Search

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. A common and disabling disease of the elderly, the standard dopamine replacement therapies do not arrest the ongoing neurodegeneration, thus calling for new treatment strategies. The present study aimed to clarify the functional relevance of the hypoxia inducible factor-1α (HIF-1α)/microRNA-128-3p (miR-128-3p) axis in hippocampal neurodegeneration in a PD mouse model obtained by intraperitoneal injection of MPTP. Targeting relationship between miR-128-3p and Axin1 was verified, so we probed the roles of Hif1a, miR-128-3p, and Axin1 in apoptosis of hippocampal neurons with gain- and loss-of function experiments using flow cytometry and TUNEL staining. We found that Axin1 was upregulated in hippocampal tissues and cells of the MPTP-lesioned mouse model of PD, while Hif1a and miR-128-3p were downregulated. Elevation of HIF-1α/miR-128-3p inhibited apoptosis of hippocampal neurons via Wnt/β-catenin signaling pathway activation due to the suppression of Axin1 in PD. In addition, forced overexpression of Hif1a could ameliorate motor dysfunction and pathological changes in the model. Collectively, activation of the HIF-1α/miR-128-3p axis could repress hippocampal neurodegeneration in MPTP-lesioned mice through an activated Wnt/β-catenin pathway due to Axin1 downregulation.

Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M protein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cyprini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the virus-infected EPC cells, the virions were mainly located in the cytoplasm, whereas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heart were higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of β-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for β-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-β-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between β-lactoglobulin and phenolic acids.

Red-spotted grouper nervous necrosis virus (RGNNV) is a highly infectious, explosive, and lethal virus that mainly infects marine fish larvae. Currently, there is no effective method to mitigate the impact of RGNNV on aquaculture production. Previous studies have shown that yolk immunoglobulin (IgY) can neutralize RGNNV in grouper fin-1 (GF-1) cells. In this study, RGNNV propagated in GF-1 cells was harvested and purified successfully. The purified RGNNV was used as an immunogen to immunize laying hens. Water-soluble fractions were separated from egg yolk, and IgY was harvested using a two-step precipitation method with saturated ammonium sulfate salt. In vitro analysis showed that the purified IgY could neutralize RGNNV. An in vivo protection assay with groupers revealed that fish fed with 10% lyophilized egg yolk powder from immunized hens were significantly protected during RGNNV challenge. The relative protection rates of groupers under treatment after RGNNV infection and continuous feeding during RGNNV infection were 68% and 70%, respectively, when fed with 10% specific egg yolk powder. This is the first study to validate that anti-RGNNV IgY can effectively protect groupers from RGNNV infection, laying a foundation for the development of anti-RGNNV products.

The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3e of Japanese flounder (Paralichthys olivaceus) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3e polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3e of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3+ T lymphocytes could be identified specifically by the mouse anti-rCD3e polyclonal antibodies, which didn't cross-react with the sIgM+ lymphocytes. Immunohistochemistry showed that CD3+ T lymphocytes could be detected in gill, skin, stomach, intestine, spleen, liver, head-kidney and mid-kidney. Flow cytometric analysis showed the percentages of CD3+ T lymphocytes in the PBL, spleen lymphocytes (SL) and head-kidney lymphocytes (HKL) of Japanese flounder increased rapidly after immunization with formalin-inactivated Edwardsiella tarda, and reached their peak levels at 5th day with 12.6%, 9.7% and 8.7%, respectively, and then decreased gradually. These results suggested that CD3+ T lymphocytes play important roles in mucosal and cell-mediated immunity, and the results would deepen our understanding on the roles of teleost T lymphocytes in the immune response.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. A common and disabling disease of the elderly, the standard dopamine replacement therapies do not arrest the ongoing neurodegeneration, thus calling for new treatment strategies. The present study aimed to clarify the functional relevance of the hypoxia inducible factor-1α (HIF-1α)/microRNA-128-3p (miR-128-3p) axis in hippocampal neurodegeneration in a PD mouse model obtained by intraperitoneal injection of MPTP. Targeting relationship between miR-128-3p and Axin1 was verified, so we probed the roles of Hif1a, miR-128-3p, and Axin1 in apoptosis of hippocampal neurons with gain- and loss-of function experiments using flow cytometry and TUNEL staining. We found that Axin1 was upregulated in hippocampal tissues and cells of the MPTP-lesioned mouse model of PD, while Hif1a and miR-128-3p were downregulated. Elevation of HIF-1α/miR-128-3p inhibited apoptosis of hippocampal neurons via Wnt/β-catenin signaling pathway activation due to the suppression of Axin1 in PD. In addition, forced overexpression of Hif1a could ameliorate motor dysfunction and pathological changes in the model. Collectively, activation of the HIF-1α/miR-128-3p axis could repress hippocampal neurodegeneration in MPTP-lesioned mice through an activated Wnt/β-catenin pathway due to Axin1 downregulation.

Nervous necrosis virus (NNV) infects more than 120 fish species worldwide and causes high mortality and significant economic losses to the aquaculture industry. Among different genotypes of NNV, the red-grouper nervous necrosis virus (RGNNV) genotype is the most widely reported with the highest number of susceptible species. Interferon (IFN) is a powerful cytokine driving host to establish an antiviral response. RGNNV may have an elaborate strategy to overcome the host's IFN-based antiviral response. In this study, we demonstrated that the B2 protein of RGNNV blocked IFN-mediated antiviral response by inhibiting host transcription directed by RNA polymerase II (RNAP II) complex. We firstly found that the upregulation of IFNφ1 promoter activity stimulated by poly(I:C) was suppressed by RGNNV infection. By systematically screening the structural and nonstructural proteins of RGNNV, we identified B2 protein as the virus's most potent antihost protein, which inhibited the IFNφ1 promoter activities stimulated by MDA5, MAVS, TBK1, IRF3, and IRF7 and decrease their protein expression. Importantly, B2 also suppressed the constitutively active cytomegalovirus (CMV) promoter, which indicated that B2 might be a general inhibitor of gene transcription. Moreover, western blotting showed that B2 could decrease RNAP II expression and RNAP II phosphorylation. All these results showed RGNNV blocked host's IFN response by B2 protein directly dysregulating RNAP II expression. To the best of our knowledge, this is the first report about the mechanism of RGNNV for anti-host IFN response. These findings will provide a new insight for further research on the viral infection mechanism and effective strategies to control RGNNV.

With the sharp development of present aquaculture, outbreaks of viral diseases have become a major obstacle for the sustainable development of the aquaculture industry with massive economic losses. Viral encephalopathy and retinopathy (VER) represents one of the main viral threats to world aquaculture, which is caused by nervous necrosis virus (NNV). In the present study, a new RGNNV strain named RGNNV-HL was isolated from cultured adult hybrid Hulong grouper (Epinephelus fuscoguttatus(♀) × Epinephelus lanceolatus(♂)) in Guangdong province, China. Cell culture-based virus isolation, RT-PCR detection, tissue section, electron microscopy, and complete genome sequence were used for virus identification. Furthermore, an artificial infection experiment was conducted for virulence testing. Gross examination of affected fish showed no noticeable lesions, no parasites in the gills or on the body surfaces, and no pathogenic bacteria. Histopathological examination revealed severe vacuolations in the brain of diseased groupers. Inoculation of the tissue homogenates of diseased fish induced obvious cytopathic effects in SSN-1, GF-1, and GS cell cultures. Electron microscopic observation revealed the presence of small-sized icosahedral-shaped viral particles (~30 nm) with no viral envelope, similar to betanodaviruses. RT-PCR and sequencing analysis confirmed the presence of red-spotted grouper nervous necrosis virus (RGNNV) capsid protein sequences. The virus clustered with RGNNV genotype, genus Betanodavirus within the Nodaviridae family on an evolutionary tree constructed based on the complete genome sequence. Thus, this newly isolated virus from adult Hulong grouper is tentatively designated RGNNV-HL. The artificial infection experiment showed that groupers began to die at 4 and 5 days after intraperitoneal injection and bath immersion, respectively. The cumulative mortality rates by injection and bath were 90% and 83.33%, respectively. To the best of our acknowledge, this is the first report of the isolation and characterization of RGNNV from Hulong grouper. Findings from this study will provide a candidate material and useful epidemiology information for further study on the infection mechanism and prevention and control strategies for RGNNV.

In this study, 111 phenolic acids and their derivatives were chosen to investigate their structure-affinity relationships when binding to human serum albumin (HSA), and effects on their antioxidant activity. A comprehensive mathematical model was employed to calculate the binding constants, using a fluorescence quenching method, and this was corrected for the inner-filter effect to improve accuracy. We found that a hydroxy group at the 2-position of the benzene ring exerted a positive effect on the affinities, while a 4-hydroxy substituent had a negative influence. Both methylation of the hydroxy groups and replacing the hydroxy groups with methyl groups at the 3- and 4-positions of the benzene ring enhanced the binding affinities. Hydrophobic force and hydrogen bonding were binding forces for the phenolic acids, and their methyl esters, respectively. The antioxidant activity of the HSA-phenolic acid interaction compounds was higher than that of the phenolic acids alone.

The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3ε of Japanese flounder (Paralichthys olivaceus) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3ε polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3ε of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3