As a traditional bulk Chinese medicinal herb, Salvia miltiorrhiza's phenolic acids are the core bioactive components responsible for its pharmacological effects. Environmental stress can regulate relevant metabolic pathways to promote the accumulation of phenolic acids. To identify key transcription factor genes in S.miltiorrhiza under H₂O₂ stress, we first used aseptic seedlings cultured under different H₂O₂ concentrations as experimental materials, performed RNA-seq analysis following standard protocols with three biological replicates, and conducted untargeted metabolomics analysis via UHPLC-QE-MS with six biological replicates. All assays were commissioned to Gene Denovo Biotechnology Co., Ltd. The results showed a concentration-dependent stress effect: Low concentrations of H₂O₂ slightly inhibited seedling growth but activated genes associated with photosynthesis and phenolic acid biosynthesis. In contrast, high concentrations of H₂O₂ strongly suppressed growth and redirected metabolic resources from growth to defense processes. This response was accompanied by the upregulation of key enzyme genes in the phenylpropanoid and tyrosine pathways, including PAL, RAS and CYP98A2, thereby promoting the accumulation of ferulic acid (FA), rosmarinic acid (RA) and salvianolic acid B (Sal B). Meanwhile, oxidative stress repressed the expression of genes related to phytohormone synthesis and photosynthesis, reducing resource allocation for growth, and maintained intracellular redox homeostasis by activating genes such as GRXC1. Transcriptional regulatory analysis revealed that WRKY28 expression was significantly and positively correlated with salvianolic acid content, while MYB305 was inhibited as a negative regulator. These findings elucidate the regulatory network underlying phenolic acid accumulation in S. miltiorrhiza under H₂O₂ stress from the perspectives of metabolic resource allocation and transcriptional regulation, providing a preliminary theoretical basis for improving the quality of S. miltiorrhiza medicinal materials through molecular breeding or environmental regulation. It should be noted that exogenous H₂O₂ treatment is a controllable experimental method, and its conclusions may differ from those under natural stress in terms of signal initiation and network integration. Further verification through multi-dimensional approaches is required for refinement.

Bupleurum giraldii (H. Wolff) Koso-Pol. is a perennial herbaceous plant belonging to the Bupleurum L. (Apiaceae). It is recognized as a provincially protected rare medicinal plant in Shaanxi with the whole plant has acknowledged medicinal value. In this study, the complete plastid genome sequence of B. giraldii was sequenced and characterized, and comparative and phylogenetic analyses were then conducted alongside other published Bupleurum plastid genome sequences. The whole plastid genome of B. giraldii was 155 761 bp in length, which exhibited the conserved circular quadripartite structure consisting of a large single copy (LSC, 85 663 bp), a small single copy (SSC, 17 492 bp), and a pair of inverted repeats (IRa/IRb, 26 303 bp). A total of 129 genes were annotated, including 84 protein-coding genes (PCGs), 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes, of which, 6 protein-coding genes, 7 tRNA genes and 4 rRNA genes were duplicated in the IR regions. A total of 66 simple sequence repeats (SSRs), 34 tandem repeats, and 31 dispersed repeats were detected. In the plastid genome of B. giraldii, codons with the relative synonymous codon usage (RSCU) value greater than 1 showed a tendency to end in adenine (A) or thymine (T), whereas those with an RSCU value less than 1 tended to end in cytosine (C) or guanine (G). Collectively, neutral plotting, ENC-plot, and PR2-plot analyses all revealed that natural selection might be the predominant factor shaping this codon usage bias. The plastid genome of Bupleurum species exhibited highly conserved gene order and content, along with little variation in overall size and GC content, no significant expansion or contraction of the IR regions was detected. Interspecific sequence variation mainly occurred in the non-coding intergenic regions, and sequence divergence in the LSC and SSC regions was significantly higher than that in the IR regions. Eleven divergence hotspot sequences were identified, located in the LSC and SSC regions respectively. Phylogenetic inferences inferred that all Bupleurum species clustered into a monophyletic clade with BS_ML = 100%, PP = 1, and which could be further divided into two independent lineages corresponding to subgenus Bupleurum and subgenus Penninervia. Bupleurum giraldii located in subgenus Bupleurum and showed the closest relationship to B. falcatum L. The complete plastid genome of B. giraldii was comprehensively investigated for the first time and its phylogenetic position in Bupleurum was elucidated with high resolution in this study, these results will provide basis for the conservation and sustainable utilization of the germplasm resources of medicinal Bupleurum species.

Agarwood is an aromatic resin produced by Aquilaria sinensis and other plants of the Thymelaeaceae family in response to external injury. It is valued both medicinally and as a fragrance material. Sesquiterpenoids are key components responsible for the characteristic aroma and bioactivity of agarwood, but their biosynthetic pathway remains incompletely elucidated. In this study, three sesquiterpene synthase genes AsTPS2, AsTPS10, and AsTPS41, were cloned and identified from methyl jasmonate (MeJA)-induced callus of A. sinensis. Recombinant proteins were obtained by expression and purification in Escherichia coli. In vitro enzymatic assays revealed that AsTPS2 and AsTPS10 exhibit similar functions: Both catalyze farnesyl diphosphate (FPP) to produce α-humulene and β-caryophyllene, and also convert geranyl diphosphate (GPP) into acyclic monoterpenes (geraniol and nerol) as well as monocyclic monoterpenes (α-terpineol and isopulegol). AsTPS41 catalyzes FPP to form β-elemene, α-guaiene, and δ-guaiene, while also acting on GPP to generate geraniol, nerol, and α-terpineol. Protein modeling and docking of AsTPS2 and AsTPS10 using AlphaFold3 and AutoDock Vina indicated that, despite sharing 83.5% sequence identity, residues lining their active cavities are completely identical, supporting their overlapping catalytic activities. Further site-directed mutagenesis was performed on key amino acids lining the active cavity of AsTPS2. The single-site mutant Y411A generated two new products, β-elemene and shyobunol, while retaining the functionality of AsTPS2. Building upon Y411A, the double-site mutant G410A-Y411A produced four additional new products: α-Elemol, nerolidol, trans-farnesol, and pogostol. In the single-site mutant C449A, the proportion of β-caryophyllene increased from 7.2% in wild-type AsTPS2 to 55.1%. Based on these findings, a Saccharomyces cerevisiae expression system was established for AsTPS2, its mutant AsTPS2^C449A, and AsTPS41. Following isolation and structural analysis by nuclear magnetic resonance (NMR), the products α-humulene, β-caryophyllene, and δ-guaiene were identified. These results provide insights into the molecular mechanisms underlying agarwood formation and lay a foundation for further research on enzymes involved in the post-modification steps of agarwood sesquiterpenoid biosynthesis.

Lonicera japonica is a well-known traditional medicinal plant in China, and its floral volatiles are also used in the food, cosmetic, and horticultural industries. The artificially bred variety "Jiu Feng 2" is widely cultivated as a raw material for herbal teas and health products. However, its aromatic profile and underlying biosynthetic basis remain poorly understood. Here, we performed comparative volatile metabolomic and transcriptomic analyses of "Jiu Feng 2" and its parental variety "Da Mao Hua". Metabolomic analysis showed that terpenoids are the predominant volatiles in both varieties, and the golden flowers are the organs with the highest accumulation of volatile metabolites in both varieties. Comparative profiling of golden flowers identified 131 differentially accumulated metabolites (DAMs). β-Ocimene and α-farnesene were enriched in "Da Mao Hua", whereas α-terpinene and perillene were enriched in "Jiu Feng 2". These DAMs were mainly enriched in terpenoid-related secondary metabolic pathways, including monoterpenoid biosynthesis and sesquiterpenoid/triterpenoid biosynthesis. Transcriptome sequencing generated 268.61 Gb of high-quality data and identified 34 281 differentially expressed genes (DEGs). In golden flowers, DEGs were significantly enriched in pathways related to terpenoid, aliphatic, and phenylpropanoid metabolism. Integrated analysis revealed that both DAMs and DEGs were significantly co-enriched in terpenoid biosynthetic pathways. Quantitative real-time PCR further confirmed that two terpene synthase (TPS) genes involved in β-ocimene and α-farnesene formation, LjTPS15 and LjTPS16, showed significantly lower expression in "Jiu Feng 2", consistent with the reduced accumulation of these volatiles. Collectively, this study reveals the major differences in aromatic compound accumulation and differentially expressed genes between two L. japonica varieties, laying the foundation for subsequent in-depth analysis of their molecular mechanism and providing a theoretical basis for breeding of L. japonica.

Recombinant human arginase 1 (rhArg1) has significant therapeutic potential for the treatment of arginase 1 deficiency (ARG1-D) and arginine-auxotrophic cancers. However, its relatively low catalytic efficiency and limited in vivo stability restrict its clinical application. To enhance the catalytic performance of rhArg1, we employed the protein language model SaProt to perform zero-shot global mutational effect prediction across the entire rhArg1 sequence. Based on the predicted scores, the top 37 candidate variants were selected for experimental validation. Enzyme activity assays revealed that 16 of the 37 variants exhibited higher activity than the wild-type (WT) enzyme. Among them, the V30A, C45F, A56V, and H312P variants showed the most pronounced improvements, with catalytic activities increased by 33%, 23%, 22%, and 90%, respectively, compared with WT. Further kinetic characterization demonstrated that the H312P variant increased by 131% in catalytic efficiency (k_cat/K_M) relative to WT. These results indicate that the protein language model SaProt can effectively guide the rational engineering of arginase. The high-activity variants identified in this study provide promising candidate molecules for the treatment of ARG1-D and arginine-auxotrophic cancers.

Centipeda minima (L.) A. Braun et Aschers. is a traditional medicinal plant. Sesquiterpenoids are its core bioactive components, and sesquiterpene synthases (STSs) are the key enzymes in sesquiterpene biosynthesis. In this study, using C. minima as material, nine full-length candidate CmTPSs genes were screened from published transcriptome data by local Blast alignment combined with phylogenetic analysis. Bioinformatics approaches were used to analyze their sequence features, phylogenetic relationships, and conserved motifs. TBtools was employed to construct gene expression heatmaps for investigating their tissue-specific expression patterns. The catalytic functions of CmTPS1, CmTPS3, CmTPS4, and CmTPS5 were further verified by gene cloning, heterologous expression in Saccharomyces cerevisiae, and GC-MS analysis. Results showed that the nine CmTPSs genes fell into five TPS subfamilies, namely TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g, and displayed distinct tissue-specific expression in roots, stems, leaves, and flowers. CmTPS1 and CmTPS4 were specific caryophyllene synthases, while CmTPS3 and CmTPS5 were multifunctional synthases. This study provides key gene resources for elucidating the molecular mechanism of sesquiterpene biosynthesis in C. minima, and offers theoretical and technical support for the efficient production of its bioactive constituents and the development of its medicinal resources.

The dried roots of Phytolacca acinosa Roxb. are widely utilized in traditional Chinese medicine for their potent diuretic, anti-edematous, and detoxifying properties. Esculentosides, the primary bioactive constituents of P. acinosa, exhibit various pharmacological activities, including anti-inflammatory, anti-tumor, and antifungal effects. However, their clinical potential is hampered by low natural abundance and the inherent challenges of chemical synthesis due to their complex structural scaffolds, while synthetic biology represents a promising strategy for the sustainable and green production of esculentoside. However, the biosynthetic pathways remain unclear, and their key functional enzymes have not been reported yet. Herein, through activity-guided enzyme mining and transcriptome analyses, three novel glycosyltransferases (PaUGT2, PaUGT15, and PaUGT25) were functionally characterized from P. acinosa callus. These enzymes were capable of catalyzing the regio-selective glycosylation of 28-COOH of phytolaccagenin. Notably, PaUGT2 could efficiently catalyze 28-O-glycosylation of esculentoside A and esculentoside B, and PaUGT15 and PaUGT25 also exhibited catalytic activities toward these substrates. These findings not only preliminarily clarified the crucial enzymes for 28-O-glycosylation and elucidated the molecular basis for the structural diversity of oleanane-type esculentosides in Phytolacca plants, but also paved the way for further deciphering the biosynthetic pathway and constructing an artificial pathway of these glycosides.

Metabolic diseases such as obesity, type 2 diabetes, metabolic dysfunction-associated steatotic liver disease, have become major global public health challenges. Their pathogenesis is closely linked to dysregulation of immune and inflammatory responses. This review summarizes the central role of immunoinflammation underlying metabolic diseases, with emphasis on the interactive mechanisms between the immune system and metabolic system. It is broadly appreciated that metabolic disturbances trigger chronic low-grade inflammation, which exacerbates insulin resistance, adipose tissue dysfunction, and multi-organ damage through the involvement of both innate and adaptive immune cells. Among these, metabolic reprogramming of macrophages, the dual role of neutrophil extracellular traps in vascular injury and fibrosis, and the regulatory functions of Tregs represent critical nodes in diseases progression. From this metaflammation perspective, intervention strategies are proposed at multiple levels: Lifestyle modifications serve as a foundation to effectively improve immune-inflammatory status; exploring the immunomodulatory potential of existing drugs for repurposing in metabolic disease treatment, such as lansoprazole; and emerging therapies, such as GLP-1 receptor agonists and 11β-HSD1 inhibitors also demonstrate strong effect on alleviating inflammatory responses. Collectively, these important advances not only deepen our understanding of the immunological mechanisms underlying metabolic diseases, but also lay a theoretical foundation for developing novel strategies for the prevention and treatment of metabolic diseases, highlighting promising potential in extending healthspan.

Innate lymphoid cells (ILCs) mainly consist of natural killer (NK) cells, type 1 ILC (ILC1), type 2 ILC (ILC2), and type 3 ILC (ILC3), all exhibiting rapid responsiveness to pathogens and tumor cells. NK cells execute antitumor and antiviral functions through cytotoxic activity, cytokine secretion, and antibody-dependent cell-mediated cytotoxicity (ADCC), whereas ILC1, ILC2, and ILC3 modulate immune regulation and inflammatory responses within tissue microenvironments. Emerging evidence highlights the pivotal role of metabolic reprogramming in the development, activation, and effector functions of ILCs. Recent studies have further revealed that metabolic pathways are the core link regulating the development, maturation, activation and functional execution of ILCs. By precisely regulating the metabolic flux of substances such as glucose, amino acids and lipids, ILCs can achieve functional transition from a quiescent state to an effector state, while metabolic imbalance directly leads to abnormalities in their immune effects. This review systematically summarizes the metabolic characteristics of NK cells and other ILC subsets, and also discusses their underlying molecular mechanisms. Furthermore, we discuss how metabolic dysregulation in NK cells and ILCs contributes to pathological conditions such as obesity, cancer, and chronic viral infections, alongside potential therapeutic interventions. Finally, we elaborate on novel immunotherapeutic strategies targeting the metabolic reprogramming of ILCs, addressing unresolved questions in this rapidly evolving field. These insights establish a theoretical framework for developing next-generation ILC-based immunotherapies.

Aortic aneurysm (AA), an irreversible pathological dilation of the aorta exceeding 50% of its normal diameter, significantly increases the risk of aortic dissection and rupture-related sudden death, causing approximately 200 000 global deaths annually. AA can occur in various aortic segments and is primarily categorized into thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm. TAA, though less common, has a worse prognosis. While the etiology and molecular mechanisms differ between the two types, both are closely linked to inflammatory responses and the activation of various proteases. This review will focus on the general cellular and molecular events applicable to both conditions. AA is usually asymptomatic, and even when detected via ultrasonography, it is usually monitored due to not meeting surgical criteria. As no effective medication currently exists to halt its growth or reduce rupture risk, this review will briefly cover animal and clinical drug trials to explore potential therapeutic strategies.