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De novo RNA-sequencing of Wolfiporia cocos mycelia cultured with filter paper composed of cellulose as the sole carbon source revealed a total of five expressed β-glucosidase genes. Among these, the β-glucosidase named Wcbg1B-1, which is composed of 539 amino acid residues and belongs to the GH1 family, had the highest mRNA abundance, accounting for 65 % of the total mRNA of the five expressed β-glucosidases. The recombinant Wcbg1B-1 was successfully expressed in Escherichia coli, with an optimal pH of 6.0 and an optimal temperature of 40°C using p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate. Recombinant Wcbg1B-1 has a K_m value of 0.32 mmol/L, a V_max of 416 μmol/min/mg and specific activity of 461.5 U/mg. Recombinant Wcbg1B-1 has strict β-glycosidic bond specificity, the highest hydrolysis activity to cellobiose, followed by lactose, and the lowest hydrolysis activity to gentian. Recombinant Wcbg1B-1 exhibits hydrolytic activity towards macromolecular cellulose such as sodium carboxymethyl cellulose, microcrystalline cellulose and filter paper. Additionally, its high tolerance to high concentrations of glucose and salt makes it more practical for cellulose hydrolysis. Recombinant Wcbg1B-1 can hydrolyze lactose at low temperatures, indicating that it could also be a potential biocatalyst for lactose-reduced dairy industry.

Four previously undescribed steroids, named sarmutogenin diginoside, sarmutogenin oleandroside, 16-hydroxy-17βH sarmutogenin digitaloside and 12-dehydroxy sarmutogenin diginoside (1-4), along with eight known ones (5-12) were isolated from the stems and leaves of Strophanthus divaricatus. The chemical structures of the new compounds including their absolute configurations, were characterized by extensive spectroscopic methods, electronic circular dichroism (ECD), X-ray diffraction, as well as comparison with literature data. Most of the isolated compounds demonstrated superior antiproliferative activities against A549 non-small cell lung cancer (NSCLC) cells compared to cisplatin, a first-line treatment regimen for NSCLC. Among these, compounds 2 and 12 can potently inhibited the growth of a panel of NSCLC cells by inducing apoptosis and G2/M cell cycle arrest.

Glial cell-induced neuroinflammation in the spinal cord is the critical pathology underlying complete Freund's adjuvant (CFA)-induced inflammatory pain. Previously, we showed that spinal glial cells undergo ferroptosis after CFA injection, which may contribute to the development of neuroinflammation and inflammatory pain. However, the mechanism underlying the occurrence of ferroptosis during inflammatory pain remains unclear. The aim of this study was to investigate the molecular factors involved in the occurrence of ferroptosis during the development of inflammatory pain. Bulk and single-cell RNA sequencing were performed to identify the key genes involved in the ferroptosis of spinal astrocytes, microglia, and oligodendrocytes in rats. We identified nuclear receptor 4A1 (NR4A1) as a common ferroptosis-related gene present in all three types of glial cells. Western blotting and immunostaining revealed increased NR4A1 levels in the spinal glial cells of the CFA-treated rats. Moreover, intrathecal injection of DIM-C-pPhOH (an NR4A1 inhibitor) effectively alleviated mechanical and thermal hypersensitivity in the CFA-treated rats by attenuating ferroptosis and neuroinflammation in spinal glial cells. Proteomic analysis revealed that mitogen-activated protein kinase 3 (MAPK3) may be the target protein of NR4A1. In addition, the combined results of chromatin immunoprecipitation and dual-luciferase assays indicated that NR4A1 can bind to the promoter region and promote transcription of MAPK3, ultimately leading to lipid peroxidation. In conclusion, this study demonstrated that increased expression of NR4A1 promotes the progression of CFA-induced inflammatory pain by enhancing ferroptosis through the transcriptional activation of MAPK3 and subsequent lipid peroxidation. Furthermore, inhibition of NR4A1 was found to suppress ferroptosis and reduce the release of pro-inflammatory cytokines in the spinal cord of rats with inflammatory pain. Collectively, these findings outline a novel pathological mechanism and identify potential therapeutic targets for the treatment of inflammatory pain.