Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

IMPORTANCE: The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

Transcriptome Sequencing Reveals Salmonella Flagellin Activation of Interferon-ß-Related Immune Responses in Macrophages.

The flagellin (FliC) of Salmonella typhimurium is a potential vaccine adjuvant as it can activate innate immunity and promote acquired immune responses. Macrophages are an important component of the innate immune system. The mechanism of flagellin's adjuvant activity has been shown to be related to its ability to activate macrophages. However, few studies have comprehensively investigated the effects of Salmonella flagellin in macrophages using transcriptome sequencing. In this study, RNA-Seq was used to analyze the expression patterns of RAW264.7 macrophages induced by FliC to identify novel transcriptomic signatures in macrophages. A total of 2204 differentially expressed genes were found in the FliC-treated group compared with the control. Gene ontology and KEGG pathway analyses identified the top significantly regulated functional classification and canonical pathways, which were mainly related to immune responses and regulation. Inflammatory cytokines (IL-6, IL-1ß, TNF-α, etc.) and chemokines (CXCL2, CXCL10, CCL2, etc.) were highly expressed in RAW264.7 cells following stimulation. Notably, flagellin significantly increased the expression of interferon (IFN)-ß. In addition, previously unidentified IFN regulatory factors (IRFs) and IFN-stimulated genes (ISGs) were also significantly upregulated. The results of RNA-Seq were verified, and furthermore, we demonstrated that flagellin increased the expression of IFN-ß and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-ß-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.

Investigation of the CEBPA gene expression pattern and association analysis of its polymorphisms with meat quality traits in chickens.

Meat quality is closely related to the fat deposition which is regulated by a cascade of transcription factors. As a transcription factor, the CCAAT/enhancer binding protein alpha (CEBPA) is considered as one of the key molecules regulating adipogenesis. Therefore, the objective of this study was to detect the expression pattern of the CEBPA gene and evaluate whether its single nucleotide polymorphisms (SNPs) were associated with the meat quality traits in Wuliang Mountain Black-bone (WLMB) chickens. The results showed that the chicken CEBPA mRNA was widely expressed in the 11 tissues, and the expression pattern of it might be tissue- and time-specific different. The locus of g.74C > G was not significantly associated with chicken meat quality. For the locus of g.552G > A, chickens with the GG genotype showed higher pH (p < 0.01), lower drip loss (p < 0.01) and higher intramuscular fat (p < 0.05) than those with other genotypes. It suggested that polymorphisms of the CEBPA gene were significantly associated with the meat quality traits of WLMB chickens. The results of this study contribute to the functional research of the CEBPA gene and lay the foundation for improving meat quality based on the marker-assisted selection in chickens.

Analysis of the physical meat quality in partridge (Alectoris chukar) and its relationship with intramuscular fat.

This study was undertaken to assess the effect of various levels of intramuscular fat (IMF: <0.5%, 0.5-0.99%, 1.0-1.49%, 1.5-1.99%, 2.0-2.49%, and >2.5%) on the physical meat quality of partridge. Physical characteristics such as moisture, pH, shear force, water-holding capacity (WHC), and color, along with IMF, were measured on 414 partridges (pectoral muscle). In this study, partridge meat was described as a kind of tender and moderately juicy meat with a nice color. Additionally, tenderness was significantly different between IMF < 0.5% and IMF > 0.5% levels (P < 0.01). IMF >0.5% also obtained higher value of WHC than IMF <0.5% although the difference was statistically nonsignificant. IMF levels did not affect pH or color. The results showed that IMF content was not significantly correlated with physical meat quality. However, in terms of eating experience, the minimum IMF level for better meat quality for consumer was 0.5%. Pearson correlation coefficients indicated that IMF content had significant negative correlations with moisture content but not with pH (r = -0.066), shear force (r = -0.072), WHC (r = 0.085), or color (L*(r = -0.049), a*(r = -0.028) and b*(r = 0.045)). Besides, meat pH had significant negative correlations with WHC (r = -0.036; P < 0.01) and lightness (L*) (r = -0.292; P < 0.01). Consequently, we consider pH to be one of the most important factors in evaluating meat quality of partridge.

Polymorphisms of pro-opiomelanocortin gene and the association with reproduction traits in chickens.

Pro-opiomelanocortin (POMC) is a member of prohormone family and has important functions in stress response, skin pigmentation, thermoregulation and reproduction. In this study, the single nucleotide polymorphisms (SNPs) of POMC gene exons were detected by direct sequencing in 317 Zhenning yellow chickens. The sequencing results indicated there were seven mutation sites (g.1140C > T, g.1185 T > C, g.2085 T > C, g.3566A > C, g.3572 G > A, g.3594 G > A and g.3628 G > A) and all of these were synonymous. Furthermore, seven haplotypes were formed and sixteen diplotypes were obtained. The associations between the POMC gene polymorphisms or diplotypes and reproduction traits were also analyzed. The association analysis results indicated that the SNP of g.1140C > T was associated with egg production at 300 d of age (E300), fertilization rate (FR), hatching rate of hatching eggs (HEHR) and hatching rate of fertilized eggs (FEHR; P < 0.05). The SNP of g.3566A>C was associated with FR (P < 0.05), SNP of g.3594G>A was associated with egg weight at 300d of age (EW300; P < 0.05), and SNP of g.3628G>A was associated with HEHR and FEHR (P < 0.01), respectively. Furthermore, chickens with H2H3 diplotype had greater EW300 and FR than those with H1H7 and H3H4 diplotypes (P < 0.05). These results indicate the expression of the POMC gene had significant genotype effects on the reproduction traits of Zhenning yellow chickens, and that the H2H3 diplotype could be used as a potential genetic marker to improve the reproduction traits in chicken breeding.

A New Flavonoid Glycoside from Lysionotus pauciflorus.

Ten flavonoids (1-10), including a new glycoside (nevadensin-7-sambubioside, 7), together with a phenylpropanoid glycoside (11) were isolated from Lysionotus pauciflorus. Their structures were elucidated by a combination of spectroscopic methods and comparing with literature data. Five compounds (1, 3, 4, 8, and 9) were obtained from the family Gesneriaceae for the first time. The new compound was evaluated in vitro for anticholinesterase activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but was found to be inactive.

Anticholinesterases and antioxidant alkamides from Piper nigrum fruits.

The anticholinesterase and antioxidant effects of five different extracts of Piper nigrum were evaluated. Twenty-one known alkamides were isolated from active ethyl acetate extract and investigated for their cholinesterase inhibitory and antioxidant effects. Among them, piperine (2), piperettine (5) and piperettyline (20) exhibited dual inhibition against AChE and BChE, and feruperine (18) was the most potent selective inhibitor of BChE. Molecular docking simulation was performed to get insight into the binding interactions of the ligands and enzymes. In addition, N-trans-feruloyltyramine (3) contributed to the strongest DPPH radical-scavenging activity. The self-induced Aß aggregation inhibition of 2, 5 and 18 was further evaluated. Results indicated that some alkamides could be multifunctional lead candidates for Alzheimer's disease therapy.

A new sesquiterpene glucoside from Lysionotus pauciflorus.

A new acorane sesquiterpene glucoside, 1R,3S,4R,5R,10R-3,10-dihydroxyacoronene-3-O-beta-D-gluc-oside (1), was isolated from the EtOAc-soluble partition of the ethanol extract of Lysionotus pauciflorus, together with six known compounds, namely p-hydroxybenzoic acid (2), vanillic acid (3). caffeic acid (4). beta-hydroxypropiosyringone (5), alpha,beta-dihydroxypropiosyringone (6), and lyoniresinol (7). The structure of the new compound was elucidated on the basis of spectroscopic methods, including 1D and 2D NMR, and high-resolution MS analysis. The absolute configuration of 1 was determined from CD spectra. When evaluated against several bacterial and fungal strains, and human cancer cell lines, compound 1 and its aglycone were inactive.

Phenanthrenes from Juncus effusus.

A chemical investigation of the EtOAc-soluble fraction from the ethanol extract of the medullae of Juncus effusus led to the isolation of three new 9,10-dihydrophenanthrenes, juncuenins E-G (1-3); two new phenanthrenes, dehydrojuncuenins D-E (4-5); one new feruloylated glycoside (6); and one known 9,10-dihydrophenanthrene (7). The structures of these compounds were determined by analyzing their spectroscopic data. Metabolites 1-4 and 7 were further evaluated for their in vitro cytotoxic activities against seven human cancer lines (A549, MCF-7, BEL-7402, HeLa, COLO205, BGC-823, and SK-OV-3). Among them, compound 1 exhibited weak cytotoxicity against MCF-7 and HeLa cell lines. Compound 7 showed moderate cytotoxicity against MCF-7 and HeLa cell lines, with IC50 values of 9.17 and 19.6 µM, respectively.