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Find video protocols related to scientific articles indexed in Pubmed.
Entry of a Novel Marine DNA Virus, Singapore Grouper Iridovirus, into Host Cells Occurs via Clathrin-Mediated Endocytosis and Macropinocytosis in a pH-Dependent Manner.
J. Virol.
PUBLISHED: 08-27-2014
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Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in the aquaculture industry but also show significant threat to global biodiversity. However, a lack of host cells has resulted in poor progress in clarifying iridovirus behavior. We investigated the crucial events during virus entry using a combination of single-virus tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na(+)/H(+) exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC), and myosin II, significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to colocalize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-?-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis but not via caveola-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to be transported along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only the clathrin-dependent pathway but also macropinocytosis are involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses.
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Involvement of fish signal transducer and activator of transcription 3 (STAT3) in SGIV replication and virus induced paraptosis.
Fish Shellfish Immunol.
PUBLISHED: 07-25-2014
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Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor which plays crucial roles in immune regulation, inflammation, cell proliferation, transformation, and other physiological processes of the organism. In this study, a novel STAT3 gene from orange spotted grouper (Ec-STAT3) was cloned and characterized. Bioinformatic analysis revealed that full-length of Ec-STAT3 was 3105-bp long and contained a 280-bp 5'UTR, a 470-bp 3'UTR, and a 2355-bp open reading frame (ORF) that encoded a 784-amino acid peptide. The deduced protein of Ec-STAT3 showed 98% identity to that of turbot (Scophthalmus maximus). Amino acid alignment showed that Ec-STAT3 contained four conserved domains, including a protein interaction domain, a coiled coil domain, a DNA binding domain, and an SH2 domain. Quantitative real-time PCR analysis showed that the highest expression level was detected in the liver, followed by skin and spleen. After injection with Singapore grouper iridovirus (SGIV), the transcript of Ec-STAT3 in spleen was increased significantly. To further explore the function of Ec-STAT3, we investigated the roles of Ec-STAT3 in SGIV infection in vitro. Immune fluorescence analysis indicated that SGIV infection altered the distribution of phosphorylated Ec-STAT3 in nucleus, and a small part of phosphorylated Ec-STAT3 was associated with virus assembly sites, suggesting that Ec-STAT3 might be important for SGIV infection. Using STAT3 specific inhibitor, S3I-201, we found that inhibition of Ec-STAT3 activation decreased the SGIV replication significantly. Moreover, inhibition of Ec-STAT3 activation obviously altered SGIV infection induced cell cycle arrest and the expression of pro-survival genes, including Bcl-2, Bcl-xL and Bax inhibitor. Together, our results firstly demonstrated the critical roles of fish STAT3 in DNA virus replication and virus induced paraptosis, but also provided new insights into the mechanism of iridovirus pathogenesis.
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Complete genome sequence analysis of goatpox virus isolated from China shows high variation.
Vet. Microbiol.
PUBLISHED: 07-23-2014
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Goatpox virus (GTPV), a member of the Capripoxvirus genus of the Poxviridae family, is the causative agent of variolo caprina (goatpox). GTPV can cause significant economic losses of domestic ruminants in endemic regions and can threaten breeding stocks. In this study, we report on the compilation of the complete genomic sequence of an isolated GTPV field strain FZ (GTPV_FZ). The 150,194bp GTPV genome consists of a central coding region bounded by two identical 2301bp inverted terminal repeats and contains 151 putative genes. Comparative genomic analysis reveals the apparent genetic relationships among Capripoxviruses are close, but sufficient genomic variants in the field isolate strain FZ have been identified to distinguish it from other GTPV strains and other Capripoxvirus species. Phylogenetic analysis based on the p32 and complete GTPV genome can be used to differentiate SPPVs, GTPVs and LSDVs. These data may contribute to the epidemiological study of the Chinese capripoxvirus and help to develop more specific detection methods to distinguish GTPVs, SPPVs and LSDVs.
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A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial.
Lancet Respir Med
PUBLISHED: 06-24-2014
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The phe508del CFTR mutation causes cystic fibrosis by limiting the amount of CFTR protein that reaches the epithelial cell surface. We tested combination treatment with lumacaftor, an investigational CFTR corrector that increases trafficking of phe508del CFTR to the cell surface, and ivacaftor, a CFTR potentiator that enhances chloride transport of CFTR on the cell surface.
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Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.
Fish Shellfish Immunol.
PUBLISHED: 04-24-2014
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Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-?B, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.
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Molecular cloning and characterization of two types of I?B? orthologues in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 02-19-2014
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Inhibitors of kappa B (I?Bs) are the members of primary regulators of NF-?B, which can inhibit NF-?B activity by blocking the NF-?B in an inactive state in the cytoplasm. In this study, two types of I?B? orthologues (EcI?B?A and EcI?B?B) from orange-spotted grouper, Epinephelus coioides, were cloned and characterized. EcI?B?A and EcI?B?B encoded putative proteins containing 308 and 318 amino acids, which shared 59% and 53% identity to I?B?A and I?B?B of Danio rerio, respectively. Amino acid sequence alignment showed that both EcI?B?A and EcI?B?B contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcI?B?A and EcI?B?B contained 5 and 6 ankyrin repeats, respectively. The genomic DNA of EcI?B?A and EcI?B?B consisted of 6 exons and 5 introns. Both of their transcripts were widely distributed in different tissues, and the expression levels were different in response to various stimuli, including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Dual-luciferase reporter assay suggested that both EcI?B?A and EcI?B?B were able to inhibit Ecc-Rel and Ecp65 induced NF-?B promoter activity in grouper spleen (GS) cells. Subcellular localization analysis showed that EcI?B?B was present predominantly in the cytoplasm, while EcI?B?A was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcI?B?A and EcI?B?B in GS cells inhibited the viral gene transcriptions of MCP, ORF019 and ORF162 of SGIV. Taken together, our findings suggested that both EcI?B?A and EcI?B?B were involved in grouper innate immunity against virus.
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Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
PUBLISHED: 01-22-2014
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Lysozyme acts as an innate immunity molecule against pathogen infection. In this study, a new G-type lysozyme gene with a typical G-type lysozyme domain (designated as Ec-lysG) was cloned and characterized from the orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysG cDNA contains 1419 bp and encodes a 256-residue protein containing a 25-residue signal peptide at the N-terminus. BLAST analysis reveals Ec-lysG shares 64% identity with Siniperca chuatsi, but 63% to another reported G-type lysozyme from orange-spotted grouper (OSG-lysG). The genomic DNA of Ec-lysG contains four exons and three introns, with a total length of 2062 bp. An amino acid sequence alignment showed that Ec-lysG shares the fundamental structural features of G-type lysozyme, including the catalytic residues, substrate binding sites, and soluble lytic transglycosylase domain. Quantitative PCR showed that Ec-lysG transcript is most abundant in the head kidney, and less abundant in the heart. The expression of Ec-lysG was differentially upregulated in the head kidney after stimulation with lipopolysaccharide, Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). A subcellular localization analysis showed that Ec-lysG is distributed predominantly in the cytoplasm. Recombinant Ec-lysG (rEc-lysG) has optimal activity at pH 7.5 and 35°C. rEc-lysG showed lytic activities against Gram-positive bacterium Streptococcus iniae, Staphylococcus aureus, and Micrococcus lysodeikticus, and the Gram-negative bacterium V. alginolyticus. Scanning electron microscopy (SEM) showed that rEc-lysG acts on M. lysodeikticus cell walls. The overexpression of Ec-lysG in grouper cells did not significantly delay the occurrence of the cytopathic effect (CPE) induced by SGIV, and did not inhibit viral gene transcription. In conclusion, Ec-lysG might be a potent antibacterial protein, with a role in innate immunity.
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Isolation and characterization of a new class of DNA aptamers specific binding to Singapore grouper iridovirus (SGIV) with antiviral activities.
Virus Res.
PUBLISHED: 01-13-2014
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The Singapore grouper iridovirus (SGIV), a member of the genus Ranavirus, is a major viral pathogen that has caused heavy economic losses to the grouper aquaculture industry in China and Southeast Asia. No efficient method of controlling SGIV outbreaks is currently available. Systematic evolution of ligands by exponential enrichment (SELEX) is now widely used for the in vitro selection of artificial ssDNA or RNA ligands, known as aptamers, which bind to targets through their stable three-dimensional structures. In our current study, we generated ssDNA aptamers against the SGIV, and evaluated their ability to block SGIV infection in cultured fish cells and cultured fish in vivo. The anti-SGIV DNA aptamers, LMB-761, LMB-764, LMB-748, LMB-439, LMB-755, and LMB-767, were selected from a pool of oligonucleotides randomly generated using a SELEX iterative method. The analysis of the secondary structure of the aptamers revealed that they all formed similar stem-loop structures. Electrophoretic mobility shift assays showed that the aptamers bound SGIV specifically, as evidenced by a lack cross-reactivity with the soft shell turtle iridovirus. The aptamers produced no cytotoxic effects in cultured grouper spleen cells (GS). Assessment of cytopathic effects (CPE) and viral titer assays showed that LMB-761, LMB-764, LMB-748, LMB-755, and LMB-767 significantly inhibited SGIV infection in GS cells. The in vivo experiments showed that LMB-761 and LMB-764 reduced SGIV-related mortality, and no negative effects were observed in orange-spotted grouper, Epinephelus coioides, indicating that these DNA aptamers may be suitable antiviral candidates for controlling SGIV infections in fish reared in marine aquaculture facilities.
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Sarilumab, a fully human monoclonal antibody against IL-6R? in patients with rheumatoid arthritis and an inadequate response to methotrexate: efficacy and safety results from the randomised SARIL-RA-MOBILITY Part A trial.
Ann. Rheum. Dis.
PUBLISHED: 12-02-2013
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To evaluate safety and efficacy of weekly (qw) and every other week (q2w) dosing of sarilumab, a fully human anti-interleukin 6 receptor ? (anti-IL-6R?) monoclonal antibody, for moderate-to-severe rheumatoid arthritis (RA).
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Missing radiographic data handling in randomized clinical trials in rheumatoid arthritis.
J Biopharm Stat
PUBLISHED: 10-22-2013
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In recent years, there has been increasing interest in compounds that have potential to slow down the structural joint damage in rheumatoid arthritis (RA) patients. Radiographs are instrumental in assessing structure damage in RA. Radiographic analyses results have become essential in establishing a "delay in structural progression" claim in newly developed agents for the treatment of RA. It is well known that the radiographic progression data generally follow a nonnormal distribution that is loaded with excessive zeros. A special concern about the radiographic data analyses is the handling of the seemingly high rate of missing values due to dropout or unreadable images. There are no uniform ways to handle missing radiographic data, and such data usually show considerable sensitivity to the imputation method chosen under the complexity of the nonnormal data and the unique missing mechanism. In this research, we proposed both an innovative multiple-imputation algorithm and a novel method called the mean rank imputation method under the nonparametric framework for sensitivity analyses. A simulation study was designed using rank analysis of covariance (ANCOVA) to extensively assess and compare the finite performance of these two new methods along with four other missing data handling methods previously used in the RA trials, namely, linear extrapolation, last observation carried-forward (LOCF), median quartile bin imputation, and median imputation under various settings. Our simulation results suggest that the multiple-imputation algorithm, providing an mITT analysis population, yields an inflated type I error and artificially good power. The proposed mean rank imputation method, following a true ITT principle, both is powerful and maintains type I error at the nominal level.
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Long-term clinical efficacy and safety of adding cilostazol to dual antiplatelet therapy for patients undergoing PCI: a meta-analysis of randomized trials with adjusted indirect comparisons.
Curr Med Res Opin
PUBLISHED: 10-18-2013
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Abstract Objective: To assess the long-term clinical efficacy and safety of adding cilostazol to aspirin plus clopidogrel (triple antiplatelet therapy, TAT) in patients undergoing percutaneous coronary intervention (PCI) and explore its role in the era of new generation adenosine diphosphate (ADP)-receptor antagonists. Methods: PUBMED, EMBASE, and the Cochrane Central Register of Controlled Trials were searched for randomized controlled trials (RCTs) comparing TAT versus dual antiplatelet therapy (DAT), followed by a manual search. Then, a meta-analysis of RCTs comparing TAT versus standard DAT in patients undergoing PCI was performed. Furthermore, indirect comparisons of TAT versus new generation ADP-receptor antagonist based DAT (prasugrel or ticagrelor based DAT) were undertaken, with standard DAT as a common comparator. The included end-points were major adverse cardiovascular event (MACE), target lesion revascularization (TLR), target vessel revascularization (TVR), death, myocardial infarction (MI), stent thrombosis, bleeding and other drug adverse events. Results: Twelve RCTs with a total of 31,789 patients were included. Compared with standard DAT (n?=?2551), TAT (n?=?2545) significantly reduced the incidence of MACE (OR: 0.56, 95% CI: 0.47-0.68, P?
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An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.
J. Gen. Virol.
PUBLISHED: 09-23-2013
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Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.
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Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.
Fish Shellfish Immunol.
PUBLISHED: 09-19-2013
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The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.
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Characterization of an envelope gene VP19 from Singapore grouper iridovirus.
Virol. J.
PUBLISHED: 09-17-2013
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Viral envelope proteins are always proposed to exert important function during virus infection and replication. Vertebrate iridoviruses are enveloped large DNA virus, which can cause great economic losses in aquaculture and ecological destruction. Although numerous iridovirus envelope proteins have been identified using bioinformatics and proteomic methods, their roles in virus infection remained largely unknown.
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Identification and characterization of Rab7 from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 07-19-2013
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Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication.
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Medaka haploid embryonic stem cells are susceptible to Singapore grouper iridovirus as well as to other viruses of aquaculture fish species.
J. Gen. Virol.
PUBLISHED: 07-04-2013
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Viral infection is a challenge in high-density aquaculture, as it leads to various diseases and causes massive or even complete loss. The identification and disruption of host factors that viruses utilize for infection offer a novel approach to generate viral-resistant seed stocks for cost-efficient and sustainable aquaculture. Genetic screening in haploid cell cultures represents an ideal tool for host factor identification. We have recently generated haploid embryonic stem (ES) cells in the laboratory fish medaka. Here, we report that HX1, one of the three established medaka haploid ES cell lines, was susceptible to the viruses tested and is thus suitable for genetic screening to identify host factors. HX1 cells displayed a cytopathic effect and massive death upon inoculation with three highly infectious and notifiable fish viruses, namely Singapore grouper iridovirus (SGIV), spring viremia of carp virus (SVCV) and red-spotted grouper nervous necrosis virus (RGNNV). Reverse transcription-PCR and Western blot analyses revealed the expression of virus genes. SGIV infection in HX1 cells elicited a host immune response and apoptosis. Viral replication kinetics were determined from a virus growth curve, and electron microscopy revealed propagation, assembly and release of infectious SGIV particles in HX1 cells. Our results demonstrate that medaka haploid ES cells are susceptible to SGIV, as well as to SVCV and RGNNV, offering a unique opportunity for the identification of host factors by genetic screening.
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How do spin waves pass through a bend?
Sci Rep
PUBLISHED: 06-24-2013
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Spin-wave devices hold great promise to be used in future information processing. Manipulation of spin-wave propagation inside the submicrometer waveguides is at the core of promoting the practical application of these devices. Just as in todays silicon-based chips, bending of the building blocks cannot be avoided in real spin-wave circuits. Here, we examine spin-wave transport in bended magnonic waveguides at the submicron scale using micromagnetic simulations. It is seen that the impact of the bend is relevant to the frequency of the passing spin wave. At the lowest frequencies, the spin wave continuously follows the waveguide in the propagation process. At the higher frequencies, however the bend acts as a mode converter for the passing spin wave, causing zigzag-like propagation path formed in the waveguide behind the bend. Additionally, we demonstrate a logic-NOT gate based on such a waveguide, which could be combined to perform logic-NAND operation.
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Identification and characterization of a tumor necrosis factor receptor like protein encoded by Singapore grouper iridovirus.
Virus Res.
PUBLISHED: 06-23-2013
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Virus encoded tumor necrosis factor receptors (TNFRs) have been demonstrated to facilitate virus to escape from apoptosis or other host immune response for viral replication. Singapore grouper iridovirus (SGIV), a large DNA virus which belongs to genus Ranavirus, is a major pathogen resulting in heavy economic losses to grouper aquaculture. Here, SGIV ORF096 (VP96) encoding a putative homolog of TNFR was identified and characterized. Multiple sequence alignment indicated that SGIV-VP96 contained two extracellular cysteine-rich domains (CRDs) with conserved four or six cysteine residues, but lacked the transmembrane domain at the C-terminus. SGIV-VP96 was identified as an early (E) gene and localized in the cytoplasm in transfected or infected cells. Overexpression of SGIV-VP96 in vitro enhanced cell proliferation, and improved cell survival against SGIV infection. Furthermore, virus infection induced apoptosis and caspase-3 activity were inhibited in SGIV-VP96 expressing FHM cells compared to the control cells. Taken together, our results suggested that SGIV might utilize virus encoded TNFR like genes to modulate the host apoptotic response for effective virus replication.
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Characterization of LPS-induced TNF? factor (LITAF) from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 06-13-2013
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Lipopolysaccharide-induced TNF? factor (LITAF) is an important transcription factor that mediates cell apoptosis and inflammatory response. In the present study, we cloned and characterized a LITAF gene from orange-spotted grouper (Epinephelus coioides) (Ec-LITAF). Ec-LITAF encoded a predicted 142 amino acid protein which shared 74% identity to sablefish (Anoplopoma fimbria) LITAF homolog. Multiple amino acid alignment showed that Ec-LITAF contained a typical LITAF domain with two CXXC motifs. Phylogenetic analysis indicated that Ec-LITAF was closely related to that of sablefish. Ec-LITAF mRNA was widely expressed in different tissues and its expression level in spleen was up-regulated after Singapore grouper iridovirus (SGIV) infection. Subcellular localization analysis revealed that the distribution of Ec-LITAF showed diffuse and aggregated patterns in cytoplasm. Interestingly, the distribution of Ec-LITAF overlayed with a viral LITAF homolog (vLITAF) encoded by SGIV. Overexpression of Ec-LITAF in vitro up-regulated the expression of tumor necrosis factors (TNF1 and TNF2) and TNF receptors (TNFR1 and TNFR2), and the expression of itself initiated apoptosis in fish cells. In addition, overexpression of Ec-LITAF not only accelerated SGIV infection induced CPE and cell death, but also increased viral gene transcription. Taken together, our data suggested that Ec-LITAF might play crucial roles during SGIV replication.
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Cloning, expression and molecular characterization of a 14-3-3 gene from a parasitic ciliate, Cryptocaryon irritans.
Vet. Parasitol.
PUBLISHED: 05-03-2013
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Cryptocaryon irritans is a parasitic ciliate and responsible for cryptocaryosis of ocean teleostean. In this paper, one gene homologous to 14-3-3 was isolated from cDNA library of C. irritans trophont/protomont stage and designated as Ci14-3-3. The full-length cDNA of the gene was 892bp with an open reading frame of 744bp, which encoded a polypeptide of 247 amino acids with a predicted molecular weight of 28.4kDa. After modification of the non-universal genetic codes, the open reading frame of Ci14-3-3 was inserted into plasmid pGEX-4T-1, transformed into Escherichia coli DH5? strain and then expressed as a glutathione S transferase fusion protein (rCi14-3-3). The result of western blot analysis showed that the rCi14-3-3 had antigenicity and the Ci14-3-3 gene in C. irritans was expressed at all stages of life cycle. The endogenous Ci14-3-3 not only distributed in cytoplasm, but also presented on the plasma membrane and the front end of cytostome in newly hatched theronts. However, when theronts were dying the protein appeared as dot-like aggregates around the nucleuses. The murine anti-rCi14-3-3 sera were capable of causing agglutination/immobilization of theronts, suggesting its potential for vaccine development.
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Identification of two distinct carcinoma-associated fibroblast subtypes with differential tumor-promoting abilities in oral squamous cell carcinoma.
Cancer Res.
PUBLISHED: 04-18-2013
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Heterogeneity of carcinoma-associated fibroblasts (CAF) has long been recognized, but the functional significance remains poorly understood. Here, we report the distinction of two CAF subtypes in oral squamous cell carcinoma (OSCC) that have differential tumor-promoting capability, one with a transcriptome and secretome closer to normal fibroblasts (CAF-N) and the other with a more divergent expression pattern (CAF-D). Both subtypes supported higher tumor incidence in nonobese diabetic/severe combined immunodeficient (NOD/SCID) Il?2(null) mice and deeper invasion of malignant keratinocytes than normal or dysplasia-associated fibroblasts, but CAF-N was more efficient than CAF-D in enhancing tumor incidence. CAF-N included more intrinsically motile fibroblasts maintained by high autocrine production of hyaluronan. Inhibiting CAF-N migration by blocking hyaluronan synthesis or chain elongation impaired invasion of adjacent OSCC cells, pinpointing fibroblast motility as an essential mechanism in this process. In contrast, CAF-D harbored fewer motile fibroblasts but synthesized higher TGF-?1 levels. TGF-?1 did not stimulate CAF-D migration but enhanced invasion and expression of epithelial-mesenchymal transition (EMT) markers in malignant keratinocytes. Inhibiting TGF-?1 in three-dimensional cultures containing CAF-D impaired keratinocyte invasion, suggesting TGF-?1-induced EMT mediates CAF-D-induced carcinoma cell invasion. TGF-?1-pretreated normal fibroblasts also induced invasive properties in transformed oral keratinocytes, indicating that TGF-?1-synthesizing fibroblasts, as well as hyaluronan-synthesizing fibroblasts, are critical for carcinoma invasion. Taken together, these results discern two subtypes of CAF that promote OSCC cell invasion via different mechanisms.
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Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcohol-related cirrhosis.
Hepatology
PUBLISHED: 02-01-2013
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Patients with alcohol-related liver disease (ALD) have antibodies directed to alcohol dehydrogenase (ADH), anti-ADH titers being associated with disease severity and active alcohol consumption. ADH-specific T-cell responses have not been characterized. We aimed to define anti-ADH cellular immune responses and their association with active alcohol consumption and disease severity. Using cultures of peripheral blood mononuclear cells (PBMCs) from 25 patients with alcohol-related cirrhosis (ARC; 12 were actively drinking or abstinent for <6 months, and 13 were abstinent for >6 months) and hepatic mononuclear cells (HMCs) from 14 patients with ARC who were undergoing transplantation, we investigated T-cell reactivity to 25 overlapping peptides representing the full human ADH protein (beta 1 subunit). ADH-specific peripheral T-cell responses were assessed by the quantification of T-cell proliferation and cytokine production and were correlated with the clinical course. In active alcohol consumers, proliferative T-cell responses targeted ADH31-95 and other discontinuous sequences in the ADH peptide, whereas only one sequence was targeted in abstinents. ADH peptides induced the production of interferon-? (IFN-?), interleukin-4 (IL-4), and IL-17. IL-4 production was lower in active drinkers versus abstinents, and IL-17 production was higher. Peptides inducing IFN-? production outnumbered those inducing T-cell proliferation. The intensity of the predominantly T helper 1 (Th 1) responses directly correlated with disease severity. Similar to PBMCs in abstinents, ADH peptides induced weak T-cell proliferation and a similar level of IL-4 production in HMCs but less vigorous Th 1 and T helper 17 responses. Conclusion: This suggests that Th 1 responses to ADH in ARC are induced by alcohol consumption. A Th 1/T helper 2 imbalance characterizes T-cell responses in active drinkers with ARC, whereas IL-4 production prevails in abstinents. This identifies new targets for immunoregulatory therapies in ALD patients for halting detrimental effector T-cell responses, which may encourage liver fibrogenesis and progression to end-stage liver disease.
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Molecular cloning, expression and functional analysis of ISG15 in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 01-20-2013
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Interferon-stimulated gene 15 (ISG15) is an ubiquitin homolog that is significantly induced by type I interferons or viral infections. Groupers, Epinephelus spp. being maricultured in China and Southeast Asian countries, always suffer from virus infection, including iridovirus and nodavirus. To date, the roles of grouper genes, especially interferon related genes in virus infection remained largely unknown. Here, the ISG15 homolog (EcISG15) was cloned from grouper Epinephelus coioides and its immune response to Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV) was investigated. The full-length EcISG15 cDNA was composed of 948 bp and encoded a polypeptide of 155 amino acids with 37-68% identity with the known ISG15 homologs from other fish species. Amino acid alignment analysis indicated that EcISG15 contained two ubiquitin-like (UBL) domains and an Ub-conjugation domain (LRGG). Expressional analysis showed that EcISG15 was dramatically induced by GNNV infection, poly I:C or poly dA-dT treatment, but no obvious changes were observed during SGIV infection. Immunofluorescence assay showed that EcISG15 localized mainly in the cytoplasm of grouper cells in response to poly I:C stimulation or GNNV infection, but not in mock or SGIV infected cells. Western blot analysis indicated that the ISGylation was absent in SGIV-infected cells, but significantly enhanced in GNNV-infected or poly I:C transfected cells, suggesting that EcISG15 might play different roles in SGIV and GNNV infection. Furthermore, overexpression of EcISG15 in vitro inhibited the transcription of GNNV genes significantly. Taken together, the results indicated that fish ISG15 might exert important roles against RNA virus infection.
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Molecular characterization of an actin depolymerizing factor from Cryptocaryon irritans.
Parasitology
PUBLISHED: 01-04-2013
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Actin depolymerizing factors regulate actin dynamics involved in cellular processes such as morphogenesis, motility, development and infection. Here, a novel actin depolymerizing factor gene (CiADF 2 ) was cloned from the cDNA library of Cryptocaryon irritans, a parasitic ciliate causing cryptocaryonosis. The full-length cDNA of CiADF 2 was 531 bp. Its open reading frame (ORF) was 417 bp, encoding a polypeptide of 138 aa with typical features of the ADF/cofilin family. Reverse transcription-PCR suggested that CiADF 2 is expressed in all stages of the life cycle. After site-directed mutagenesis of a non-universal genetic code, the ORF was subcloned in Escherichia coli. The bacteria were induced with the addition of isopropylthio-?-D-galactoside to express a fusion protein of recombinant CiADF2 (rCiADF2) with glutathione S transferase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed the predicted molecular mass of rCiADF2 of 16·2 kDa. A mouse antibody against rCiADF2 recognized native CiADF2, and rCiADF2 reacted with mouse antisera against C. irritans trophonts. CiADF2 was abundant in the plasma around cytostomes, suggesting that CiADF2 is involved in ciliate movement. Moreover, rCiADF2 showed F-actin binding and depolymerizing activity. This study will help to clarify the pathogenic biology of the parasite and develop effective control measures for cryptocaryonosis.
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miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death.
PLoS ONE
PUBLISHED: 01-01-2013
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Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-? (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.
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High-maintenance-dose clopidogrel in patients undergoing percutaneous coronary intervention: a systematic review and meta-analysis.
PLoS ONE
PUBLISHED: 01-01-2013
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Despite routine use of clopidogrel, adverse cardiovascular events recur among some patients undergoing percutaneous coronary intervention (PCI). To optimize antiplatelet therapies, we performed a meta-analysis to quantify the efficacy of high versus standard-maintenance-dose clopidogrel in these patients.
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Heterogeneity among orf virus isolates from goats in Fujian Province, Southern China.
PLoS ONE
PUBLISHED: 01-01-2013
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Orf virus is a parapoxvirus that causes recurring contagious ecthyma or orf disease in goat, sheep and other wild and domestic ruminants. Infected animals show signs of pustular lesions on the mouth and muzzle and develop scabs over the lesions. Although the infection is usually cleared within 1-2 months, delayed growth and associated secondary infections could still impact the herds. Orf virus can also infect humans, causing lesions similar to the animals in pathological histology. Prior infection of orf virus apparently offers little protective immunity against future infections. Several gene products of orf virus have been identified as responsible for immunomodulatory functions. In our recent study of orf virus isolates from an area along the Minjiang River in northern Fujian Province, we found a high heterogeneity among isolates from 10 farms within a 120-kilometer distance. Only two isolates from locations within 1 km to each other had same viral genes. There is no correlation between the geographical distance between the corresponding collection sites and the phylogenetic distance in ORFV011 or ORV059 genes for any two isolates. This finding suggests that there are diverse populations of orf virus present in the environment. This may in part contribute to the phenomenon of recurring outbreaks and heighten the need for better surveillance.
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Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.
Fish Shellfish Immunol.
PUBLISHED: 07-30-2011
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p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38?) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38? showed high homologies to other fish p38a MPAK, p38b MAPK and p38? MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38? are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38? were different, and Ec-p38? was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38? in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38? inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38? played a crucial role in regulating apoptosis and virus replication during iridovirus infection.
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Transcriptome analysis of orange-spotted grouper (Epinephelus coioides) spleen in response to Singapore grouper iridovirus.
BMC Genomics
PUBLISHED: 07-12-2011
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Orange-spotted grouper (Epinephelus coioides) is an economically important marine fish cultured in China and Southeast Asian countries. The emergence of infectious viral diseases, including iridovirus and betanodavirus, have severely affected food products based on this species, causing heavy economic losses. Limited available information on the genomics of E. coioides has hampered the understanding of the molecular mechanisms that underlie host-virus interactions. In this study, we used a 454 pyrosequencing method to investigate differentially-expressed genes in the spleen of the E. coioides infected with Singapore grouper iridovirus (SGIV).
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Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling.
Apoptosis
PUBLISHED: 06-10-2011
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Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (??m) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.
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Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.
Virus Res.
PUBLISHED: 04-17-2011
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Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.
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Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis.
Apoptosis
PUBLISHED: 03-29-2011
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Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus-host interaction.
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Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells.
J. Gen. Virol.
PUBLISHED: 03-14-2011
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Stress-activated protein kinases (SAPKs), including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), are usually activated in response to different environmental stimuli, including virus infection. In the present study, the roles of SAPKs during Singapore grouper iridovirus (SGIV) infection were investigated in fish cells. The results showed that increased phosphorylation of JNK1/2 and p38 MAPK occurred during active replication of SGIV in grouper cell cultures. Moreover, downstream effectors (c-Jun, MAPK-activated protein kinase 2, p53, activator protein 1, Myc and nuclear factor of activated T cells) were activated after SGIV infection, suggesting that SGIV replication activated the JNK and p38 MAPK signalling pathways. Notably, using specific inhibitors, it was found that viral gene transcripts, protein expression and viral titres were not affected by inhibition of p38 MAPK but were suppressed significantly by inhibiting JNK1/2 activation. In addition, transcription of grouper immune genes including interferon regulatory factor 1, interleukin-8 and tumour necrosis factor alpha (TNF-?) were regulated by JNK, whilst only TNF-? was regulated by p38 MAPK. It is proposed that the JNK pathway is important for SGIV replication and modulates the inflammatory responses during virus infection.
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Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088.
Proteomics
PUBLISHED: 02-23-2011
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Singapore grouper iridovirus (SGIV) is an enveloped virus causing heavy economic losses to marine fish culture. The envelope fractions of SGIV were separated from the purified virions by Triton X-100 treatment, and subjected to 1-DE-MALDI-TOF/TOF-MS/MS and LC-MALDI-TOF/TOF-MS/MS analysis. A total of 19 virus-encoded envelope proteins were identified in this study and 73.7% (13/17) of them were predicted to be membrane proteins. Three viral envelope proteins were uniquely identified by 1-DE-MALDI, whereas another ten proteins were identified only by LC-MALDI, with six proteins identified by both workflows. VP088 was chosen as a representative of proteomic identification and characterized further. VP088 was predicted to be a viral transmembrane envelope protein which contains two RGD (Arg-Gly-Asp) motifs, three transmembrane domains, and five N-glycosylation sites. VP088 gene transcript was first detected at 12?h p.i. and reached the peak at 48?h p.i. Combined with the drug inhibition assay, VP088 gene was identified as a late (L) gene. Recombinant VP088 (rVP088) was expressed in Escherichia coli, and the specific antiserum against rVP088 was raised. VP088 was proved to be a viral envelope protein by Western blot and immunoelectron microscopy (IEM). Furthermore, rVP088 can bind to a 94?kDa host cell membrane protein, suggesting that VP088 might function as an attaching protein. Neutralization assay also suggested that VP088 is involved in SGIV infection. This study will lead to a better understanding of molecular mechanisms of the iridoviral pathogenesis and virus-host interactions.
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Identification and functional characterization of an interferon regulatory factor 7-like (IRF7-like) gene from orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
PUBLISHED: 01-26-2011
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Interferon regulatory factor (IRF) 7 plays a crucial role in modulating cellular responses to viral infection and cytokines, including interferons (IFNs). In the present study, a novel IRF7 gene (designated as EcIRF7) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcIRF7 cDNA is composed of 2089 bp and encodes a polypeptide of 433 amino acids with 81% identity to IRF7 of Siniperca chuatsi, and the genomic DNA of EcIRF7 consists of 9 exons and 8 introns, with a length of approximately 5629 bp. EcIRF7 contains three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain, all of which are highly conserved across species. Recombinant EcIRF7 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcIRF7 serum preparation. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcIRF7, with a relative strong expression in spleen, kidney, skin and intestine. The expression of EcIRF7 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus and Singapore grouper iridovirus (SGIV). EcIRF7 showed similar intracellular localization pattern to those of mammalian and chicken, and translocated into nucleus after SGIV infection. Further more, EcIRF7 was proved to be capable of activating zebrafish type I IFN promoter and inhibiting the replication of SGIV in grouper spleen (GS) cells. These results suggest that EcIRF7 is potentially involved in grouper immune responses to invasion of viral and bacterial pathogens.
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Identification of a novel marine fish virus, Singapore grouper iridovirus-encoded microRNAs expressed in grouper cells by Solexa sequencing.
PLoS ONE
PUBLISHED: 01-25-2011
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MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms.
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Estimation of treatment effect for the sequential parallel design.
Stat Med
PUBLISHED: 01-07-2011
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The sequential parallel clinical trial is a novel clinical trial design being used in psychiatric diseases that are known to have potentially high placebo response rates. The design consists of an initial parallel trial of placebo versus drug augmented by a second parallel trial of placebo versus drug in the placebo non-responders from the initial trial. Statistical research on the design has focused on hypothesis tests. However, an equally important output from any clinical trial is the estimate of treatment effect and variability around that estimate. In the sequential parallel trial, the most important treatment effect is the effect in the overall population. This effect can be estimated by considering only the first phase of the trial, but this ignores useful information from the second phase of the trial. We develop estimates of treatment effect that incorporate data from both phases of the trial. Our simulations and a real data example suggest that there can be substantial gains in precision by incorporating data from both phases. The potential gains appear to be greatest in moderate-sized trials, which would typically be the case in phase II trials.
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Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice.
Vaccine
PUBLISHED: 07-21-2010
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Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.
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Cloning, characterization, and expression analysis of orange-spotted grouper (Epinephelus coioides) ILF2 gene (EcILF2).
Fish Shellfish Immunol.
PUBLISHED: 06-10-2010
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Interleukin-2 enhancer binding factor 2 (ILF2), also named as nuclear factor 45 (NF45), plays important roles in regulating interleukin-2 expression in mammals. In the present study, a novel ILF2 gene (designated EcILF2) was cloned and well characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcILF2 cDNA is composed of 1544 bp and encodes a polypeptide of 387 amino acids with 98% identity to ILF2 of Atlantic salmon. The genomic DNA of EcILF2 consists of 14 exons and 13 introns, with a length of approximately 6.9 kb. EcILF2 contains two conserved domains including an RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. Recombinant EcILF2 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcILF2 serum preparation. Subcellular localization analysis revealed that EcILF2 was distributed predominantly in the nucleus. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcILF2, with a relative strong expression in skin, liver, brain, head kidney and spleen. The expression of EcILF2 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Furthermore, EcILF2 was able to activate human IL-2 promoter in different cell lines and promote the endogenous IL-2 transcription in human H9 T cells. These results suggest that EcILF2 is potentially involved in grouper immune responses to invasion of bacterial and viral pathogens.
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Interaction of functional NPC1 gene polymorphism with smoking on coronary heart disease.
BMC Med. Genet.
PUBLISHED: 02-22-2010
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The protein of Niemann-pick type C1 gene (NPC1) is known to facilitate the egress of cholesterol and other lipids from late endosomes and lysosomes to other cellular compartments. This study aims to investigate whether the genetic variation in NPC1 is associated with risk of coronary heart disease (CHD) and to detect whether NPC1 might interact with smoking on the risk of CHD.
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Molecular cloning, characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 06-16-2009
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C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell-cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys(71), Cys(152), Cys(167), Cys(175)) involved in the formation of disulphide bridges and the potential Ca(2+)/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca(2+)-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca(2+)-dependent manner.
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Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae.
BMC Genomics
PUBLISHED: 05-14-2009
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Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis.
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Characterization of Singapore grouper iridovirus (SGIV) ORF086R, a putative homolog of ICP18 involved in cell growth control and virus replication.
Arch. Virol.
PUBLISHED: 03-17-2009
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Singapore grouper iridovirus (SGIV), as a causative agent of serious systemic disease, causes significant economic losses in grouper aquaculture. In this study, a novel ICP18 homolog encoded by SGIV ORF086R was identified and characterized. Strikingly, ICP18 homologs can be found in all ranaviruses, but not in other sequenced large DNA viruses. SGIV ICP18 is an immediate-early gene and begins to be transcribed as early as 2 h post-infection (p.i.). Western blotting indicated that SGIV ICP18 is translated as early as 6 h p.i. and is a viral non-envelope protein. Subcellular localization analysis revealed that the SGIV ICP18 displays a finely punctate cytoplasmic pattern. Furthermore, overexpression of SGIV ICP18 can promote the growth of grouper embryonic cells (GP) and contribute to SGIV replication. These results should offer important insights into the pathogenesis of ranaviruses.
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The Kunitz-like modulatory protein haemangin is vital for hard tick blood-feeding success.
PLoS Pathog.
PUBLISHED: 03-13-2009
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Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of angiogenesis and wound healing processes.
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Cloning and expression characterization of the serum transferrin gene in the Chinese black sleeper (Bostrichthys sinensis).
Gene
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In this study, the gene encoding serum transferrin (STF) in the Chinese black sleeper (Bostrichthys sinensis) was cloned and sequenced. The full BsSTF cDNA is 2324 bp in length with an open reading frame (ORF) of 2067 bp encoding 688 amino acids and has 47.1%-65.7% identity with other fish homologues. The BsSTF genomic DNA is composed of 17 exons and 16 introns and is well conserved with respect to other vertebrate species in the number of exons and in the position of introns. The expression of the BsSTF mRNA was examined in all tissues tested including liver, blood, muscle, spleen, stomach, skin, intestine, brain, head kidney, fin, gill and gonad, with the highest expression levels found in the liver. Temporal transcriptional changes of BsSTF during embryonic development of the Chinese black sleeper were detected, and the results showed a substantial decrease in the early stages, from the cleavage stage to the blastula stage, reaching minimum levels at the blastula stage and increasing gradually until the pre-hatching stage, indicating that BsSTF is likely an important factor in early embryonic development. After infection with Vibrio harveyi, BsSTF expression was found to increase mainly in the liver and stomach, acting as a positive acute protein, suggesting that BsSTF is involved in the immune response.
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A PCR based survey of Babesia ovata in cattle from various Asian, African and South American countries.
J. Vet. Med. Sci.
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Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.
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Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV).
Fish Shellfish Immunol.
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Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.
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Regulatory effect of hypoxia-inducible factor-1? on hCG-stimulated endothelin-2 expression in granulosa cells from the PMSG-treated rat ovary.
J. Reprod. Dev.
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Endothelin (ET)-2 plays a crucial role in ovarian ovulation in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1?-mediated transcriptional activation contributes to the increased expression of ET-2 gene in response to hCG in rat ovarian granulosa cells (GCs) during gonadotropin-induced superovulation. By real-time RT-PCR analysis, ET-2 mRNA expression was found to significantly increase in cultured ovarian GCs after treatment with hCG, or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while this increased ET-2 mRNA expression could also be blocked by ferrous ammonium sulfate (FAS) under human chorionic gonadotropin (hCG) treatment. Further analysis also found that these changes of ET-2 mRNA were consistent with HIF-1? expression or HIF-1 activity, and HIF-1? inhibitor echinomycin inhibited ovulation in rats. Taken together, these results indicate that ET-2 is transcriptionally activated by hCG through HIF-1?-mediated mechanism in GCs. This HIF-1?-induced transcriptional activation may be one of the important mechanisms mediating the increase of ET-2 expression in GCs during the gonadotropin-induced mammalian ovulatory process in vivo.
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Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
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Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including ?-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.
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Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins.
Exp. Mol. Med.
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Echinomycin is a small-molecule inhibitor of hypoxia- inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1?-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1? mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1? and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1? and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1? directly mediated the transcriptional activation of ET-2 during gonadotropin- induced superuvulation. Taken together, these results demonstrated that HIF-1?-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.
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Single nucleotide polymorphisms in i-type lysozyme gene and their correlation with vibrio-resistance and growth of clam Meretrix meretrix based on the selected resistance stocks.
Fish Shellfish Immunol.
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I-type lysozyme is considered to play crucial roles in both anti-bacteria and digestion function of the bivalve, which signifies that it is related to both immunity and growth. In this study, based on the principle of case-control association analysis, using the stock materials with different vibrio-resistance profile obtained by selective breeding, single nucleotide polymorphisms (SNPs) in the DNA partial sequence of an i-type lysozyme of Meretrix meretrix (MmeLys) were discovered and examined for their association with vibrio-resistance and growth. Twenty-seven SNPs were detected and fifteen of them were genotyped in clam stocks with different resistance to Vibrio harveyi (09-C and 09-R) and to Vibrio parahaemolyticus (11-S and 11-R). Allele frequency distribution among different stocks was compared. And wet weight of clams with different genotype at each SNP locus was compared. The results indicated that SNP locus 9 was associated with V. harveyi and V. parahaemolyticus resistance and growth of M. meretrix. Loci 12 and 14 were associated with both V. parahaemolyticus-resistance and growth, and also have the potential to be related with V. harveyi-resistance of M. meretrix. Therefore these three SNPs especially locus 9 were the potential markers which may be involved in assisting resistance selective breeding. In addition, this study showed evidence that improvements in clam resistance to vibriosis could be achieved through selective breeding. All results provided encouragement for the continuation of the selective breeding program for vibrio-resistance gain in clam M. meretrix and the application of polymorphisms in MmeLys to the future marker assisted selection.
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Selection and identification of Singapore grouper iridovirus vaccine candidate antigens using bioinformatics and DNA vaccination.
Vet. Immunol. Immunopathol.
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In this study, we described a rapid and efficient method which integrated the bioinformatic prediction and DNA vaccine technology to identify vaccine candidates against Singapore grouper iridovirus (SGIV). The 162 previously defined open reading frames (ORFs) of SGIV were subjected to extensive sequence similarity searches, as well as motif, cellular location, and domain prediction. Based on our analysis, 13 genes were chosen and cloned into the eukaryotic expression vector pcDNA 3.1. In vitro and in vivo expression of these DNA vaccine constructs was examined in Epinephelus akaara spleen cells (EAGS) and immunized fish by Western blot and RT-PCR analysis, respectively. Three weeks after the second booster, immunized fish were challenged with SGIV and the level of protection and survival was assessed. Fish vaccinated with plasmid DNA encoding viral ORF072, ORF039 and ORF036 (designated as pcDNA-72, pcDNA-39 and pcDNA-36, respectively) exhibited 66.7%, 66.7% and 58.3% relative percent survival rates, respectively, in comparison with the control fish. These three DNA vaccines induced innate immune responses, raising significantly high level of Mx expression relative to the fish vaccinated with the empty plasmid at 3 days post-vaccination. Furthermore, recombinant protein from ORF072 was also used to immunize another set of fish and similar protective effect was obtained. Taken together, our results validated the applicability of bioinformatics in genome mining, resulting in the identification of three protective antigens. The promising results obtained in the present study have prompted further testing to improve the immunogenicity of these potential DNA vaccines.
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Cloning, characterization, and expression analysis of a thioredoxin from orange-spotted grouper (Epinephelus coioides).
Dev. Comp. Immunol.
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Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.
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Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
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Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
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Antiviral effects of ?-defensin derived from orange-spotted grouper (Epinephelus coioides).
Fish Shellfish Immunol.
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Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a ?-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1?. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.
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Cloning and characterization of a surface antigen CiSA-32.6 from Cryptocaryon irritans.
Exp. Parasitol.
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Cryptocaryon irritans is a ciliated parasite causing cryptocaryosis in marine fish. To isolate functional genes, a cDNA library of C. irritans trophonts was constructed and a gene designated CiSA-32.6 (GenBank ID: JF812643) was cloned and characterized. The full-length cDNA (1158 bp) encoded a deduced polypeptide of 330 amino-acid (aa) with a signal peptide of 22 aa. To express the ciliate gene, a truncated open reading frame (CiSA-32.6t) was synthesized to remove fragments encoding the signal peptide and hydrophobic C-terminal and to modify non-universal genetic codes. CiSA-32.6t was subcloned into Escherichia coli DH5? strain using the pGEX-4T-1 vector and then expressed as a glutathione S transferase fusion protein (rCiSA-32.6t). Western blotting analysis showed that sera from mice immunized with rCiSA-32.6t reacted specifically with a native protein (32.6 kDa) in parasite lysates. Moreover, rCiSA-32.6t reacted specifically with sera from mice immunized with a C. irritans trophont lysate. Expression of the CiSA-32.6 gene in C. irritans was detected at all developmental stages by reverse transcriptase PCR and Western blotting analysis. This study provides the basis of further investigations into the pathogenic biology of C. irritans and the control of cryptocaryosis.
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