Objective To investigate the effects of cucurbitacin B (CucB) on alleviating skin lesions and inflammation in psoriasis mice via the cGAS-STING signaling pathway. Methods The expression of genes associated with the cGAS-STING signaling pathway in psoriatic lesions and non-lesional skin was analyzed, and hallmark gene set enrichment analysis was performed. The cytotoxicity of CucB on BMDMs was evaluated using the CCK-8 assay. The expression levels of genes and proteins related to the cGAS-STING signaling pathway, along with the secretion of inflammatory cytokines, were measured at different concentrations of CucB using quantitative PCR, Western blotting, and ELISA. Imiquimod-induced psoriasis BALB/c mice were divided into four groups: normal group, model group, low-dose CucB group [0.1 mg/ (kg.d)], and high-dose CucB group [0.4 mg/ (kg.d)], with five mice per group. PASI scoring was performed to assess the severity of psoriasis after 6 days of treatment, and HE staining was conducted to observe pathological damage. Meanwhile, the mRNA levels of inflammatory cytokines and their secretion were detected by qPCR and ELISA. Results Most cGAS-STING signaling-related genes were upregulated in lesional skin of psoriasis patients, and the hallmark gene set enrichment analysis revealed that the most significantly upregulated genes were primarily associated with immune response signaling pathways. CucB inhibited dsDNA-induced phosphorylation of interferon regulatory factor 3 (IRF3) and STING proteins in both bone-marrow derived macrophages(BMDMs) and THP-1 cells. CucB also suppressed dsDNA-induced mRNA expression of IFNB1, TNF, IFIT1, CXCL10, ISG15, and reduced the secretion of cytokines such as IFN-β, IL-1β, and TNF-α in THP-1 cells. In the imiquimod-induced psoriasis mouse model, CucB treatment reduced psoriatic symptoms, alleviated skin lesions, and attenuated inflammation. ELISA and qPCR results showed that CucB significantly reduced serum secretion levels of IL-6, TNF-α, and IL-1β, as well as the mRNA levels of IL23A, IL1B, IL6, TNF, and IFNB1. Conclusion CucB inhibits cytoplasmic DNA-induced activationc of the GAS-STING pathway. CucB significantly attenuates skin lesions and inflammation in IMQ-induced psoriatic mice, and the potential molecular mechanism may be related to the down-regulation of the cGAS-STING pathway.
Animals ; Psoriasis/pathology* ; Signal Transduction/drug effects* ; Membrane Proteins/genetics* ; Mice ; Nucleotidyltransferases/genetics* ; Disease Models, Animal ; Mice, Inbred BALB C ; Skin/metabolism* ; Triterpenes/therapeutic use* ; Humans ; Cytokines/metabolism* ; Inflammation/drug therapy* ; Male

OBJECTIVES: This study aims to explore the potential relationships of cell-free DNA (cfDNA) in gingival crevicular fluid (GCF) with periodontal clinical indicators and the expression of DNA receptor pathway cyclic guanosine phosphate-adenosine phosphate synthase (cGAS)-stimulator of interferon genes (STING) in gingival tissues and human gingival fibroblasts (HGFs). METHODS: GCF and gingival tissue samples were collected from periodontally healthy individuals and patients diagnosed with periodontitis. Periodontal clinical indicators were recorded, including plaque index (PLT), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). The concentration of cfDNA in GCF was quantified, and the correlation between GCF and periodontal clinical indicators was analyzed. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the distribution of cGAS, STING, and p-STING in gingival tissues. Additionally, the mRNA expression levels of the key components of the cGAS-STING signaling pathway, namely, cGAS, STING, inhibitory of kappa-B kinase (IKK), nuclear factor kappa-B p65 (NF-κB p65), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured. Furthermore, cfDNA extracted from GCF was employed to stimulate HGFs in the healthy control and periodontitis groups, and the mRNA expression levels of the key molecules of cGAS-STING signaling pathway were detected through Western blot and RT-qPCR. RESULTS: The concentration of cfDNA in GCF was found to be significantly elevated in the periodontitis group compared with the control group. Moreover, cfDNA concentration demonstrated a strong positive correlation with the periodontal clinical indicators. Immunofluorescence analysis revealed considerably increased percentage of fluorescence co-localization of cGAS, STING, and p-STING with the gingival fibroblast FSP-1 marker in the gingival tissues of the periodontitis group. The mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6,and TNF-α were significantly higher in the periodontitis group. In vitro stimulation of HGFs with GCF-derived cfDNA resulted in increased protein expression of cGAS and p-STING and considerably upregulated the mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6, and TNF-α in the healthy and periodontitis groups compared with the blank group. Correlation analysis showed that the concentration of cfDNA at the sampling site was positively correlated with the mRNA expression levels of cGAS, STING, NF-κB p65, and IL-6 in gingival tissues. CONCLUSIONS cfDNA concentrations in the GCF of patients with periodontitis are considerably elevated, and are associated with the activation of the cGAS-STING signaling pathway in HGFs. These findings suggest that cfDNA contributes to the progression of periodontitis.
Humans ; Gingival Crevicular Fluid/metabolism* ; Signal Transduction ; Gingiva/cytology* ; Nucleotidyltransferases/genetics* ; Membrane Proteins/genetics* ; Cell-Free Nucleic Acids/analysis* ; Fibroblasts/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Periodontitis/metabolism* ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Adult ; RNA, Messenger/metabolism* ; Male ; Female

This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.
Animals ; Swine ; African Swine Fever Virus/metabolism* ; Membrane Proteins/metabolism* ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction/genetics*

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics* ; Animals ; DNA ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals ; Cells, Cultured ; Circovirus ; Interferon Type I/genetics* ; Macrophages, Alveolar/virology* ; Membrane Proteins/metabolism* ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
Base Sequence ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.
DNA, Bacterial ; immunology ; metabolism ; DNA, Viral ; immunology ; metabolism ; Gene Expression Regulation ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3 ; genetics ; immunology ; Interferon Type I ; biosynthesis ; immunology ; Membrane Proteins ; genetics ; immunology ; Models, Molecular ; NF-kappa B ; genetics ; immunology ; Nucleotides, Cyclic ; biosynthesis ; immunology ; Nucleotidyltransferases ; genetics ; immunology ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction

To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.
Amino Acid Sequence ; Cloning, Molecular ; Erythritol ; analogs & derivatives ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Nucleotidyltransferases ; chemistry ; genetics ; metabolism ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Protein Structure, Secondary ; Sequence Alignment ; Sugar Phosphates ; metabolism ; Tripterygium ; chemistry ; enzymology ; genetics

The systematic position of Fritillaria hupehensis has been in dispute. Phylogentic analyses were conducted on sequences of ITS, rpl16, matK sequences for species of F. hupehensis and allies. Lilium davidii was designed as outgroup. The analyses were performed using MP and ML methods. Conclusions could be achieved as follow. The topologies of MP and ML are consistent. The samples of F. hepehensis from different places form a supported clade with a strong bootstrap. And then form a strongly supported clade with F. anhuiensis, F. monantha. The results suggests that although F. hupehensis has a closet relation with the two ones, it exists some difference.
DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Endoribonucleases ; genetics ; Fritillaria ; classification ; genetics ; Molecular Sequence Data ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Leaves ; genetics ; Ribosomal Proteins ; genetics ; Sequence Analysis, DNA ; Species Specificity

Abstract: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.
Antimalarials ; metabolism ; supply & distribution ; Artemisinins ; metabolism ; supply & distribution ; Biosynthetic Pathways ; Cytochrome P-450 Enzyme System ; genetics ; Escherichia coli ; metabolism ; Gene Dosage ; Genetic Engineering ; Isoenzymes ; genetics ; RNA Nucleotidyltransferases ; genetics ; Retinal Dehydrogenase ; genetics ; Saccharomyces cerevisiae ; metabolism ; Synthetic Biology