Cyclopamine, a typical isosteroidal alkaloid identified as a hedgehog pathway inhibitor, serves as a crucial molecular scaffold for the semi-synthesis of drugs treating nevoid basal cell carcinoma. Verazine is a key intermediate in the biosynthetic pathway of cyclopamine. In this study, we reconstructed the biosynthetic pathway of verazine in engineered Saccharomyces cerevisiae BY-SQ1, obtaining a strain that produced verazine with a yield of 265.38 μg/L. Furthermore, during a 1,000-ns classical MD trajectory, cholesterol retained a broadly consistent heme-facing pose within VcCYP90B27. The modeled pro-R C22 hydrogen remained closer to the heme Fe center on average than the pro-S hydrogen (4.01 vs 4.65 Å), revealing a persistent orientational bias in the bound ensemble. MM-GBSA and contact analyses further indicated that hydrophobic packing around the steroid scaffold and side chain may help maintain this asymmetric presentation. This research not only lays a foundation for the biosynthesis of cyclopamine and other steroidal alkaloids, but also provides a structural rationale for how cholesterol recognition by VcCYP90B27 may contribute to the observed 22R hydroxylation and identifies candidate residues for experimental evaluation.
The integration of light and cold signaling is critical for cold acclimation in plants, but the regulatory complexity of the underlying transcriptional network remains poorly understood. To systematically dissect this interplay in tomato (Solanum lycopersicum), we constructed a computational model that merges the core cold-responsive inducer of CBF expression 1 (ICE1)–C-repeat binding factor (CBF)–cold regulated factors (COR) pathway with light-sensitive modules, including the constitutive photomorphogenic 1 (COP1)–elongated hypocotyl 5 (HY5)–MYB domain protein 15 (MYB15) cascade and the phytochrome–phytochrome interacting factor 4 (PIF4)–GA-INSENSITIVE 4 (GIA4) regulatory axis. Simulations successfully reproduced the experimentally observed gene expression dynamics across different photoperiods and revealed that phytochrome activity is co-modulated by both light quality and temperature. Our model predicts that a low red/far-red ratio enhances the expression of CBF, whereas cold treatment stabilizes phyA and phyB proteins, jointly promoting cold tolerance. Additionally, the PIF4–GAI4 negative feedback loop is shown to generate sustained oscillations in SlPIF4's expression, and the cold-responsive gene SlCOR413 is predicted to exhibit low-temperature-induced oscillatory behavior. This integrative framework provides a systems-level tool for dissecting light–cold crosstalk and offers a basis for rationally engineering cold tolerance in horticultural crops through targeted modulation of transcriptional networks.
Mature adipocytes (ACs) are essential for modeling human adipose tissue physiology in vitro, yet their large size, buoyancy, and shear sensitivity make them challenging to incorporate into engineered three-dimensional (3D) constructs. This study introduces an agarose support-bath (SB) extrusion workflow for bioprinting geometrically defined adipose constructs using a gellan gum (GG) bioink. We examined the viscosity of GG solutions and SBs to identify a low-shear printability window suitable for lipid-laden cells. SB printing significantly enhanced filament stability, pore uniformity, and construct accuracy compared to free extrusion. Viability assays showed that ACs remained intact and metabolically active after 3 d post-printing, with minimal membrane damage and maintained unilocular morphology. Immunofluorescence staining revealed uniform lipid-filled cells and expression of perilipin A, while basal and stimulated glycerol release indicated retained metabolic activity. These findings demonstrate that the established SB extrusion protocol enables gentle, precise deposition of ACs using a GG-based bioink. This workflow offers a reproducible and accessible platform for generating physiologically relevant adipose cell models, supporting early-stage biological assessment, preliminary drug testing, and soft tissue engineering.
