The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

PD-L1 engagement on T cells promotes self-tolerance and suppression of neighboring macrophages and effector T cells in cancer.

Programmed cell death protein 1 (PD-1) ligation delimits immunogenic responses in T cells. However, the consequences of programmed cell death 1 ligand 1 (PD-L1) ligation in T cells are uncertain. We found that T cell expression of PD-L1 in cancer was regulated by tumor antigen and sterile inflammatory cues. PD-L1+ T cells exerted tumor-promoting tolerance via three distinct mechanisms: (1) binding of PD-L1 induced STAT3-dependent 'back-signaling' in CD4+ T cells, which prevented activation, reduced TH1-polarization and directed TH17-differentiation. PD-L1 signaling also induced an anergic T-bet-IFN-γ- phenotype in CD8+ T cells and was equally suppressive compared to PD-1 signaling; (2) PD-L1+ T cells restrained effector T cells via the canonical PD-L1-PD-1 axis and were sufficient to accelerate tumorigenesis, even in the absence of endogenous PD-L1; (3) PD-L1+ T cells engaged PD-1+ macrophages, inducing an alternative M2-like program, which had crippling effects on adaptive antitumor immunity. Collectively, we demonstrate that PD-L1+ T cells have diverse tolerogenic effects on tumor immunity.

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Implications of a multiscale structure of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.

Superconductivity in pressurized trilayer La4Ni3O10-δ single crystals.

The pursuit of discovering new high-temperature superconductors that diverge from the copper-based model1-3 has profound implications for explaining mechanisms behind superconductivity and may also enable new applications4-8. Here our investigation shows that the application of pressure effectively suppresses the spin-charge order in trilayer nickelate La4Ni3O10-δ single crystals, leading to the emergence of superconductivity with a maximum critical temperature (Tc) of around 30 K at 69.0 GPa. The d.c. susceptibility measurements confirm a substantial diamagnetic response below Tc, indicating the presence of bulk superconductivity with a volume fraction exceeding 80%. In the normal state, we observe a strange metal behaviour, characterized by a linear temperature-dependent resistance extending up to 300 K. Furthermore, the layer-dependent superconductivity observed hints at a unique interlayer coupling mechanism specific to nickelates, setting them apart from cuprates in this regard. Our findings provide crucial insights into the fundamental mechanisms underpinning superconductivity, while also introducing a new material platform to explore the intricate interplay between the spin-charge order, flat band structures, interlayer coupling, strange metal behaviour and high-temperature superconductivity.

Collective motion in hcp-Fe at Earth's inner core conditions.

Earth's inner core is predominantly composed of solid iron (Fe) and displays intriguing properties such as strong shear softening and an ultrahigh Poisson's ratio. Insofar, physical mechanisms to explain these features coherently remain highly debated. Here, we have studied longitudinal and shear wave velocities of hcp-Fe (hexagonal close-packed iron) at relevant pressure-temperature conditions of the inner core using in situ shock experiments and machine learning molecular dynamics (MLMD) simulations. Our results demonstrate that the shear wave velocity of hcp-Fe along the Hugoniot in the premelting condition, defined as T/Tm (Tm: melting temperature of iron) above 0.96, is significantly reduced by ~30%, while Poisson's ratio jumps to approximately 0.44. MLMD simulations at 230 to 330 GPa indicate that collective motion with fast diffusive atomic migration occurs in premelting hcp-Fe primarily along [100] or [010] crystallographic direction, contributing to its elastic softening and enhanced Poisson's ratio. Our study reveals that hcp-Fe atoms can diffusively migrate to neighboring positions, forming open-loop and close-loop clusters in the inner core conditions. Hcp-Fe with collective motion at the inner core conditions is thus not an ideal solid previously believed. The premelting hcp-Fe with collective motion behaves like an extremely soft solid with an ultralow shear modulus and an ultrahigh Poisson's ratio that are consistent with seismic observations of the region. Our findings indicate that premelting hcp-Fe with fast diffusive motion represents the underlying physical mechanism to help explain the unique seismic and geodynamic features of the inner core.

A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.

Single domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, as well as therapeutic and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research - i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat IgG. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

Translatome analysis in acute ischemic stroke: Astrocytes and microglia exhibit differences in poststroke alternative splicing of expressed transcripts.

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo-Tag-seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT-PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

Intracellular infection by symbiotic bacteria requires the mitotic kinase AURORA1.

The subcellular events occurring in cells of legume plants as they form transcellular symbiotic-infection structures have been compared with those occurring in premitotic cells. Here, we demonstrate that Aurora kinase 1 (AUR1), a highly conserved mitotic regulator, is required for intracellular infection by rhizobia in Medicago truncatula. AUR1 interacts with microtubule-associated proteins of the TPXL and MAP65 families, which, respectively, activate and are phosphorylated by AUR1, and localizes with them within preinfection structures. MYB3R1, a rhizobia-induced mitotic transcription factor, directly regulates AUR1 through two closely spaced, mitosis-specific activator cis elements. Our data are consistent with a model in which the MYB3R1-AUR1 regulatory module serves to properly orient preinfection structures to direct the transcellular deposition of cell wall material for the growing infection thread, analogous to its role in cell plate formation. Our findings indicate that the eukaryotically conserved MYB3R1-TPXL-AUR1-MAP65 mitotic module was conscripted to support endosymbiotic infection in legumes.

Two-Dimensional M-Chalcogene Family with Tunable Superconducting, Topological, and Magnetic Properties.

An interesting question is whether chalcogen atoms can emulate the role of carbon or boron elements stabilized between two transition metal layers, as observed in MXenes or MBenes. Here, we predict a new family of two-dimensional ternary compounds M4XY2 (where M = Pd, Y, Zr, etc.; X = S, Se, Te; and Y = Cl, Br, I), named M-chalcogene. Through first-principles calculations, we reveal diverse physical properties in these compounds, including superconducting, topological, and magnetic characteristics, where the bilayer transition metals play crucial roles. Moreover, the expected helical edge states and superconducting transition temperatures in Pd4SCl2 can be finely tuned by strains. Additionally, the Ti4SCl2 is predicted to be a topological insulator and shows promise as a gas sensor candidate for certain exotic gases. Our findings expand two-dimensional material families and provide promising platforms for diverse physical phenomena with efficient tunability by external stimuli for various applications.

Quantum-Confined Tunable Ferromagnetism on the Surface of a Van der Waals Antiferromagnet NaCrTe2.

The surface of three-dimensional materials provides an ideal and versatile platform to explore quantum-confined physics. Here, we systematically investigate the electronic structure of Na-intercalated CrTe2, a van der Waals antiferromagnet, using angle-resolved photoemission spectroscopy and ab initio calculations. The measured band structure deviates from the calculation of bulk NaCrTe2 but agrees with that of ferromagnetic monolayer CrTe2. Consistently, we observe unexpected exchange splitting of the band dispersions, persisting well above the Néel temperature of bulk NaCrTe2. We argue that NaCrTe2 features a quantum-confined 2D ferromagnetic state in the topmost surface layer due to strong ferromagnetic correlation in the CrTe2 layer. Moreover, the exchange splitting and the critical temperature can be controlled by surface doping of alkali-metal atoms, suggesting the feasibility of tuning the surface ferromagnetism. Our work not only presents a simple platform for exploring tunable 2D ferromagnetism but also provides important insights into the quantum-confined low-dimensional magnetic states.

Precious-Metal-Free Mo-MXene Catalyst Enabling Facile Ammonia Synthesis Via Dual Sites Bridged by H-Spillover.

To date, NH3 synthesis under mild conditions is largely confined to precious Ru catalysts, while nonprecious metal (NPM) catalysts are confronted with the challenge of low catalytic activity due to the inverse relationship between the N2 dissociation barrier and NHx (x = 1-3) desorption energy. Herein, we demonstrate NPM (Co, Ni, and Re)-mediated Mo2CTx MXene (where Tx denotes the OH group) to achieve efficient NH3 synthesis under mild conditions. In particular, the NH3 synthesis rate over Re/Mo2CTx and Ni/Mo2CTx can reach 22.4 and 21.5 mmol g-1 h-1 at 400 °C and 1 MPa, respectively, higher than that of NPM-based catalysts and Cs-Ru/MgO ever reported. Experimental and theoretical studies reveal that Mo4+ over Mo2CTx has a strong ability for N2 activation; thus, the rate-determining step is shifted from conventional N2 dissociation to NH2* formation. NPM is mainly responsible for H2 activation, and the high reactivity of spillover hydrogen and electron transfer from NPM to the N-rich Mo2CTx surface can efficiently facilitate nitrogen hydrogenation and the subsequent desorption of NH3. With the synergistic effect of the dual active sites bridged by H-spillover, the NPM-mediated Mo2CTx catalysts circumvent the major obstacle, making NH3 synthesis under mild conditions efficient.

Biomimetic Design of a Dynamic M-O-V Pyramid Electron Bridge for Enhanced Nitrogen Electroreduction.

Electrochemical nitrogen reduction reaction (eNRR) offers a sustainable route for ammonia synthesis; however, current electrocatalysts are limited in achieving optimal performance within narrow potential windows. Herein, inspired by the heliotropism of sunflowers, we present a biomimetic design of Ru-VOH electrocatalyst, featuring a dynamic Ru-O-V pyramid electron bridge for eNRR within a wide potential range. In situ spectroscopy and theoretical investigations unravel the fact that the electrons are donated from Ru to V at lower overpotentials and retrieved at higher overpotentials, maintaining a delicate balance between N2 activation and proton hydrogenation. Moreover, N2 adsorption and activation were found to be enhanced by the Ru-O-V moiety. The catalyst showcases an outstanding Faradaic efficiency of 51.48% at -0.2 V (vs RHE) with an NH3 yield rate exceeding 115 µg h-1 mg-1 across the range of -0.2 to -0.4 V (vs RHE), along with impressive durability of over 100 cycles. This dynamic M-O-V pyramid electron bridge is also applicable to other metals (M = Pt, Rh, and Pd).

Centrosymmetry-Breaking Morphotropic Phase Boundary: A Pathway to Highly Sensitive and Strong Pressure-Responsive Nonlinear Optical Switches.

The quest for high-performance piezoelectric materials has been synonymous with the pursuit of the morphotropic phase boundary (MPB), yet the full potential of MPBs remains largely untapped outside of the realm of ferroelectrics. In this study, we reveal a new class of MPB by creating continuous molecular-based solid solutions between centro- and noncentrosymmetric compounds, exemplified by (tert-butylammonium)1-x(tert-amylammonium)xFeCl4 (0 ≤ x ≤ 1), where the MPB is formed due to disorder of molecular cations. Near the MPB, we discovered an exceptionally sensitive nonlinear optical material in the centrosymmetric phase, capable of activation at pressures as low as 0.12-0.27 GPa, and producing tunable second-harmonic generation (SHG) signals from zero to 18.8 times that of KH2PO4 (KDP). Meanwhile, synchrotron diffraction experiments have unveiled a third competing phase (P212121) appearing at low pressure, forming a triple-phase point near the MPB, thereby providing insight into the mechanism underpinning the nonlinear optical (NLO) switch behavior. These findings highlight the opportunity to harness exceptional physical properties in symmetry-breaking solid solution systems by strategically designing novel MPBs.

Comparative Venom Multiomics Reveal the Molecular Mechanisms Driving Adaptation to Diverse Predator-Prey Ecosystems in Closely Related Sea Snakes.

Predator-prey arms races are ideal models for studying the natural selection and adaptive evolution that drive the formation of biological diversity. For venomous snakes, venom is a key bridge linking snakes with their prey, but whether and how venom evolves under the selection of diet remains unclear. Here, we focused on two closely related sea snakes, Hydrophis cyanocinctus and Hydrophis curtus, which show significant differences in prey preferences. Data-independent acquisition (DIA)-based proteomic analysis revealed different degrees of homogeneity in the venom composition of the two snakes, which was consistent with the differential phylogenetic diversity of their prey. By investigating the sequences and structures of three-finger toxins (3FTx), a predominant toxin family in elapid venom, we identified significant differences between the two sea snakes in the binding activity of 3FTx to receptors from different prey populations, which could explain the trophic specialization of H. cyanocinctus. Furthermore, we performed integrated multiomic profiling of the transcriptomes, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and proteomes of the venom glands; constructed venom-related mRNA-miRNA-lncRNA networks; and identified a series of noncoding RNAs involved in the regulation of toxin gene expression in the two species. These findings are highly informative for elucidating the molecular basis and regulatory mechanisms that account for discrepant venom evolution in response to divergent diets in closely related snakes, providing valuable evidence for the study of coselection and coevolution in predator-prey ecosystems.

A multicenter study on deep learning for glioblastoma auto-segmentation with prior knowledge in multimodal imaging.

A precise radiotherapy plan is crucial to ensure accurate segmentation of glioblastomas (GBMs) for radiation therapy. However, the traditional manual segmentation process is labor-intensive and heavily reliant on the experience of radiation oncologists. In this retrospective study, a novel auto-segmentation method is proposed to address these problems. To assess the method's applicability across diverse scenarios, we conducted its development and evaluation using a cohort of 148 eligible patients drawn from four multicenter datasets and retrospective data collection including noncontrast CT, multisequence MRI scans, and corresponding medical records. All patients were diagnosed with histologically confirmed high-grade glioma (HGG). A deep learning-based method (PKMI-Net) for automatically segmenting gross tumor volume (GTV) and clinical target volumes (CTV1 and CTV2) of GBMs was proposed by leveraging prior knowledge from multimodal imaging. The proposed PKMI-Net demonstrated high accuracy in segmenting, respectively, GTV, CTV1, and CTV2 in an 11-patient test set, achieving Dice similarity coefficients (DSC) of 0.94, 0.95, and 0.92; 95% Hausdorff distances (HD95) of 2.07, 1.18, and 3.95 mm; average surface distances (ASD) of 0.69, 0.39, and 1.17 mm; and relative volume differences (RVD) of 5.50%, 9.68%, and 3.97%. Moreover, the vast majority of GTV, CTV1, and CTV2 produced by PKMI-Net are clinically acceptable and require no revision for clinical practice. In our multicenter evaluation, the PKMI-Net exhibited consistent and robust generalizability across the various datasets, demonstrating its effectiveness in automatically segmenting GBMs. The proposed method using prior knowledge in multimodal imaging can improve the contouring accuracy of GBMs, which holds the potential to improve the quality and efficiency of GBMs' radiotherapy.

Fluorinated 1,7-DO2A-Based Iron(II) Complexes as Sensitive 19F MRI Molecular Probes for Visualizing Renal Dysfunction in Living Mice.

Kidney diseases have become an important global health concern due to their high incidence, inefficient diagnosis, and poor prognosis. Devising direct methods, especially imaging means, to assess renal function is the key for better understanding the mechanisms of various kidney diseases and subsequent development of effective treatment. Herein, we developed a fluorinated ferrous chelate-based sensitive probe, 1,7-DO2A-Fe(II)-F18 (Probe 1), for 19F magnetic resonance imaging (MRI). This highly fluorinated probe (containing 18 chemically equivalent 19F atoms with a fluorine content at 35 wt %) achieves a 15-time enhancement in signal intensity compared with the fluorine-containing ligand alone due to the appropriately regulated 19F relaxation times by the ferrous ion, which significantly increases imaging sensitivity and reduces acquisition time. Owing to its high aqueous solubility, biostability, and biocompatibility, this probe could be rapidly cleared by kidneys, which provides a means for monitoring renal dysfunction via 19F MRI. With this probe, we accomplish in vivo imaging of the impaired renal dysfunction caused by various kidney diseases including acute kidney injury, unilateral ureteral obstruction, and renal fibrosis at different stages. Our study illustrates the promising potential of Probe 1 for in vivo real-time visualization of kidney dysfunction, which is beneficial for the study, diagnosis, and even stratification of different kidney diseases. Furthermore, the design strategy of our probe is inspiring for the development of more high-performance 19F MRI probes for monitoring various biological processes.

Integrated phenotypic, transcriptomics and metabolomics: growth status and metabolite accumulation pattern of medicinal materials at different harvest periods of Astragalus Membranaceus Mongholicus.

BACKGROUND: Astragalus membranaceus var. mongholicus (Astragalus), acknowledged as a pivotal "One Root of Medicine and Food", boasts dual applications in both culinary and medicinal domains. The growth and metabolite accumulation of medicinal roots during the harvest period is intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the harvest date during the transition from conventional yield content of medicinal materials to high and to identify the core regulators governing such a critical transition. To solve this problem, we performed a correlation analysis of phenotypic, transcriptome, and metabolome dynamics during the harvesting of Astragalus roots. RESULTS: First, our analysis identified stage-specific expression patterns for a significant proportion of the Astragalus root genes and unraveled the chronology of events that happen at the early and later stages of root harvest. Then, the results showed that different root developmental stages can be depicted by co-expressed genes of Astragalus. Moreover, we identified the key components and transcriptional regulation processes that determine root development during harvest. Furthermore, through correlating phenotypes, transcriptomes, and metabolomes at different harvesting periods, period D (Nov.6) was identified as the critical period of yield and flavonoid content increase, which is consistent with morphological and metabolic changes. In particular, we identified a flavonoid biosynthesis metabolite, isoliquiritigenin, as a core regulator of the synthesis of associated secondary metabolites in Astragalus. Further analyses and experiments showed that HMGCR, 4CL, CHS, and SQLE, along with its associated differentially expressed genes, induced conversion of metabolism processes, including the biosynthesis of isoflavones and triterpenoid saponins substances, thus leading to the transition to higher medicinal materials yield and active ingredient content. CONCLUSIONS: The findings of this work will clarify the differences in the biosynthetic mechanism of astragaloside IV and calycosin 7-O-ß-D-glucopyranoside accumulation between the four harvesting periods, which will guide the harvesting and production of Astragalus.