrane of hepatocytes leading to a diminution of glucose uptake. PTP4A1 prevented hepatosteatosis by activating the transcription factor cyclic adenosine monophosphate-responsive element-binding protein H (CREBH)/fibroblast growth factor 21 (FGF21) axis. Liver-specific PTP4A1 or systemic FGF21 overexpression in Ptp4a1 -/- mice fed an HF diet restored the disorder of hepatosteatosis and glucose homeostasis. Finally, liver-specific PTP4A1 expression ameliorated an HF diet-induced hepatosteatosis and hyperglycemia in wild-type mice. Conclusions: Hepatic PTP4A1 is critical for regulating hepatosteatosis and glucose homeostasis by activating the CREBH/FGF21 axis. Our current study provides a novel function of PTP4A1 in metabolic disorders; hence, modulating PTP4A1 may be a potential therapeutic strategy against hepatosteatosis-related diseases., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.

Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 10 3.7 -10 6.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 10 4 RSIV genome copies/fish, RSIV was not detected at 21-30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3-5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.

Obesity is a growing global epidemic that can cause serious adverse health consequences, including insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). Obesity development can be attributed to energy imbalance and metabolic inflexibility. Here, we demonstrated that lack of Kelch-like protein 3 (KLHL3) mitigated the development of obesity, IR, and NAFLD by increasing energy expenditure. KLHL3 mutations in humans cause Gordon's hypertension syndrome; however, the role of KLHL3 in obesity was previously unknown. We examined differences in obesity-related parameters between control and Klhl3 -/- mice. A significant decrease in body weight concomitant with fat mass loss and improved IR and NAFLD were observed in Klhl3 -/- mice fed a high-fat (HF) diet and aged. KLHL3 deficiency inhibited obesity, IR, and NAFLD by increasing energy expenditure with augmentation of O 2 consumption and CO 2 production. Delivering dominant-negative (DN) Klhl3 using adeno-associated virus into mice, thereby dominantly expressing DN-KLHL3 in the liver, ameliorated diet-induced obesity, IR, and NAFLD. Finally, adenoviral overexpression of DN-KLHL3, but not wild-type KLHL3, in hepatocytes revealed an energetic phenotype with an increase in the oxygen consumption rate. The present findings demonstrate a novel function of KLHL3 mutation in extrarenal tissues, such as the liver, and may provide a therapeutic target against obesity and obesity-related diseases., (© 2022. The Author(s).)

The consumption of cultured crustaceans has been steadily increasing, and Pacific whiteleg shrimp ( Litopenaeus vannamei ) are major cultivated invertebrates worldwide. However, shrimp productivity faces a variety of challenges, mainly related to outbreaks of lethal or growth retardation-related diseases. In particular, hepatopancreatic microsporidiosis caused by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an important disease associated with growth retardation in shrimp. Here, we report the detection of EHP through histopathological, molecular and electron microscopy methods in the hepatopancreas of Pacific whiteleg shrimp with growth disorder in a South Korean farm. Phylogenetic analysis showed a clade distinct from the previously reported EHP strains isolated in Thailand, India, China and Vietnam. An EHP infection was not associated with inflammatory responses such as hemocyte infiltration. Although EHP infection has been reported worldwide, this is the first report in the shrimp aquaculture in Korea. Therefore, an EHP infection should be managed and monitored regularly for effective disease control and prevention.

The objective of this study was to demonstrate the pharmacokinetic-pharmacodynamic profile, bioavailability, and withdrawal time of tylosin tartrate (TT) administered to olive flounder via intramuscular (IM, 10 or 20 mg/kg, n = 240) and intravascular (IV, 10 mg/kg, n = 90) injections. Serum concentrations of tylosin were determined using a validated liquid chromatography-tandem mass spectrometry method. According to the non-compartmental analysis, the bioavailability of TT was 87%. After the IV injection, the terminal half-life, total body clearance, volume of distribution, and mean residence time of TT were 21.07 h, 0.07 L/kg/h, 2.15 L/kg, and 16.39 h, respectively. Rapid absorption (T max 0.25 h), prolonged action (terminal half-life, 33.96 and 26.04 h; MRT, 43.66 and 33.09 h), and linear dose-response relationship (AUC 0-inf , 123.55 and 246.05 µg/mL*h) were monitored following 10 and 20 mg/kg IM injection. The withdrawal time of TT from muscle (water temperature, 22 °C) was 9.84 days, rounded up to 10 days (220 degree days). Large C max /MIC 90 , AUC 0-inf /MIC 90 , and T > MIC 90 values were obtained for Streptococcus isolates and these PK/PD indices satisfied the criteria required for efficacy evaluation. This study lays a foundation for the optimal use of TT and provides valuable information for establishing therapeutic regimens.

A graphene photodetector decorated with Bi 2 Te 3 nanowires (NWs) with a high gain of up to 3 × 10 4 and wide bandwidth window (400-2200 nm) has been demonstrated. The photoconductive gain was improved by two orders of magnitude compared to the gain of a photodetector using a graphene/Bi 2 Te 3 nanoplate junction. Additionally, the position of photocurrent generation was investigated at the graphene/Bi 2 Te 3 NWs junction. Eventually, with low bandgap Bi 2 Te 3 NWs and a graphene junction, the photoresponsivity improved by 200% at 2200 nm (~0.09 mA/W).

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine., Competing Interests: The authors have declared that no competing interests exist.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.

The self-renewal properties of human pluripotent stem cells (hPSCs) contribute to their efficacy in tissue regeneration applications yet increase the likelihood of teratoma formation, thereby limiting their clinical utility. To address this issue, we developed a tool to specifically target and neutralize undifferentiated hPSCs, thereby minimizing tumorigenicity risk without negatively affecting regenerated and somatic tissues. Specifically, we conjugated a monoclonal antibody (K6-1) previously generated in our laboratory against desmoglein 2 (Dsg2), which is highly differentially expressed in undifferentiated hPSCs versus somatic tissues, to the chemotherapeutic agent doxorubicin (DOX). The K6-1-DOX conjugates were selectively targeted and incorporated into Dsg2-positive hPSCs, leading to pH-dependent endosomal release and nuclear localization of DOX with subsequent cytotoxicity via an apoptotic caspase cascade. Conversely, Dsg2-negative fibroblasts showed minimal conjugate uptake or cytotoxicity, suggesting that K6-1-DOX treatment would yield few side effects owing to off-target effects. Selective removal of undifferentiated stem cells was also supported by in vivo studies using a mouse xenograft model, wherein hIgG-DOX- but not K6-1-DOX-pretreated-hPSC injection led to teratoma development. Together, these results validated the ability of the Dsg2-targeted antibody-anticancer drug conjugate to facilitate the safety of stem cell therapies., (Copyright © 2020. Published by Elsevier Ltd.)

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22-89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Obesity is recognized as the cause of multiple metabolic diseases and is rapidly increasing worldwide. As obesity is due to an imbalance in energy homeostasis, the promotion of energy consumption through browning of white adipose tissue (WAT) has emerged as a promising therapeutic strategy to counter the obesity epidemic. However, the molecular mechanisms of the browning process are not well understood. In this study, we investigated the effects of the GATA family of transcription factors on the browning process., Methods: We used qPCR to analyze the expression of GATA family members during WAT browning. In order to investigate the function of GATA3 in the browning process, we used the lentivirus system for the ectopic expression and knockdown of GATA3. Western blot and real-time qPCR analyses revealed the regulation of thermogenic genes upon ectopic expression and knockdown of GATA3. Luciferase reporter assays, co-immunoprecipitation, and chromatin immunoprecipitation were performed to demonstrate that GATA3 interacts with proliferator-activated receptor-γ co-activator-1α (PGC-1α) to regulate the promoter activity of uncoupling protein-1 (UCP-1). Enhanced energy expenditure by GATA3 was confirmed using oxygen consumption assays, and the mitochondrial content was assessed using MitoTracker. Furthermore, we examined the in vivo effects of lentiviral GATA3 overexpression and knockdown in inguinal adipose tissue of mice., Results: Gata3 expression levels were significantly elevated in the inguinal adipose tissue of mice exposed to cold conditions. Ectopic expression of GATA3 enhanced the expression of UCP-1 and thermogenic genes upon treatment with norepinephrine whereas GATA3 knockdown had the opposite effect. Luciferase reporter assays using the UCP-1 promoter region showed that UCP-1 expression was increased in a dose-dependent manner by GATA3 regardless of norepinephrine treatment. GATA3 was found to directly bind to the promoter region of UCP-1. Furthermore, our results indicated that GATA3 interacts with the transcriptional coactivator PGC-1α to increase the expression of UCP-1. Taken together, we demonstrate that GATA3 has an important role in enhancing energy expenditure by increasing the expression of thermogenic genes both in vitro and in vivo., Conclusion: GATA3 may represent a promising target for the prevention and treatment of obesity by regulating thermogenic capacity., Competing Interests: Declaration of competing interest The authors have no conflicts of interest and declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Vibrio parahaemolyticus is a gram-negative bacterium, which is responsible for acute hepatopancreatic necrosis disease (AHPND) in shrimp and has various virulent factors. So, to intensify the knowledge on pathogenic mechanism, the heterogeneous V.parahaemolyticus strains genome are indeed. Here, genome of seven V.parahaemolyticus strains, which are virulent to shrimps were sequenced by PacBio platform and the virulence was confirmed through the presence of plasmid (∼69 Kb) with binary toxin genes (i.e., pirA and pirB) with PCR method., (© 2020 Published by Elsevier Inc.)

Whole-genome next-generation sequencing was used to investigate the local evolution of viral haemorrhagic septicaemia virus, a serious pathogen affecting economically important fish such as rainbow trout and turbot in Europe and olive flounder in Asia. Sequence analysis showed that all isolates were genotype IVa, but could be classified further into four subgroups (K1-K4). In addition, genomic regions encompassing the nucleoprotein, phosphoprotein, matrix protein and non-virion protein genes, as well as the seven non-coding regions, were relatively conserved, whereas glycoprotein and RNA-dependent RNA polymerase genes were variable in the coding region. Taken together, the data demonstrate that whole-genome next-generation sequencing may be useful for future surveillance, prevention and control strategies against viral haemorrhagic septicaemia., (© 2020 John Wiley & Sons Ltd.)

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases., (© 2019 Federation of American Societies for Experimental Biology.)

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) are known to play a role as a first line of defence against microbial invasion. Liver Expressed Antimicrobial Peptides-2 (LEAP-2) is one of the AMPs. LEAP-2 includes four highly conserved cysteine residues and belongs to a cysteine-rich peptides group. We identified and characterized the molecular properties of LEAP-2 in rock bream. The expression levels of rock bream LEAP-2 (RbLEAP-2) in the 12 different tissues of healthy fish and the RbLEAP-2 expression pattern after infections with Edwardsiella piscicida ( E. piscicida ), Streptococcus iniae ( S. iniae ) and red seabream iridovirus (RSIV) were examined. This data provide that RbLEAP-2 plays an important role in innate immunity when rock bream is infected with a pathogen., (© 2019 The Author(s).)

Nuclear factor-kappaB (NF-κB) repressing factor (NKRF) specifically inhibits the transcriptional activity of NF-κB protein. The PmNKRF cDNA is composed of 757 amino acid residues. Alignment analysis revealed that the G-patch and R3H domains are conserved in different organisms. We aimed to analyse red sea bream NKRF (PmNKRF) gene expression after infection with pathogens [ Streptococcus iniae or red sea bream iridovirus (RSIV)] and in healthy individuals. In healthy individuals, PmNKRF was ubiquitously expressed in all 12 tested tissues, predominantly in the head kidney and spleen. Expression of PmNKRF was significantly up-regulated in the gills, kidney, liver and spleen after RSIV infection. After S. iniae infection, PmNKRF expression was significantly down-regulated in the gills and significantly up-regulated in the kidney, liver and spleen.

CXCL10 plays an important role in angiogenesis and inhibits the differentiation of endothelial cells into capillaries. It also plays an important role in the generation and transmission of effector T cell responses and the recruitment of T cells to inflammatory sites. In this article, we constructed cDNAs to identify and analyse the CXCL10 domain, and performed multiple alignments and a phylogenetic analysis to determine homology with other animals. Real-time PCR was performed to confirm construction and expression after bacterial and viral infection.

Background: Runners who participate in endurance sports such as marathons or ultramarathons have superior physical capabilities and health benefits compared to others. However, they may suffer long-term effects of the negative physiological changes during long-distance running. This study aimed to examine the effects of an ultramarathon on hormone levels, and the associated risks., Methods: Ten participants who completed a 622-km ultramarathon were included. Blood was collected prerace, at the 300-km mark, the 622-km mark, and on the 3rd day of recovery (RD3) and the 6th day of recovery (RD6) and analyzed for human growth hormone (HGH), cortisol, beta-endorphin, serotonin, testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels., Results: HGH and beta-endorphin showed the highest increase at the 300-km mark compared to prerace levels, with recovery on RD3 and RD6, respectively. LH, cortisol, and serotonin showed the highest levels of increase at the 622-km mark, with recovery on RD6 for LH, and RD3 for cortisol and serotonin. FSH showed the highest level of decrease at both 300-km and 622-km marks compared to prerace levels, with recovery on RD3. Testosterone decreased the most at the 300-km mark compared to the prerace level and this decrease was below the normal levels; however, it recovered to normal levels on RD3., Conclusions: Hormone levels after the 622-km ultramarathon were within their normal ranges, except for testosterone. However, all the hormones recovered to prerace levels on RD3 or RD6. This study showed that running ultramarathons does not cause abnormal hormone levels.

This article provides efficacy and safety data of ceftiofur antibiotics against streptococcal infection in starry flounder. Ceftiofur, which is a veterinary antibiotics, is effective against fishery bacteria. Ceftiofur can be prescribed and sold by veterinarians. However, it is illegal in South Korea for fishery disease managers to prescribe and sell ceftiofur. Therefore, in order to utilize available antibiotics and prevent illegal use of veterinary antibiotics, it is necessary to perform research to determine the recommended effective dose and administration methods of antibiotics for fisheries. In this article, the appropriate concentration and injection method of antibiotics to treat starry flounder infected with S. parauberis PH0710 were provided. In addition, histopathological examination results were provided to confirm the effect of antibiotics on the host tissue. Accordingly, these data could be used as basic data for the application of ceftiofur antibiotics in disease management for fisheries.

Peptidoglycan recognition protein 2 (PGRP2) is a Zn 2+ -dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6-53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn 2+ . These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection., (Copyright © 2018. Published by Elsevier Ltd.)

Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPβ or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Various photodetectors showing extremely high photoresponsivity have been frequently reported, but many of these photodetectors could not avoid the simultaneous amplification of dark current. A gate-controlled graphene-silicon Schottky junction photodetector that exhibits a high on/off photoswitching ratio (≈10 4 ), a very high photoresponsivity (≈70 A W -1 ), and a low dark current in the order of µA cm -2 in a wide wavelength range (395-850 nm) is demonstrated. The photoresponsivity is ≈100 times higher than that of existing commercial photodetectors, and 7000 times higher than that of graphene-field-effect transistor-based photodetectors, while the dark current is similar to or lower than that of commercial photodetectors. This result can be explained by a unique gain mechanism originating from the difference in carrier transport characteristics of silicon and graphene., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10 -5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV., (Copyright © 2018. Published by Elsevier Ltd.)

Olive flounder (Paralichthys olivaceus) is one of the most economically important aquaculture fish. However, its production is often affected by various diseases, especially viral hemorrhagic septicemia virus (VHSV) that cause serious economic losses. In this study, we sequenced the whole transcriptome of the P. olivaceus using Illumina RNA-sEq. De novo assembly of control and virus-infected cDNA libraries of head kidney at 13 and 20 °C was accomplished with 2,007,532,438 raw reads, resulting in 244,578 unigenes with an average length of 533 bp and found 65,535 candidate coding unigenes with homology to other species by BLAST analysis. DEG analysis among control and virus-infected head kidney samples of 13 and 20 °C revealed that 1290 up-regulated and 162 down-regulated genes (p ≤ 0.01), linked to metabolism, virulence factors, adhesion and immune-response. We constructed an expressed gene catalog for the P. olivaceus to serve as a resource for marine environmental genomic and immuno-genetic/genomic studies focused on uncovering the molecular mechanisms underlying the responses of P. olivaceus to VHSV under different temperature.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H 2 O 2 and H 2 O. Here, we identified a homolog of MnSOD from big belly seahorse (Hippocampus abdominalis; HaMnSOD) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of HaMnSOD was observed in the ovary, followed by the heart and the brain. Further, immune related responses of HaMnSOD towards pathogenic stimulation were observed through bacterial and viral challenges. Highest HaMnSOD expression in response to stimulants Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS-scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of HaMnSOD in the big belly seahorse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The iron-withholding strategy of innate immunity is an effective antimicrobial defense mechanism that combats microbial infection by depriving microorganisms of Fe 3+ , which is important for their growth and propagation. Transferrins (Tfs) are a group of iron-binding proteins that exert their antimicrobial function through Fe 3+ sequestration. The current study describes both structural and functional characteristics of a transferrin ortholog from rock bream Oplegnathus fasciatus (RbTf). The RbTf cDNA possesses an open reading frame (ORF) of 2079 bp encoding 693 amino acids. It has a molecular mass of approximately 74 kDa and an isoelectric point of 5.4. In silico analysis revealed that RbTf has two conserved domains: N-terminal domain and C-terminal domain. Pairwise homology analysis and phylogenetic analysis revealed that RbTf shared the highest identity (82.6%) with Dicentrarchus labrax Tf. According to the genomic analysis, RbTf possesses 17 exons and 16 introns, similar to the other orthologs. Here, we cloned the N terminal and C terminal domains of RbTf to evaluate their distinct functional features. Results obtained through the CAS (chrome azurol S) assay confirmed the iron-binding ability of the RbTf, and it was further determined that the iron-binding ability of rRbTfN was higher than that of rRbTfC. The antimicrobial functions of the rRbTfN and the rRbTfC were confirmed via the iron-dependent bacterial growth inhibition assay. Tissue distribution profiling revealed a ubiquitous expression with intense expression in the liver. Temporal assessment revealed that RbTf increased after stimulation of LPS, Edwardsiella tarda, and Streptococcus iniae post injection (p.i.). These findings demonstrated that RbTf is an important antimicrobial protein that can combat bacterial pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >10 3 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Viral hemorrhagic septicemia (VHS) is a highly contagious disease of cultured flounder caused by VHS virus (VHSV). To develop effective VHSV vaccines, it is essential to understand the molecular mechanisms underlying the host's protective response against VHSV. The purpose of this study is to clarify which genes are involved in the protective response of olive flounder after VHSV vaccination. We first injected olive flounder intraperitoneally with 10 7 TCID 50 heat-inactivated VHSV vaccine and evaluated the vaccine efficacy at 20 °C. Fish vaccinated with heat-inactivated VHSV were significantly protected compared to non-vaccinated fish, with a relative percentage survival of 83%. To analyze the vaccination-induced changes in the expression profiles of genes, kidneys were collected from control and vaccinated fish at days 1, 3, and 7 after vaccination and global gene expression profiling was carried out by RNA sequencing. The analysis revealed that 15,001 genes were differentially expressed by at least 2-fold between vaccinated fish and non-vaccinated controls. Of these, 58 genes clustered into the acute phase response, Toll-like receptor, interferon-inducible/regulatory proteins, and apoptosis pathways. These data provided insights into the molecular mechanisms underlying the protective immune response of olive flounder against heat-inactivated VHSV vaccine and might aid future studies to develop a highly immunogenic vaccine against VHSV in flounder., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vaccination by immersion is suitable for mass vaccination of small size fish. However, no viral vaccine has been developed for immersion applications, because of low efficacy. In this study, we evaluated the efficacy and safety of immersion vaccine against viral hemorrhagic septicemia (VHS) containing Montanide IMS 1312 VG adjuvant in olive flounder (Paralichthys olivaceus). Healthy fish were vaccinated by an immersion method with a heat-inactivated FP-VHS2010-1 strain of VHS virus (VHSV) in combination with Montanide IMS 1312 VG for 5 min at 20 ± 2 °C. The control group was vaccinated with sterile PBS. No toxicity of immersion vaccine with Montanide IMS 1312 VG adjuvant was observed by hematological and histopathological analysis. Immersion vaccine with adjuvant enhanced gene expression of immune-associated genes, i.e., genes encoding interleukin (IL)-1β, IL-6, IL-8, and Toll-like receptor (TLR) 3. Relative percent survival (RPS) of fish was measured on weeks 4 and 8 post vaccination. In fish vaccinated with adjuvant, RPS was significantly higher than that of fish vaccinated without adjuvant. The results of the present study provide evidence that the VHSV immersion vaccine with Montanide IMS 1312 VG induces protective immunity in olive flounder against VHS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), α-globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV., (Copyright © 2016 Elsevier B.V. All rights reserved.)