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.

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.

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.

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.

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.

Functional characterization of BmOVOs in silkworm, Bombyx mori.

BACKGROUND: In our previous study, we identified four isoforms of the Bmovo gene, Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 from the silkworm ovary and verified that ovarian development was regulated by the BmOVO proteins. RESULTS: To understand the regulatory mechanisms of ovarian development, the regulation of four BmOVO isoforms on the B. mori ovarian tumor (Bmotu) promoter activity was investigated with luciferase reporter assays. The results showed the Bmotu promoter activity was positively regulated by BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4 in a dose-dependent manner, of which BmOVO-2 had the highest transcriptional activation. However, the first (A1) and third acidic domains (A3) at the N-terminus of BmOVO-1 are transcriptional repression domains, while the fourth (A4) and fifth acidic domains (A5) are transcriptional activation domains. A recombinant BmOVO zinc-finger domain was found to bind to the GTACCGTTGTA sequence located at the Bmotu promoter. Furthermore, the Bmotu promoter activity was negatively regulated by 'Tal-like' peptide, which can trigger BmOVO-1 degradation at the N-terminus. CONCLUSIONS: These results will help us to further understand the regulatory mechanisms of BmOVO isoforms on Bmotu promoter activity and ovarian development in the silkworm.

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.

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.

Bombyx mori bidensovirus infection alters the intestinal microflora of fifth instar silkworm (Bombyx mori) larvae.

Bacterial diseases can occur as a result of disruption of the intestinal microbial population in the silkworm, Bombyx mori, and are often induced by bidensovirus (BmBDV) infection. We investigated the effects of BmBDV infection on intestinal microbes and immune gene responses in fifth instar silkworm larvae. Midgut contents were collected from BmBDV-infected and uninfected silkworms at 48, 96, and 144 h post-infection (hpi) and the intestinal flora were analyzed using 16S rRNA gene Illumina MiSeq sequencing technology. The abundance of intestinal bacteria differed between BmBDV-infected and uninfected silkworms. There were no significant differences in bacterial diversity at 48 and 96 hpi, but bacterial diversity in infected larvae was lower at 144 hpi compared with that of uninfected larvae. At the phylum level, the ratio of Proteobacteria was higher in infected larvae than in uninfected larvae at 48 and 96 hpi, but was lower after 144 hpi, while the ratio of Firmicutes had increased relative to uninfected silkworms. At the genus level, the ratio of Enterococcus increased gradually in infected silkworms, however, proportion of bacteria genera Incertae sedis were increased at 96 hpi, and the proportion of Lactococcus had decreased at 96 hpi. Principal component analysis showed that the proportion of Enterococcus species present was negatively correlated with most dominant genera. Increases in the abundances of the genera Anderseniella, Simplicispira, Enterococcus and, genera Incertae sedis, were associated with BmBDV infection. Quantitative reverse transcription-polymerase chain reaction indicated that expression levels of genes associated with immune deficiency (IMD), Toll, and JAK/STAT pathways were higher at 144 hpi with BmBDV infection. Enterococcus abundance was higher and was positively correlated with the expression level of spatzle-1, peptidoglycan recognition protein LE, and peptidoglycan recognition protein LB genes, suggesting that an increase in the abundance of Enterococcus leads to activation of the Toll and IMD immune pathways.

Identification and characterization of circular RNAs in the silkworm midgut following Bombyx mori cytoplasmic polyhedrosis virus infection.

The pathogenesis of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is unclear, although accumulating evidence indicates that circular RNAs (circRNAs), which act as competing endogenous RNAs or positive regulators, play important roles in regulating gene expression in eukaryotes and, thus, may play a role in BmCPV infections. To explore the expression and biological functions of circRNAs in the silkworm midgut following BmCPV infection, silkworm circRNA expression profiles of normal midgut tissue (control) and BmCPV-infected midgut tissue (test) were determined using high-through sequencing. A total of 9,753 and 7,475circRNAs were detected from the control and test samples, respectively. The two samples shared 6,085 circRNAs, while 646 and 737 circRNAs were expressed specifically in the control and test samples, respectively. A total of 3,638 circRNAs were shown to be differentially expressed, and 400 circRNAs were substantially differentially expressed with a fold-change ≥ 2.0 (p< 0.05 and a false discover rate < 0.05), of which 294 were up-regulated and 106 were down-regulated following infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to determine the principal functions of the substantially differentially regulated genes. circRNA-miRNA interaction networks were constructed based on a correlation analysis between the differentially expressed circRNAs and the nature of their microRNA (miRNA) binding sites. The network inferred that 13 miRNAs interacting with 193 circRNAs were among the 300 most abundant relationships. bmo-miR-3389-5p, bmo-miR-745-3p, and bmo-miR-3262 were related to 30, 34, and 34 circRNAs, respectively. circRNA_8115, circRNA_9444, circRNA_4553, circRNA_0827, and circRNA_6649 contained six, five, four, four, and four miRNA binding sites, respectively. We further found that alternative circularization of circRNAs is a common feature in silkworms and that the junction sites of many silkworm circRNAs are flanked by canonical GT/AG splicing signals. Our study is the first to show the circRNA response to virus infection. Thus, it provides a novel perspective on circRNA-miRNA interactions during BmCPV pathogenesis, and it lays the foundation for future research of the potential roles of circRNAs in BmCPV pathogenesis.

Functions and impact of tal-like genes in animals with regard to applied aspects.

A large number of DNAs in eukaryote genomes can code for atypical transcripts, and their functions are controversial. It has been reported that the transcripts contain many small open reading frames (sORFs), which were originally considered as non-translatable RNAs. However, increasing evidence has suggested that some of these sORFs can encode for small peptides and some are conserved across large evolutionary distances. It has been reported that the small peptides have functions and may be involved in varieties of cellular processes, playing important roles in development, physiology, and metabolism. Among the sORFs, studies of the non-canonical gene polished rice/tarsal-less (pri/tal) in Drosophila and mille-pattes(mlpt) in Tribolium have been more thoroughly studied. The genes similar to pri/tal in other species have been defined as the tarsal-less-related gene family, tal-like gene. In this review, we described recent progress in the discovery and functional characterization of the small peptides encoded by the tal-like gene and their possible functional potentials.

Exogenous gene can be expressed by a recombinant Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitro-transcribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.

Identification of tarsal-less peptides from the silkworm Bombyx mori.

The polycistronic and non-canonical gene tarsal-less (tal, known as pri) was reported to be required for embryonic and imaginal development in Drosophila; however, there are few reports of the tal gene in the silkworm Bombyx mori. Here, we cloned a tal-like (Bmtal) gene, and a sequence analysis showed that the Bmtal cDNA (1661 bp) contains five small open reading frames (smORFs) (A1, A2, A3, A4, and B) that encode short peptides of 11-12 (A1-A4) amino acid residues containing an LDPTG(E)L(Q)(V)Y motif that is conserved in Drosophila Tal, as well as a 32-amino-acid B peptide. Reverse transcription-quantitative polymerase chain reaction showed that the expression of the Bmtal gene was highest in the trachea, followed by the silk gland and Malpighian tubule, in day 3 fifth-instar larvae. Subcellular localization showed that BmTal localized in the nucleus. By regulating the expression of the full-length Bmtal gene and the functional smORFs of Bmtal, we showed that the expression levels of the Bmovo gene and genes related to the Notch, transforming growth factor-ß, and Hippo signaling pathways changed with changes in BmTal peptide expression. A co-immunoprecipitation assay showed that BmTal interacts with polyubiquitin, which triggered degradation and/or processing of the 14-3-3 protein zeta. A comparative transcriptome analysis showed that 2843 (2045) genes were up- (down)-regulated after Bmtal gene expression was up-regulated. The up- (down)-regulated differentially expressed genes were enriched in 326 (278) gene ontology terms (P ≤ 0.05) and 54 (59) Kyoto Encyclopedia of Genes and Genomes pathways (P ≤ 0.05), and the results indicated that the BmTal peptides could function as mediators of hormone levels or the activities of multiple pathways, including the peroxisome proliferator-activated receptor, Hedgehog, mitogen-activated protein kinase, adipocytokine, and gonadotropin-releasing hormone signaling pathways, as well as the innate immune response. These results increase our understanding of the function and mechanism of BmTal at the genome-wide level.

Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-ß-cyclodextrin (MßCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

Tunable AIEE fluorescence constructed from a triphenylamine luminogen containing quinoline - application in a reversible and tunable pH sensor.

Herein, tunable emissions in aggregation processes of triphenylamine derivatives (TPAQs) and their protonated cations, as well as protonated processes have been described. In this study, three triphenylamine-based compounds (TPAQs) were synthesized and their optical properties were investigated. Initially, the TPAQs displayed aggregation-induced emission enhancement (AIEE) properties via the restricted intramolecular charge transfer (ICT) state. Interestingly, the single-branched fluorophore (STPAQ) and its protonated cation emitted different color fluorescence in the solution and aggregation state. They emitted green fluorescence, which originated from the intramolecular charge transfer (ICT) state in a strong polar solvent, but the fluorescence bands turned blue, which was attributed to the LE state in the aggregated state. However, the cations of triple-branched fluorophores (TTPAQs) exhibited an inverse tunable emission process from bluish violet fluorescence of the LE state in a weak polar solvent (e.g., THF) to green fluorescence of the ICT state in the aggregated state. In a THF/water mixture solution (fw = 10%), the STPAQ could switch its emission between blue and green in the pH range of 10.0-0.5. This phenomenon enabled STPAQ to serve as a fluorescent pH sensor in solution. In the powder state, double-branched fluorophores (DTPAQs) could be used as a fluorescent sensor for the detection of acidic and basic organic vapors in the solid state.

Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

Receptor-mediated endocytosis using a ß1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

Effect of BBX-B8 overexpression on development, body weight, silk protein synthesis and egg diapause of Bombyx mori.

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5-3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56-14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.

Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp.

Grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in China. This study was aimed to develop a novel oral vaccine against grass carp reovirus (GCRV). GCRV vp6 and vp7 genes with ß-actin promoter of Megalobrama amblycephala and polyhedrin promoter (Ph10) of baculovirus, respectively, were cloned into plasmid pFast™-Dual to construct a vector pFast-PHVP7-AVP6, which was used to generate a recombinant baculovirus BacFish-vp6/vp7 via Bac-to-Bac system. The VP7 expression was analyzed from freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae by western blotting, and VP6 expression was analyzed from orally vaccinated fish with the freeze-dried powder by RT-PCR. The VP6 expression was also analyzed from both CIK cells transduced with BacFish-vp6/vp7 and tissues of vaccinated fish by immunofluorescence analysis. Recombinant VP7 could be detected from the BacFish-vp6/vp7-infected silkworm pupae. Pathological changes were not observed in CIK cells transduced with BacFish-vp6/vp7, and VP6 expression was found in CIK cells. When the grass carps were orally administrated with the freeze-dried powder, vp6 gene transcription was found in blood of the vaccinated fishes and VP6 protein was observed in liver and kidney of the vaccinated fish by immunofluorescence analysis. These results indicated that vp7 gene was expressed in the BacFish-vp6/vp7-infected silkworm and vp6 gene was expressed in orally vaccinated fish with freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae, suggesting the possibility to use the powder as an orally administrated vaccine.

Aggregation-induced emission and intermolecular charge transfer effect in triphenylamine fluorophores containing diphenylhydrazone structures.

Three new chromophores incorporating acceptor-π-donor-π-acceptor structural motifs and mono-, di- and tri-branched diphenylsulfone base linked to triphenylamine through a hydrazone π-bridge were synthesized, and the photoluminescence properties of the three chromophores were studied in solutions as well as in aggregated states. All the fluorophores emitted strong blue fluorescence in THF. Mono- and di-branched triphenylamine both exhibited increasing blue fluorescence and displayed an AIEE effect in the aggregated state. Tri-branched triphenylamine emitted green fluorescence and presented the AIE effect in the aggregated state. These interesting phenomena have been interpreted by a molecular stacking mode with molecular dynamics (MD) and DFT calculations. The unique propeller shaped molecular configuration of triphenylamine prevented face to face π-π stacking and induced the hindered rotation, which resulted in the AIEE or AIE effect in the aggregated state. The enlarged coplanarity of diphenylhydrazone chains increased the conjugation of tri-branched triphenylamine, which was beneficial to the formation of ICT and AIE and resulted in emitting green ICT fluorescence in the aggregated state. Fluorescent microscope imaging and the fluorescent pictures of the powder states certified the strong AIEE effect or AIE effect in the solid.