Fish Autophagy Protein 5 Exerts Negative Regulation on Antiviral Immune Response Against Iridovirus and Nodavirus

Autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. However, little is known about the functions of fish autophagy-related genes (Atgs). In this study, we cloned and characterized Atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (Epinephelus coioides) (EcAtg5). EcAtg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (Lates calcarifer) and humans (Homo sapiens), respectively. The transcription level of EcAtg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (RGNNV). In cells infected with Singapore grouper iridovirus (SGIV), EcAtg5 expression declined during the early stage of infection and increased in the late stage. Fluorescence microscopy revealed that EcAtg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. Overexpression of EcAtg5 significantly increased the replication of RGNNV and SGIV at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. Knockdown of EcAtg5 decreased the replication of RGNNV and SGIV. Further studies showed that overexpression EcAtg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κB, IFN-sensitive response element, and IFNs. In addition, ectopic expression of EcAtg5 affected cell cycle progression by hindering the G1/S transition. Taken together, our results demonstrated that fish Atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral IFN responses, and affecting the cell cycle

Grouper RIP2 inhibits Singapore grouper iridovirus infection by modulating ASC-caspase-1 interaction

IntroductionReceptor interacting protein 2 (RIP2), serves as a vital sensor of cell stress, is able to respond to cell survival or inflammation, and is involved in antiviral pathways. However, studies on the property of RIP2 in viral infections in fish have not been reported.MethodsIn this paper, we cloned and characterized RIP2 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP2) and further discussed the relevance of EcRIP2 to EcASC, comparing the influences of EcRIP2 and EcASC on the modulation of inflammatory factors and the NF-κB activation to reveal the mechanism of EcRIP2 in fish DNA virus infection.ResultsEncoded a 602 amino acid protein, EcRIP2 contained two structural domains: S-TKc and CARD. Subcellular localization signified that EcRIP2 existed in cytoplasmic filaments and dot aggregation patterns. After SGIV infection, the EcRIP2 filaments aggregated into larger clusters near the nucleus. The infection of SGIV could notably up-regulate the transcription level of the EcRIP2 gene compared with lipopolysaccharide (LPS) and red grouper nerve necrosis virus (RGNNV). Overexpression of EcRIP2 impeded SGIV replication. The elevated expression levels of inflammatory cytokines induced by SGIV were remarkably hindered by EcRIP2 treatment in a concentration-dependent manner. In contrast, EcASC treatment could up-regulate SGIV-induced cytokine expression in the presence of EcCaspase-1. Enhancing amounts of EcRIP2 could overcome the down regulatory effect of EcASC on NF-κB. Nevertheless, increasing doses of EcASC failed to restrain the NF-κB activation in the existence of EcRIP2. Subsequently, it was validated by a co-immunoprecipitation assay that EcRIP2 dose-dependently competed with EcASC binding to EcCaspase-1. With increasing time to SGIV infection, EcCaspase-1 gradually combined with more EcRIP2 than EcASC.DiscussionCollectively, this paper highlighted that EcRIP2 may impede SGIV-induced hyperinflammation by competing with EcASC for binding EcCaspase-1, thereby suppressing viral replication of SGIV. Our work supplies novel viewpoints into the modulatory mechanism of RIP2-associated pathway and offers a novel view of RIP2-mediated fish diseases

Hybrid Phosphate-Alumina Iron-Based Core-Shell Soft Magnetic Composites Fabricated by Sol-Gel Method and Ball Milling Method

Novel Fe-based soft magnetic composites (SMCs) with hybrid phosphate-alumina layers were prepared by both sol–gel and ball-milling methods. The effects of the fabrication methods and the addition of Al2O3 particles on the microstructure and the soft magnetic performance of SMCs were studied. The formation of the hybrid phosphate-Al2O3 shell not only leads to the decrease of the total core loss, but also results in the reduction of the permeability and saturation magnetization. However, the degree of decrease caused by the different methods were not identical. The sample with 8% Al2O3 prepared by the sol–gel method showed the best magnetic performance, exhibiting a high-amplitude permeability (μa) of 85.14 and a low total core loss (Ps) of 202.3 W/kg at 50 mT and 100 kHz. The hysteresis loss factor and the eddy current loss factor were obtained by loss separation. The results showed that the samples with the same Al2O3 content prepared by different methods exhibited almost the same total core loss. However, the contribution of the hysteresis loss and the eddy current loss showed an obvious difference in behavior because of the change of the particle shapes and the refinement of the particle size during the ball-milling process

Effects of compaction and heat treatment on the soft magnetic properties of iron-based soft magnetic composites

Iron-based soft magnetic composites (SMCs) are promising substitutes for laminate steels in electromagnetic applications due to their excellent magnetic properties and productivity. However, the preparation process is a key factor in deciding the magnetic performance of SMCs. In this work, the Fe-based soft magnetic composites with improved soft magnetic properties were achieved by optimizing the compaction and the annealing process. Results showed that the core–shell structure of powders which would directly have an impact on the permeability and the core loss of the SMCs could be affected by the compaction and the annealing process. In addition, the magnetic properties were enhanced by tuning the microstructure. As a result, the optimal magnetic performance of the compact with high permeability and low total core loss was obtained. The real part of the permeability of the soft magnetic composites could reach a maximal value of 336.8 and a rather low core loss of 2.5 W Kg ^−1 (measured at 50 mT and 5 kHz). Therefore, soft magnetic composites with enhanced magnetic properties were obtained by optimizing the powder metallurgy (PM) process in this study

Effect of Surface Densification on the Microstructure and Mechanical Properties of Powder Metallurgical Gears by Using a Surface Rolling Process

Powder metallurgy (PM) components are widely used in the auto industry due to the advantage of net-shape forming, low cost, and high efficiency. Still, usage of PM components is limited in the auto industry when encountering rigorous situations, like heavy load, due to lower strength, hardness, wear resistance, and other properties compared to wrought components due to the existence of massive pores in the PM components. In this study, through combining the powder metallurgy process and rolling process, the pores in the PM components were decreased and a homogenous densified layer was formed on the surface, which resulted in the enhancement of the strength, hardness, wear resistance, and other properties, which can expand its range of application. In this paper, we study the impact of different rolling feeds on the performance of the components’ surfaces. We found that with the increase of the rolling feed, the depth of the densified layer increased

Modulation of Growth Duration, Grain Yield and Nitrogen Recovery Efficiency by EMS Mutagenesis under <i>OsNRT2.3b</i> Overexpression Background in Rice

Growth duration is an important agronomic trait that determines the season and area of crop growth. Previous experiments showed that overexpression of nitrate transporter OsNRT2.3b significantly increased rice yield, nitrogen use efficiency, and growth duration. Through screening, we obtained four ethyl methanesulfonate (EMS)-mutagenized mutants with shorter growth duration compared with O8 of OsNRT2.3b overexpression line. The nitrogen translocation efficiency and physiological nitrogen use efficiency of the mutants were not significantly different from O8, which were increased by 24.4% and 14.2%, respectively compared with WT, but the growth duration of the mutant was significantly lower than O8. Analysis of O8 and mutants showed that the growth duration positively correlated with grain weight per panicle, grain yield, and nitrogen recovery efficiency. In conclusion, our results provide a new idea for balancing rice yield and growth duration

The Effect of Rolling Temperature on the Microstructure and Mechanical Properties of Surface-Densified Powder Metallurgy Fe-Based Gears Prepared by the Surface Rolling Process

In this investigation, the surface-rolling process was performed to improve the performance of PM (powder metallurgy) parts. Different rolling temperatures were applied and the effect of rolling temperature on the microstructure and mechanical properties of the surface dense layers in the samples were investigated. In the study, room temperature and temperatures of 100 °C, 200 °C, 300 °C were studied during the rolling process. The results confirmed that the sample prepared with a pre-heated temperature of 200 °C had the lowest porosity at the surface area. It also exhibited the highest surface hardness and wear resistance. The optimum rolling temperature was determined to be 200 °C and the related mechanism was discussed

Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection

Isolation and Characterization of a Newly Discovered Phage, V-YDF132, for Lysing Vibrio&nbsp;harveyi

A newly discovered lytic bacteriophage, V-YDF132, which efficiently infects the pathogenic strain of Vibrio harveyi, was isolated from aquaculture water collected in Yangjiang, China. Electron microscopy studies revealed that V-YDF132 belonged to the Siphoviridae family, with an icosahedral head and a long noncontractile tail. The phage has a latent period of 25 min and a burst size of 298 pfu/infected bacterium. V-YDF132 was stable from 37 to 50 &deg;C. It has a wide range of stability (pH 5&ndash;11) and can resist adverse external environments. In addition, in vitro the phage V-YDF132 has a strong lytic effect on the host. Genome sequencing results revealed that V-YDF132 has a DNA genome of 84,375 bp with a GC content of 46.97%. In total, 115 putative open reading frames (ORFs) were predicted in the phage V-YDF132 genome. Meanwhile, the phage genome does not contain any known bacterial virulence genes or antimicrobial resistance genes. Comparison of the genomic features of the phage V-YDF132 and phylogenetic analysis revealed that V-YDF132 is a newly discovered Vibrio phage. Multiple genome comparisons and comparative genomics showed that V-YDF132 is in the same genus as Vibrio phages vB_VpS_PG28 (MT735630.2) and VH2_2019 (MN794238.1). Overall, the results indicate that V-YDF132 is potentially applicable for biological control of vibriosis