Heterologous expression of taxane genes confers resistance to fall armyworm in Nicotiana benthamiana.

KEY MESSAGE: Taxadiene synthase, taxadiene-5α-hydroxylase, and taxane 13α-hydroxylase genes were introduced into Nicotiana benthamiana, and the improved resistance to lepidoptera pest fall armyworm was reported. Fall armyworm (FAW) is a serious agricultural pest. Genetic engineering techniques have been used to create pest-resistant plant varieties for reducing pest damage. Paclitaxel is a diterpenoid natural metabolite with antineoplastic effects in medicine. However, the effects of taxanes on the growth and development of lepidoptera pests, such as the FAW, are unknown. Here, selected paclitaxel precursor biosynthesis pathway genes, taxadiene synthase, taxane 5α-hydroxylase, and taxane 13α-hydroxylase, were engineered in the heterologous host Nicotiana benthamiana plants. Bioassay experiments showed that the transgenic N. benthamiana plants displayed improved resistance to FAW infestation, with degeneration of gut tissues and induced expression of apoptosis-related genes. Cytotoxicity experiment showed that the paclitaxel precursor, 10-deacetylbaccatin III, is cytotoxic to Sf9 cells, causing cell cycle arrest at the G2/M phase and disorder of the cytoskeleton. Metabolome analysis showed that heterologous expression of taxane genes in N. benthamiana affected the digestive system, steroid hormone and purine metabolism pathways of FAW larvae. In summary, this study provides a candidate approach for FAW control.

HDAC3 Knockdown Dysregulates Juvenile Hormone and Apoptosis-Related Genes in Helicoverpa armigera.

Insect development requires genes to be expressed in strict spatiotemporal order. The dynamic regulation of genes involved in insect development is partly orchestrated by the histone acetylation-deacetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although histone deacetylase 3 (HDAC3) is required for mice during early embryonic development, its functions in Helicoverpa armigera (H. armigera) and its potential to be used as a target of insecticides remain unclear. We treated H. armigera with HDAC3 siRNA and RGFP966, a specific inhibitor, examining how the HDAC3 loss-of-function affects growth and development. HDAC3 siRNA and RGFP966 treatment increased mortality at each growth stage and altered metamorphosis, hampering pupation and causing abnormal wing development, reduced egg production, and reduced hatching rate. We believe that the misregulation of key hormone-related genes leads to abnormal pupa development in HDAC3 knockout insects. RNA-seq analysis identified 2788 differentially expressed genes (≥two-fold change; p ≤ 0.05) between siHDAC3- and siNC-treated larvae. Krüppel homolog 1 (Kr-h1), was differentially expressed in HDAC3 knockdown larvae. Pathway-enrichment analysis revealed the significant enrichment of genes involved in the Hippo, MAPK, and Wnt signaling pathways following HDAC3 knockdown. Histone H3K9 acetylation was increased in H. armigera after siHDAC3 treatment. In conclusion, HDAC3 knockdown dysregulated juvenile hormone (JH)-related and apoptosis-related genes in H. armigera. The results showed that the HDAC3 gene is a potential target for fighting H. armigera.

Anti-oxidative and anti-inflammatory activities of the ethanol extract of edible flower from Chimonanthus praecox.

Chimonanthi Praecocis Flos, namely wintersweet flower, is the edible flower or flower bud of Chimonanthus praecox (L.) Link which is a deciduous shrub plant originated from China and is widely cultivated as a garden or ornamental plant all over the world. However, few studies focused on its anti-inflammatory property. In the present study, we explored the anti-inflammatory and anti-oxidative activities of ethanol extract of Chimonanthi Praecocis Flos (CPE) which contained 7.980% ± 0.176% total flavonoids and 1.461% ± 0.041% total alkaloids. In LPS-stimulated RAW264.7 macrophages, CPE significantly decreased the production of NO and prostaglandin E2 (PGE2) through reducing the expressions of their synthases-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). It also suppressed the transcription and translation of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Further research revealed that CPE impeded the phosphorylation and degradation of IκBα, thus restraining the nuclear translocation of p65, and consequently dampening NF-κB signaling. In endotoxemia mice, several pro-inflammatory cytokines in serum were also decreased after CPE treatment. Besides anti-inflammatory activity, anti-oxidative activity is another important capacity of wintersweet flower. Indeed, CPE reduced LPS-elevated intracellular total reactive oxygen species (ROS) level by weakening NADPH oxidase activity in cell system. Moreover, it directly scavenged DPPH radical and superoxide anion, and exerted ferric reducing ability in cell-free system. Our findings demonstrate that wintersweet flower can be used as a beneficial natural product or an additive by virtue of its anti-oxidative and anti-inflammatory properties.

A comparative study of spike protein of SARS-CoV-2 and its variant Omicron (B.1.1.529) on some immune characteristics.

The emergence of Omicron variant raises great concerns because of its rapid transmissibility and its numerous mutations in spike protein (S-protein). S-protein can act as a pathogen-associated molecular pattern and complement activator as well as antigen. We compared some immune characteristics of trimer S-proteins for wild type (WT-S) and B.1.1.529 Omicron (Omicron-S) to investigate whether the mutations have affected its pathogenicity and antigenic shift. The results indicated that WT-S and Omicron-S directly activated nuclear factor-κB (NF-κB) and induced the release of pro-inflammatory cytokines in macrophages, but the actions of Omicron-S were weaker. These inflammatory reactions could be abrogated by a Toll-like receptor 4 antagonist TAK-242. Two S-proteins failed to induce the production of antiviral molecular interferon-ß. In contrast to pro-inflammatory effects, the ability of two S-proteins to activate complement was comparable. We also compared the binding ability of two S-proteins to a high-titer anti-WT-receptor-binding domain antibody. The data showed that WT-S strongly bound to this antibody, while Omicron-S was completely off-target. Collectively, the mutations of Omicron have a great impact on the pro-inflammatory ability and epitopes of S-protein, but little effect on its ability to activate complement. Addressing these issues can be helpful for more adequate understanding of the pathogenicity of Omicron and the vaccine breakthrough infection.

Resibufogenin, one of bufadienolides in toad venom, suppresses LPS-induced inflammation via inhibiting NF-κB and AP-1 pathways.

Toad venom is a traditional Chinese medicine that has a long history in treating infectious and inflammatory diseases, such as carbuncle, pharyngitis. As one of the major active components in toad venom, resibufogenin (RBG) possesses a variety of pharmacological activities, including lowering blood pressure, reducing proteinuria and preventing oxidative stress. But only its antitumor activity attracts widespread attention in these years. This study aimed to explore the nonnegligible anti-inflammatory activity of RBG in vivo and in vitro. In endotoxemia mice, a single intraperitoneal administration of RBG significantly lowered serum TNF-α, IL-6 and MCP-1 levels. In LPS-stimulated macrophages, RBG decreased LPS-induced pro-inflammatory mediators' productions (e.g., iNOS, IL-6, TNF-α and MCP-1) through suppressing their transcriptions. Mechanism study showed that RBG hindered IκBα phosphorylation and prevented nuclear translocation of p65, thus inactivating nuclear factor-κB (NF-κB) signaling. Concurrently, RBG also dampened activator protein-1 (AP-1) signaling through inhibiting the phosphorylation levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Besides LPS (TLR4 ligand) model, RBG also inhibited Pam3CSK4 (TLR2 ligand)- or poly I:C (TLR3 ligand)-induced inflammatory reactions, suggesting that its target(s) site is(are) not on the cytomembrane. These findings not only support the pharmacological basis for the traditional use of toad venom in inflammatory diseases, but also provide a promising anti-inflammatory candidate.

MAPKs/AP-1, not NF-κB, is responsible for MCP-1 production in TNF-α-activated adipocytes.

Obesity is associated with the infiltration of monocytes/macrophages into adipose tissue in which MCP-1 plays a crucial role. But the regulatory mechanism of MCP-1 expression in adipocytes is not well defined. Our results demonstrated that TNF-α induced abundant MCP-1 production in adipocytes, including 3T3-L1 pre- (≈ 9 to 18-fold), mature adipocytes (≈ 4 to 6-fold), and primary adipocytes(< 2-fold), among which 3T3-L1 pre-adipocytes showed the best reactiveness. Thus, 3T3-L1 pre-adipocytes were used for the most of following experiments. At the transcriptional level, TNF-α (20 ng/mL) also promoted the mRNA expression of MCP-1. It is well recognized that the engagement of TNF-α with its receptor can trigger both NF-κB and AP-1 signalling, which was also confirmed in our study (5-fold and 2-fold). Unexpectedly and counterintuitively, multiple NF-κB inhibitors with different mechanisms failed to suppress TNF-α-induced MCP-1 production, but rather the inhibitors for any one of MAPKs (JNK, ERK and p38) could do. This study, for the first time, reveals that MAPKs/AP-1 but not NF-κB signalling is responsible for MCP-1 production in TNF-α-activated adipocytes. These findings provide important insight into the role of AP-1 signalling in adipose tissue, and may lead to the development of therapeutical repositioning strategies in metaflammation.Abbreviations: AP-1, activator protein-1; CHX, cycloheximide; IR, insulin resistance; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; RT-qPCR, quantitative real-time PCR; T2DM, type 2 diabetes mellitus; TRE, triphorbol acetate-response element.

Equisetin is an anti-obesity candidate through targeting 11ß-HSD1.

Obesity is increasingly prevalent globally, searching for therapeutic agents acting on adipose tissue is of great importance. Equisetin (EQST), a meroterpenoid isolated from a marine sponge-derived fungus, has been reported to display antibacterial and antiviral activities. Here, we revealed that EQST displayed anti-obesity effects acting on adipose tissue through inhibiting adipogenesis in vitro and attenuating HFD-induced obesity in mice, doing so without affecting food intake, blood pressure or heart rate. We demonstrated that EQST inhibited the enzyme activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a therapeutic target of obesity in adipose tissue. Anti-obesity properties of EQST were all offset by applying excessive 11ß-HSD1's substrates and 11ß-HSD1 inhibition through knockdown in vitro or 11ß-HSD1 knockout in vivo. In the 11ß-HSD1 bypass model constructed by adding excess 11ß-HSD1 products, EQST's anti-obesity effects disappeared. Furthermore, EQST directly bond to 11ß-HSD1 protein and presented remarkable better intensity on 11ß-HSD1 inhibition and better efficacy on anti-obesity than known 11ß-HSD1 inhibitor. Therefore, EQST can be developed into anti-obesity candidate compound, and this study may provide more clues for developing higher effective 11ß-HSD1 inhibitors.

Salicylate Sodium Suppresses Monocyte Chemoattractant Protein-1 Production by Directly Inhibiting Phosphodiesterase 3B in TNF-α-Stimulated Adipocytes.

As a worldwide health issue, obesity is associated with the infiltration of monocytes/macrophages into the adipose tissue causing unresolved inflammation. Monocyte chemoattractant protein-1 (MCP-1) exerts a crucial effect on obesity-related monocytes/macrophages infiltration. Clinically, aspirin and salsalate are beneficial for the treatment of metabolic diseases in which adipose tissue inflammation plays an essential role. Herein, we investigated the effect and precise mechanism of their active metabolite salicylate on TNF-α-elevated MCP-1 in adipocytes. The results indicated that salicylate sodium (SAS) could lower the level of MCP-1 in TNF-α-stimulated adipocytes, which resulted from a previously unrecognized target phosphodiesterase (PDE), 3B (PDE3B), rather than its known targets IKKß and AMPK. The SAS directly bound to the PDE3B to inactivate it, thus elevating the intracellular cAMP level and activating PKA. Subsequently, the expression of MKP-1 was increased, which led to the decrease in p-EKR and p-p38. Both PDE3B silencing and the pharmacological inhibition of cAMP/PKA compromised the suppressive effect of SAS on MCP-1. In addition to PDE3B, the PDE3A and PDE4B activity was also inhibited by SAS. Our findings identify a previously unrecognized pathway through which SAS is capable of attenuating the inflammation of adipocytes.

Phaeosphspirone (1/1'), a pair of unique polyketide enantiomers with an unusual 6/5/5/6 tetracyclic ring from the desert plant endophytic fungus Phaeosphaeriaceae sp.

Phaeosphspirone, an undescribed polyketide with a unique 6/5/5/6-fused tetracyclic system, and two known analogues, herbarin and O-methylherbarin, were purified from the endophytic fungus Phaeosphaeriaceae sp. isolated from the desert plant Bassia dasyphylla. The connectivity and relative configuration of phaeosphspirone was elucidated by comprehensive HR-ESI-MS and NMR analysis together with a computer-assisted structure elucidation (CASE) method. A pair of enantiomers existing in phaeosphspirone were separated by HPLC chromatography after reacting with chiral reagents, from which the absolute configuration of phaeosphspirone was simultaneously determined based on Mosher's rule. This tandem strategy provides a useful approach for the separation and stereochemical determination of enantiomers possessing secondary hydroxyl groups. The structural feature of phaeosphspirone, herbarin and O-methylherbarin together with gene cluster analysis suggested their polyketide biosynthetic origin. Herbarin and O-methylherbarin exhibited moderate cytotoxicity against three cancer cell lines.

Combination of the endophytic manganese-oxidizing bacterium Pantoea eucrina SS01 and biogenic Mn oxides: An efficient and sustainable complex in degradation and detoxification of malachite green.

Recently, Mn oxides (MnOxs) have been attracting considerable interest in the oxidation of organic pollutants. However, the reduction of MnOx in these reactions leads to the deactivation of the catalyst, which must be frequently regenerated. We evaluated the application of a manganese-oxidizing bacterium (MOB) and MnOx in removing toxic dyes. We studied the co-function of a plant-endophytic MOB, Pantoea eucrina SS01, with its bio-generated MnOx and evaluated the detoxification activity and chemical transformation mechanisms of the complex in malachite green (MG) degradation. We found a synergistic effect between MnOx and the strain. Particularly, strain SS01 could adsorb MG but could not degrade it, whereas the addition of Mn(II) promoted MG degradation by the formation of a complex containing the bacterium and MnOx aggregates (SS01-bio-MnOx), with distinct morphology characteristics. The complex showed a marked sustainability in the degradation of MG into less toxic or non-toxic metabolites. In this process, strain SS01 might have enhanced the regeneration of MnOx, accelerating MG degradation. Our data not only contribute to understanding the mechanism of MG removal by the SS01-bio-MnOx complex, but also provide a scientific basis for the future application of MOB and MnOx.

Stereochemical determination of four 10-membered ring resorcylic acid lactones from the desert plant endophytic fungus Chaetosphaeronema hispidulum.

Four 10-membered ring resorcylic acid lactones (RALs) including a new compound hispidulactone F (1) and three known analogs hispidulactone B (2), 2 R, 4R-sonnerlactone (3), and 2 R, 4S-sonnerlactone (4) were isolated from the special bioenvironmental desert plant endophytic fungus Chaetosphaeronema hispidulum. The structure of the new compound hispidulactone F (1) was determined by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, and HMBC). Hispidulactone F (1) and hispidulactone B (2) were a pair of stereoisomers at C-3, whereas 2 R, 4R-sonnerlactone (3) and 2 R, 4S-sonnerlactone (4) were another pair of stereoisomers at C-4. The stereochemistries of the hydroxyl groups at C-3 in 1 and 2, and at C-4 in 3 and 4 were first determined by modified Mosher's reactions. Thus, the absolute configuration C-3 in hispidulactone B (2) was not right in our previous report, and was rectified to be R. Compounds 1 and 4 were evaluated for their cytotoxic effects on the proliferation of HepG2. The possible biosynthetic pathway of compounds 1-4 was also presented.

Peptide LSARLAF induces integrin ß3 dependent outside-in signaling in platelets.

INTRODUCTION: Peptide LSARLAF (LSA) can bind and activate integrin αIIbß3 in the absence of 'inside-out' signal. The active αIIbß3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbß3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation. METHOD: Ligand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing αIIbß3 or αVß3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed ß3, Src, FcRγ-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA. RESULTS: Ligand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to αIIbß3 or αVß3. LSA also enhanced CHO cells with αIIbß3 or αVß3 expression spreading on immobilized fibrinogen. ß3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLCγ2 and Syk was also ß3 dependent. Src, FcRγ-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA. CONCLUSION: LSA-induced platelet activation is ß3 dependent, and signaling molecules Src, FcRγ-chain, SLP-76 and LAT play crucial roles in LSA-induced ß3 mediated signaling.