Novel methyltransferase G9a inhibitor induces ferroptosis in multiple myeloma through Nrf2/HO-1 pathway.

Multiple myeloma (MM) is a common malignant hematologic neoplasm, and the involvement of epigenetic modifications in its development and drug resistance has received widespread attention. Ferroptosis, a new ferroptosis-dependent programmed death mode, is closely associated with the development of MM. The novel methyltransferase inhibitor DCG066 has higher cell activity, but its mechanism of action in MM has not been clarified. Here, we found that DCG066 (5µM) inhibited the proliferation and induced ferroptosis in MM cells; the intracellular levels of ROS, iron, and MDA were significantly elevated, and the level of GSH was reduced after the treatment of DCG066; The protein expression levels of SLC7A11, GPX4, Nrf2 and HO-1 were significantly reduced, and these phenomena could be reversed by ferroptosis inhibitor Ferrostatin-1 (Fer-1) and Nrf2 activator Tert-butyl hydroquinone (TBHQ). Meanwhile, the protein expression levels of Keap1 was increased, and heat shock proteins (HSP70, HSP90 and HSPB1) were reduced after DCG066 treatment. In conclusion, this study confirmed that DCG066 inhibits MM proliferation and induces ferroptosis via the Nrf2/HO-1 pathway.

Drug-Loaded Biomimetic Carriers for Non-Hodgkin's Lymphoma Therapy: Advances and Perspective.

Chemotherapy remains the mainstay of treatment for the lymphoma patient population, despite its relatively poor therapeutic results, high toxicity, and low specificity. With the advancement of biotechnology, the significance of drug-loading biomimetic materials in the medical field has become increasingly evident, attracting extensive attention from the scientific community and the pharmaceutical industry. Given that they can cater to the particular requirements of lymphoma patients, drug-loading biomimetic materials have recently become a potent and promising delivery approach for various applications. This review mainly reviews the recent advancements in the treatment of tumors with biological drug carrier-loaded drugs, outlines the mechanisms of lymphoma development and the diverse treatment modalities currently available, and discusses the merits and limitations of biological drug carriers. What is more, the practical application of biocarriers in tumors is explored by providing examples, and the possibility of loading such organisms with antilymphoma drugs for the treatment of lymphoma is conceived.

A recombinant baculovirus vector vaccine (BacMCP) against the infectious spleen and kidney necrosis virus (ISKNV).

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-ß, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Circ-Udg Derived from Cyprinid Herpesvirus 2 Promotes Viral Replication.

Cyprinid herpesvirus 2 (CyHV-2) has caused great losses to the gibel carp (Carassius auratus gibelio) industry. Previous studies showed that certain DNA viruses can encode circular RNAs (circRNAs) to regulate virus infection, which provides new clues for the treatment of viral disease. Whether CyHV-2 can encode circRNAs is still unknown. Here, 10 CyHV-2-derived circRNAs were identified, and the function of circ-udg, a circRNA derived from the CyHV-2 uracil DNA glycosylase (udg) gene, was studied. Although the expression level of circ-udg was lower than that of the parental gene, udg, its expression level was elevated in tandem with the proliferation of CyHV-2 and udg. In vitro experiments confirmed that circ-udg could promote the proliferation of CyHV-2. Moreover, circ-udg could encode a truncated UDG protein consisting of 147-amino-acid residues (termed circ-udg-P147). Both UDG and circ-udg-P147 were found to promote CyHV-2 proliferation, but the promoting effect of circ-udg on CyHV-2 proliferation was attenuated after circ-udg lost the ability to encode circ-udg-P147. Also, circ-udg-P147 could not change the transcription level of the udg gene. Interestingly, the UDG protein level was increased by circ-udg-P147. These results deepen the understanding of the genetic information carried by the genome of CyHV-2 and provide a new target for the treatment of gibel carp bleeding disease caused by CyHV-2. IMPORTANCE The outbreak of C. auratus gibelio gill hemorrhagic disease caused by CyHV-2 brought great losses to the gibel carp industry. Therefore, exploring the interaction between CyHV-2 and host and the molecular mechanism of viral infection is of great significance in preventing and treating the gibel carp gill hemorrhagic disease. Although some progress has been made in the study of CyHV-2, the mechanism of interaction between CyHV-2 and crucian carp is still unclear. In this study, we found that CyHV-2 can encode circRNA to regulate virus replication. Our study provides novel information on CyHV-2 functional genomics, a reference for research into the circRNA of other viruses, and theoretical guidance for preventing and treating gibel carp bleeding disease.

Micropeptide vsp21 translated by Reovirus circular RNA 000048 attenuates viral replication.

To date, some DNA viruses and single-stranded RNA viruses have been found to generate circRNAs. However, the reports on circRNAs produced by double-stranded RNA viruses are very limited. In this study, Bombyx mori cypovirus (BmCPV), a typical double-stranded RNA virus belonging to the Reoviridae, was demonstrated to generate viral circRNAs (vcircRNAs) and a vcircRNA_000048 whose sequence corresponds with the region 164-1245 nt on the BmCPV genomic dsRNA S5 segment (GQ294468.1) was validated by PCR, Sanger sequencing, reverse transcription-rolling circle amplification, and Northern blotting. Furthermore, we verified that vcircRNA_000048 translates a micropeptide vsp21 with 21 amino acid residues in an IRES-dependent manner, and vsp21 attenuates the viral replication. These findings provided a novel clue to understanding the regulation of viral multiplication and interaction of reovirus with the host.

ß-Arrestin 2 acts an adaptor protein that facilitates viral replication in silkworm.

ß-Arrestin 2 is known to be a widely distributed adaptor protein in mammals but its function has never been reported in Lepidoptera insects. Herein, the ß-Arrestin 2 (BmArrestin 2) gene from silkworm was cloned and characterized. The spatiotemporal expression level of BmArrestin 2 was highest in the gonads at the 3rd day of 5th instar, whereas the highest and lowest abundance of BmArrestin 2 were identified in the tracheal and testis, respectively. BmArrestin 2 is mainly distributed in the cytoplasm. Furthermore, in BmN cells，overexpression of BmArrestin 2 promoted Bombyx mori nucleopolyhedrovirus (BmNPV) and B. mori cytoplasmic polyhedrosis virus (BmCPV) replication as the increment of the concentration of plasmid transfection, whereas silencing the gene with specific siRNA inhibited viral replication. Replication of BmNPV and BmCPV also was weakened using BmArrestin 2 antiserum as the increment of the concentration. Immunofluorescent staining revealed the invasion of recombinant BmNPV or BmCPV was decreased after blocking endogenous BmArrestin 2. On the other hand, BmArrestin 2 co-localizes with recombinant BmNPV and BmCPV virions in BmN cells. These results suggest that BmArrestin 2 may represent a novel target for antiviral strategies, as it is an adaptor protein that plays a key role in virus replication.

BmCPV-Derived Circular DNA vcDNA-S7 Mediated by Bombyx mori Reverse Transcriptase (RT) Regulates BmCPV Infection.

Circular DNAs derived from single-stranded RNA viruses play important roles in counteracting viral infection. However, whether double-stranded RNA viruses generate functional circular DNAs is still unknown. Using circDNA sequencing, divergent PCR, DNA in situ hybridization and rolling circular amplification, we presently confirmed that in silkworm, Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA virus belonging to cypovirus, is prone to produce a BmCPV-derived circular DNA termed as vcDNA-S7. We have also found that vcDNA-S7 formation is mediated by endogenous reverse transcriptase (RT), and the proliferation of BmCPV can be inhibited by vcDNA-S7 in vitro and in vivo. Moreover, we have discovered that the silkworm RNAi immune pathway is activated by vcDNA-S7, while viral small interfering RNAs (vsiRNAs) derived from transcribed RNA by vcDNA-S7 can be detected by small RNA deep sequencing. These results suggest that BmCPV-derived vcDNA-S7, mediated by RT, can serve as a template for the biogenesis of antiviral siRNAs, which may lead to the repression of BmCPV infection. To our knowledge, this is the first demonstration that a circular DNA, produced by double stranded RNA viruses, is capable of regulating virus infection.

Bombyx mori cypovirus (BmCPV) induces PINK1-Parkin mediated mitophagy via interaction of VP4 with host Tom40.

The mechanism by which infection by Bombyx mori cytoplasmic nucleopolyhedrosis virus (BmCPV) causes autophagy has not been studied in detail. Herein we have observed by electron microscopy that infection with BmCPV causes autophagosome and mitochondrial structure damage in Bombyx mori midgut. In BmN cells infected with BmCPV and expressing eGFP-LC3, fluorescence spots and LC3-II levels increased, suggesting that BmCPV infection causes autophagy. Autophagy inducer rapamycin (Rap) and autophagy inhibitor 3-methyladenine (3-MA) were used to monitor the effects of mitophagy on BmCPV proliferation. It was found BmCPV proliferation to be promoted by mitophagy. Transient transfection experiments in cultured BmN cells showed that mitophagy can be triggered by expression of BmCPV structural protein VP4. Moreover, VP4 caused upregulation of p-Drp1, PINK1 and Parkin proteins in the mitophagy pathway and downregulation of mitochondrial membrane protein Tom20. Furthermore, interaction between VP4 with Tom40 was confirmed by Co-IP, western blot and colocalization experiment, and overexpression of Tom40 reduce the level of mitochondrial autophagy induced by VP4. These results suggested that VP4 induced PINK1-Parkin-mediated mitophagy interacting with Tom40. These findings deepen our understanding of the interaction between BmCPV and silkworm and also provide a molecular target for screening anti-BmCPV drugs.

Bombyx mori Akirin hijacks a viral peptide vSP27 encoded by BmCPV circRNA and activates the ROS-NF-κB pathway against viral infection.

Bombyx mori cypovirus (BmCPV), a member of the family Reoviridae, is a model of Cypovirus, has a 10 segmented double-stranded RNA genome. However, so far, only one viral small peptide vSP27 with negative regulation on viral infection was identified; the mechanisms underlying host-BmCPV interaction are still unknown. Here, we identified that vSP27 was translated from a BmCPV derived circular RNA (circRNA-vSP27). Subsequently, results showed that vSP27 induced generation of ROS activated the NF-κB signaling pathway, induced the expression of antimicrobial peptides, and suppressed BmCPV infection. On the other hand, we identified a nuclear protein Akirin that could hijack vSP27, positively regulate the NF-κB pathway, and lead to inhibiting the viral infection. Altogether, our data suggested that BmCPV derived circRNA-vSP27 with small peptide translation activity may be employed by the host immunity in defense against the BmCPV infection.

Hepatocellular carcinoma progression mediated by hepatitis B virus-encoded circRNA HBV_circ_1 through interaction with CDK1.

Hepatitis B virus (HBV) produces circular RNA (circRNA), whose functions have not yet been clearly elucidated. In this study, a novel circRNA HBV_circ_1 produced by HBV was identified in HBV-positive HepG2.2.15 cells and HBV-related hepatocellular carcinoma (HCC) tissue (HCCT). Microarray analysis of 68 HCCT samples showed that HBV_circ_1 abundance was significantly higher than that in paracancerous tissues. In addition, survival rate of HBV_circ_1-positive patients was significantly lower compared with HBV_circ_1-negative patients. Transient expression indicated that HBV_circ_1 enhanced cell proliferation, migration, and invasion and inhibited apoptosis in vitro. Furthermore, ectopical HBV_circ_1 expression increased tumor size in vivo. HBV_circ_1 was confirmed to interact with cyclin-dependent kinase 1 (CDK1) to regulate cell proliferation. These results suggest that HCC progression may be promoted by interaction of HBV_circ_1 with CDK1. Our data not only showed a novel clue to understand carcinogenesis and progress of HBV-related HCC but also provided a new target for the development of therapeutic drugs.

Tight junction protein claudin-2 promotes cell entry of Bombyx mori cypovirus.

Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: •Claudin-2 could promote BmCPV infection of cells. •Claudin-2 interacted with BmCPV during BmCPV infection. •Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.

Metatranscriptomic Analysis Reveals an Imbalance of Hepatopancreatic Flora of Chinese Mitten Crab Eriocheir sinensis with Hepatopancreatic Necrosis Disease.

Hepatopancreas necrosis disease (HPND) of the Chinese mitten crab Eriocheir sinensis causes huge economic loss in China. However, the pathogenic factors and pathogenesis are still a matter of dissension. To search for potential pathogens, the hepatopancreatic flora of diseased crabs with mild symptoms, diseased crabs with severe symptoms, and crabs without visible symptoms were investigated using metatranscriptomics sequencing. The prevalence of Absidia glauca and Candidatus Synechococcus spongiarum decreased, whereas the prevalence of Spiroplasma eriocheiris increased in the hepatopancreatic flora of crabs with HPND. Homologous sequences of 34 viral species and 4 Microsporidian species were found in the crab hepatopancreas without any significant differences between crabs with and without HPND. Moreover, DEGs in the hepatopancreatic flora between crabs with severe symptoms and without visible symptoms were enriched in the ribosome, retinol metabolism, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, biosynthesis of unsaturated fatty acids, and other glycan degradation. Moreover, the relative abundance of functions of DEDs in the hepatopancreatic flora changed with the pathogenesis process. These results suggested that imbalance of hepatopancreatic flora was associated with crab HPND. The identified DEGs were perhaps involved in the pathological mechanism of HPND; nonetheless, HPND did not occur due to virus or microsporidia infection.

Incidence of Carassius auratus Gibelio Gill Hemorrhagic Disease Caused by CyHV-2 Infection Can Be Reduced by Vaccination with Polyhedra Incorporating Antigens.

Encapsulation of antigens within protein microcrystals (polyhedra) is a promising approach for the stable delivery of vaccines. In this study, a vaccine was encapsulated into polyhedra against cyprinid herpesvirus II (CyHV-2). CyHV-2 typically infects gibel carp, Carassius auratus gibelio, causing gill hemorrhagic disease. The vaccine was constructed using a codon-optimized sequence, D4ORF, comprising the ORF72 (region 1-186 nt), ORF66 (region 993-1197 nt), ORF81 (region 603-783 nt), and ORF82 (region 85-186 nt) genes of CyHV-2. The H1-D4ORF and D4ORF-VP3 sequences were, respectively, obtained by fusing the H1-helix sequence (region 1-90 nt) ofBombyx mori cypovirus(BmCPV) polyhedrin to the 5' terminal end of D4ORF and by fusing a partial sequence (1-279 nt) of the BmCPV VP3 gene to the 3' terminal end of D4ORF. Furthermore, BmNPV-H1-D4ORF-polh and BmNPV-D4ORF-VP3-polh recombinant B. mori nucleopolyhedroviruses (BmNPVs), belonging to the family Baculoviridae, and co-expressing BmCPV polyhedrin and H1-D4ORF or D4ORF-VP3, were constructed. H1-D4ORF and D4ORF-VP3 fusion proteins were confirmed to be encapsulated into recombinant cytoplasmic polyhedra by Western blotting. Degradation of vaccine proteins was assessed by SDS-PAGE, and the results showed that the encapsulated vaccine proteins in polyhedra could be protected from degradation. Furthermore, when gibel carp were vaccinated with the purified polyhedra from BmNPV-H1-D4ORF-polh and BmNPV-D4ORF-VP3-polh via injection, the antibody titers in the serum of the vaccinated fish reached 1:6400-1:12,800 at 3 weeks post-vaccination. Therelative percentage of survival of immunized gibel carp reached 64.71% and 58.82%, respectively, following challenge with CyHV-2. These results suggest that incorporating vaccine protein into BmCPV polyhedra may be a novel approach for developing aquaculture microencapsulated vaccines.

Proteomic analysis of the exosomes secreted from Ctenopharyngodon idellus kidney cells infected with grass carp reovirus reveals their involvement in the cellular responses to viral infection.

Exosomes are small membrane-enclosed vesicles secreted by various types of cells. Exosomes not only participate in different physiological processes in cells, but also involve in the cellular responses to viral infection. Grass carp reovirus (GCRV) is a non-enveloped virus with segmented, double-stranded RNA genome. Nowadays, the exact role of exosomes in regulating the life cycle of GCRV infection is still unclear. In this study, the exosomes secreted from Ctenopharyngodon idellus kidney (CIK) cells infected or uninfected with GCRV were isolated, and the differential protein expression profiles were analyzed by proteomic technologies. A total of 1297 proteins were identified in the isolated exosomes. The differentially abundant proteins were further analyzed with functional categories, and numerous important pathways were regulated by exosomes in GCRV-infected CIK cells. These exosomal proteins were estimated to interact with the genes (proteins) of the top 10 most enriched signaling pathways. Furthermore, GW4869 exosome inhibitor suppressed the expression level of VP7 in GCRV-infected cells, suggesting that exosomes play a crucial role in the life cycle of GCRV infection. These findings could shed new lights on understanding the functional roles of exosomes in the cellular responses to GCRV infection.

AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models.

BACKGROUND: The protective efficacy of avian influenza virus (AIV) vaccines is unsatisfactory due to the presence of various serotypes generated by genetic reassortment. Thus, immunization with a polyantigen chimeric epitope vaccine may be an effective strategy for protecting poultry from infection with different AIV subtypes. METHODS: Baculovirus has recently emerged as a novel and attractive gene delivery vehicle for animal cells. In the present study, a recombinant baculovirus BmNPV-CMV/THB-P10/CTLT containing a fused codon-optimized sequence (CTLT) of T lymphocyte epitopes from H1HA, H9HA, and H7HA AIV subtypes, and another fused codon-optimized sequence (THB) of Th and B cell epitopes from H1HA, H9HA, and H7HA AIV subtypes, driven by a baculovirus P10 promoter and cytomegalovirus CMV promoter, respectively, was constructed. RESULTS: Western blotting and cellular immunofluorescence demonstrated that the CTLT (THB) can be expressed in rBac-CMV/THB-P10/CTLT-infected silkworm cells (mammalian HEK293T cells). Furthermore, the recombinant virus, rBac-CMV-THB-CTLT, was used to immunize both chickens and mice. CONCLUSIONS: The results of an indirect ELISA, immunohistochemistry, and T lymphocyte proliferation assay indicated that specific humoral and cellular responses were detected in both chicken and mice. These results suggest that rBac-CMV/THB-P10/CTLT can be developed as a potential vaccine against different AIV subtypes.

circEgg regulates histone H3K9me3 by sponging bmo-miR-3391-5p and encoding circEgg-P122 protein in the silkworm, Bombyx mori.

A large number of circular RNAs (circRNAs) have been found in different organisms; however, their function in the regulation of histone modification remains unknown. In this study, we found that the circRNA circEgg, cyclized by the 9th-13th exon of Bombyx mori histone-lysine N-methyltransferase eggless (BmEgg) gene, mainly distributes in the cytoplasm, its expression levels changed with silkworm developmental stages, and the linear transcript level of the BmEgg gene was decreased when circEgg was overexpressed. Moreover, circEgg was found to repress histone H3 lysine 9 methylation (H3K9me3), promote histone H3 lysine 9 acetylation (H3K9ac), and positively regulate histone deacetylase (HDAC) Rpd3 (BmHDAC Rpd3) gene expression by sponging the microRNA bmo-miR-3391-5p. Furthermore, circEgg encodes a circEgg-P122 protein which appears to inhibit H3K9me3. These results suggest that circEgg regulates histone modification by sponging bmo-miR-3391-5p and encoding circEgg-P122 protein. To our knowledge, this is the first report showing that a circRNA produced by BmEgg plays an important role in histone epigenetic modification.

Interleukin-17 suppresses grass carp reovirus infection in Ctenopharyngodon idellus kidney cells by activating NF-κB signaling.

The grass carp accounts for a large proportion of aquacultural production in China, but the hemorrhagic disease caused by grass carp reovirus (GCRV) infection often causes huge economic losses to the industry. Interleukin 17 (IL-17) is an important cytokine that plays a critical role in the inflammatory and immune responses. Although IL-17 family members have been extensively studied in mammals, our knowledge of the activity of IL-17 proteins in teleosts in response to viral infection is still limited. In this study, the role of IL-17 in GCRV infection and its mechanism were investigated. The expression levels of IL-17AF1, IL-17AF2, and IL-17AF3 in Ctenopharyngodon idella kidney (CIK) cells gradually increased from 6 h after infection with GCRV. The nuclear translocation of p65, which acts in the NF-κB signaling pathway, was also increased by GCRV infection. The overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 also promoted the nuclear translocation of p65 and the levels of phospho-IκBα in CIK cells, and reduced the expression of the viral structural protein VP7. An NF-κB signal inhibitor abolished the inhibition of GCRV infection by IL-17 proteins. These results suggested that the NF-κB signaling pathway was activated by the overexpression of IL-17 proteins, resulting in the inhibition of viral infection. In conclusion, in this study, we demonstrated that IL-17AF1, IL-17AF2, and IL-17AF3 acted as immune cytokines, exerting an antiviral effect by activating the NF-κB signaling pathway.

Alternative isoforms of BmYki have different transcriptional co-activator activity in the silkworm, Bombyx mori.

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.

Identification and characterization of novel type of RNAs, circRNAs in crucian carp Carassius auratus gibelio.

Circular RNAs (circRNAs) with regulatory potency activity was identified from varieties of species. Crucian carp (Carassius auratus gibelio) is one of the most freshwater aquaculture species in China. Every year, huge economic damage to the farming was caused by the virus and bacterial infection. Until now, there is any information about circRNA reported from the Crucian carp. In this study, the expression pattern of circRNA in Crucian carp was investigated with transcriptomic analysis. The results showed that only 37 circRNAs were identified from the Crucian carp, and these circRNAs biogenesis was formed with canonical GU-AG splicing mechanism with unevenly distributed on the chromosomes. Wherein, most of the circRNAs were derived from the sense overlapping strategy. Reverse transcript PCR and Sanger sequencing data indicated that these circRNAs were existed authenticity in Crucian carp. The bioinformatics analysis indicated that circRNAs identified from the Crucian carp with potential miRNA sponge regulate the expression level of mRNAs. GO annotation and KEGG pathway analysis of these circRNAs showed that more than 20% circRNAs were related with catalytic activity and binding in the category of molecular function, and these circRNAs were enriched in 9 signaling pathways, such as, Wnt signaling pathway, MAPK signaling pathway, Ubiquitin mediated proteolysis et al. 220 mRNAs would be regulated by the circRNAs via miRNAs mediation. These target mRNAs were further analyzed with functional annotation and KEGG analysis. GO annotation analysis showed that several genes were related with function of nucleotide binding, transcription regulatory activity. KEGG pathway analysis showed that 5 genes were enriched in the pathway of Endocytosis. The circRNA-miRNA-mRNA regulation network indicated that one miRNA can link one or more circRNA and one or more mRNA. Overall, these results will not only help us to further understand the novel RNA transcripts in Crucian carp, but also provide the novel clue to investigate the interaction between host and pathogens from this novel circRNA molecule.

Recombinant baculovirus BacCarassius-D4ORFs has potential as a live vector vaccine against CyHV-2.

Cyprinid herpesvirus II (CyHV-2) is highly contagious and pathogenic to Carassius auratus gibelio (gibel carp), causing enormous economic losses in aquaculture in Yancheng city, Jiangsu province, China; however, to date, there is no effective way to protect C. auratus gibelio from CyHV-2 infection. In this study, a recombinant baculovirus vector vaccine, BacCarassius-D4ORFs, containing a fused codon-optimized sequence D4ORFs comprising the ORF72 (region 1-186 nt), ORF66 (region 993-1197 nt), ORF81 (region 603-783 nt) and ORF82 (region 85-186 nt) genes of CyHV-2, driven by a Megalobrama amblycephala ß-actin promoter, was constructed. Then, qPCR, Western blotting and immunofluorescence assays showed that the fused gene D4ORFs was successfully delivered and expressed in fish cells or tissues by transduction with BacCarassius-D4ORFs. The fused gene D4ORFs could not be detected by PCR in the C. auratus gibelio injected with BacCarassius-D4ORFs after 7 weeks. Specific antibody against ORF72 could be detected in the serum of vaccinated C. auratus gibelio by injection with BacCarassius-D4ORFs. Furthermore, when C. auratus gibelio were vaccinated with BacCarassius-D4ORFs via the oral or injection route, followed by challenge with CyHV-2, the relative survival rate of immunized C. auratus gibelio reached 59.3% and 80.01%, respectively. These results suggested that BacCarassius-D4ORFs has the potential to be used as a vector-based vaccine for the prevention and treatment of disease caused by CyHV-2 infection.