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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.)

The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Infection with nervous necrosis virus (NNV) causes viral nervous necrosis, which inflicts serious economic losses in marine fish cultivation. Virus-like particles (VLPs) are protein complexes consisting of recombinant virus capsid proteins, whose shapes are similar to native virions. VLPs are considered a novel vaccine platform because they are not infectious and have the ability to induce neutralizing antibodies efficiently. However, there have been few studies of protective immune responses employing virus challenge following immunization with NNV VLPs, and this is important for evaluating the utility of the vaccine. In the present study, we produced red-spotted grouper (Epinephelus akaara) NNV (RGNNV) VLPs in Saccharomyces cerevisiae and investigated protective immune responses in convict grouper (Epinephelus septemfasciatus) following intraperitoneal injection and oral immunization with the RGNNV VLPs. The parenterally administered VLPs elicited neutralizing antibody with high efficacy, and provided the fish with full protection against RGNNV challenge: 100% of the immunized fish survived compared with only 37% of the control fish receiving phosphate-buffered saline. RGNNV VLPs administered orally provoked neutralizing antibody systemically and conferred protective immunity against virus challenge: however only 57% of the fish survived. Our results demonstrate that RGNNV VLP produced in yeast has great potential as vaccine in fish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The effects of a dietary probiotic mixture containing Lactococcus (Lc.) lactis BFE920 isolated from bean sprout and autochthonous Lactobacillus (Lb.) plantarum FGL0001 originally isolated from the hindgut of olive flounder (Paralichthys olivaceus) were investigated for the purpose of improving the probiotic effects of Lc. lactis BFE920 on the olive flounder. The immunostimulatory, disease protective, and weight gain effects of Lc. lactis BFE920 were significantly improved when olive flounder (average weight 37.5±1.26 g) were fed the probiotic mixture (log10 7.0 CFU each/g feed pellet) for 30 days. Flounder fed the mixture showed improved skin mucus lysozyme activity and phagocytic activity of innate immune cells compared to flounder fed a single probiotic agent or a control diet. While the levels of neutrophil activity in flounder fed the single probiotic agent or the mixture were similar, they were significantly higher than levels in a control group. Additionally, probiotic-fed flounder showed significantly increased expressions of IL-6, IL-8, and TNF-α in the intestine compared to the control group. Following a 30-day period of being fed probiotics or a control diet, the olive flounder were challenged with an i.p. injection of Streptococcus iniae (log10 6.0 CFU/fish). The groups fed the mixed probiotics, Lc. lactis BFE920, Lb. plantarum FGL0001, and the control diet had survival rates of 55%, 45%, 35%, and 20%, respectively. Flounder fed the probiotic mixture gained 38.1±2.8% more body weight compared to flounder fed the control diet during the 30-day study period. These data strongly suggest that a mixture of Lc. lactis BFE920 and Lb. plantarum FGL0001 may serve as an immunostimulating feed additive useful for disease protection in the fish farming industry., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

(AD). Although physical exercise and AD have received attention in the scientific literature, the mechanism through which treadmill exercise may impact the brain insulin signaling of AD has not been elucidated. This study aimed to evaluate the neuroprotective effects of treadmill exercise on apoptotic factors (Bcl-2/Bax ratio, caspase-3), HSP70, COX-2, BDNF and PI3-K/Akt signaling pathway in the cortex of NSE/hPS2m transgenic mice model of AD. Treadmill exercise ameliorated cognitive function in water maze test and significantly increased the level of Bcl-2/Bax ratio and HSP-70 in Tg-exe group compared to Tg-con group; on the other hand, it significantly decreased the expression of caspase-3 and COX-2 in Tg-exe group compared to Tg-con group. In addition, treadmill exercise significantly increased the expression of BDNF and PI3K/Akt in Tg-exe group compared to Tg-con group. Consequently, treadmill exercise improves cognitive function possibly via activating neurotrophic factor, BDNF and PI3k/Akt signaling pathway, and Aβ-induced neuronal cell death in the cortex of Tg mice was markedly suppressed following treadmill exercise. These results suggest that treadmill exercise may be beneficial in preventing or treating Alzheimer's disease.

Streptococcus iniae is a beta-hemolytic, Gram-positive coccus, which affects a broad range of freshwater and marine fish species, causing substantial economic losses in the aquaculture industry worldwide. Thus, it is very important to derive a complete genome sequence of the bacterium to aid in the development of vaccines and methods for preventing fish streptococcosis and zoonotic infections in humans. Here, we present the draft genome sequence of S. iniae KCTC 11634 (1,955,615 bp, with a G+C content of 36.6%), which contains 1,868 putative coding sequences.

Streptococcus parauberis is a coccoid, nonmotile, alpha-hemolytic, Gram-positive bacterium of the Streptococcaceae family. Streptococcus parauberis strain KCTC11980 was isolated from the kidney of a diseased olive flounder collected from an aquaculture farm on Jeju Island in 2010. The 2.12-Mb genome sequence consists of 44 large contigs in 16 scaffolds and contains 2,214 predicted protein-coding genes, with a G+C content of 35.4%.

Blood physiological responses, growth and survial rates were examined in juvenile starry flounder, Plotichthys stellatus exposed to different salinities (5, 10, 20, 33 ppt) for 90 days. At the end of the experiment, the plasma levels of Na+and osmolality were similar at 10, 20, 33 ppt, however, the values were significantly lower at 5 ppt compared to those at other salinities. Stress responses such as plasma levels of cortisol, glucose, hematocrit (Ht) and hemoglobin (Hb) levels in all groups showed no significant difference. Although no differences in growth were observed, body weight at 20 ppt tended to be higher than others. Survival in all groups was greater than 99% with no significant differences. These results suggest that starry flounder is euryhalin species, thus this fish can be reared with normal growth and survival rate at 5-33 ppt salinity without osmoregulatory disturbance and stress.

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.

Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062 bp) contained an open reading frame (ORF) of 594 bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10 μg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress.

Members of the tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies play crucial roles in both innate and adaptive immunity. In the present study, we isolated the full-length cDNA for black rockfish (Sebastes schlegelii) TNFR (BrTNFR). This cDNA is 2405 bp in length and contains a 939-bp open reading frame, a 27-bp 5' untranslated region, and a 1439-bp 3' untranslated region including a polyadenylation signal (AATAAA) and polyadenylation site. The 313-amino-acid predicted BrTNFR sequence is homologous to other TNFR sequences, contains four cysteine-rich domains and a death-effector domain (DED), and lacks a transmembrane region. Expression of BrTNFR mRNA was detected in eight different tissues from healthy black rockfish and was highest in peripheral blood lymphocytes and gills. In analyses of mitogen-stimulated BrTNFR expression in peripheral blood lymphocytes, expression of BrTNFR mRNA was observed between 1 and 24h after stimulation with lipopolysaccharide, concanavalin A/phorbol myristate acetate, or poly I:C. Although the data suggest that BrTNFR represents an ancestral member of the TNFR superfamily, the orthology of TNFR in teleost fish is difficult to establish because few TNFRs have been identified in these species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A voltametric diagnosis of trace lead was performed using macro type combination sensors of fluorine-doped graphite pencil electrodes (FPE). Two pencils were used as the reference and auxiliary instead of expensive Ag/AgCl standards and Pt counters. Under optimized conditions, a square wave (SW) stripping working curve was attained at 10-70 mg/L and a micro range of 10-70 μg/L. The developed sensor was not only inexpensive but also had a fast (90-s) accumulation time. An analytical detection limit of 10 μg/L was obtained. The results were applied to an ex vivo tissue and an artificial turf., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The transport and metabolism of the antitumour drug candidate 2'-benzoyloxycinnamaldehyde (BCA) was characterized in Caco-2 cells. BCA disappeared rapidly from the donor side without being transported to the receiver side during its absorptive transport across Caco-2 cells. Its metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid (OCA) were formed in both the donor and the receiver sides. HCA, in a separate study, also disappeared rapidly from the donor side, mostly being converted to its oxidative metabolite OCA during its absorptive transport across Caco-2 cells. OCA was transported rapidly in the absorptive direction across Caco-2 cells with a P(app) of 25.4 +/- 1.0 x 10(-6) cm s(-1) (mean +/- standard deviation (SD), n = 3). OCA was fully recovered from both the donor and the receiver side throughout the time-course of this study. Formation of HCA from BCA was inhibited almost completely by bis(p-nitrophenyl)phosphate (BNPP), a selective inhibitor of carboxylesterases (CES), and phenylmethylsulfonyl fluoride (PMSF), a broad specificity inhibitor of esterases in Caco-2 cells, suggesting that this hydrolytic biotransformation was likely mediated predominantly by CES. Conversion of HCA to OCA was inhibited significantly by isovanillin, a selective inhibitor of aldehyde oxidase (AO). Inhibitors for xanthine oxidase (XO) and aldehyde dehydrogenase (ALDH), which are known to be involved in the oxidation of aldehydes to carboxylic acids, did not have a significant effect on the biotransformation of HCA to OCA in Caco-2 cells. In summary, the present work demonstrates that BCA is hydrolysed rapidly to HCA, followed by subsequent oxidation to OCA, in Caco-2 cells. The results provide a mechanistic understanding of the poor absorption and low bioavailability of BCA after oral administration.

The pharmacokinetics and metabolism of 2'-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague-Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent. BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels. In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h. BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid. In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.