Laccase-catalyzed repolymerization of technical lignins is an emerging biocatalytic strategy for upgrading lignin streams into polymeric systems and biomaterial precursors under mild reaction conditions. Lignosulfonates are attractive substrates for enzymatic polymerization due to their high water-solubility; however, their relatively low reactivity can limit their application. In contrast, organosolv lignin typically presents low-molecular weight and high phenolic content, which increase its susceptibility to laccase-mediated oxidation; however, its limited water solubility restricts enzymatic processing. In this work, lignin blending was evaluated as a strategy to modulate laccase-catalyzed polymerization by combining lignosulfonates (LS) with organosolv lignin from vine shoots (LVS). The effect of LVS incorporation on enzymatic oxidation was investigated through oxygen consumption measurements, viscosity evolution, molecular weight analysis and characterization of LS-based films. The presence of LVS modified oxidation kinetics, leading to prolonged oxidative activity, accelerated viscosity development and higher apparent molecular weight fractions at shorter reaction times compared to LS alone. LS-LVS films were produced to evaluate whether blending affected material surface properties. FTIR analysis supported structural changes consistent with oxidative coupling, while water contact angle measurements showed increased surface hydrophobicity in LS/LVS systems. Overall, the results demonstrate that blending lignins with different structural features can modulate enzymatic polymerization and influence the properties of LS films. This work highlights lignin blending as a simple biocatalytic strategy to modulate laccase-catalyzed polymerization and upgrade heterogeneous lignin streams toward lignin-based biomaterials within biorefinery concepts.

Filamentous fungi generate chemical diversity through several evolutionary strategies, including the elaboration of conserved biosynthetic intermediates into structurally distinct secondary metabolites. Emodin and its corresponding anthrone are central intermediates in fungal aromatic polyketide metabolism, yet the enzymatic logic that remodels these precursors into more complex scaffolds remains incompletely understood. To investigate this process, we examined an infection-responsive non-reducing polyketide synthase biosynthetic gene cluster (barc) from the entomopathogenic fungus Metarhizium anisopliae predicted to encode an emodin-derived aromatic polyketide pathway. Heterologous reconstruction of the barc cluster in Aspergillus nidulans led to the production of the previously reported diphenyl ether barceloneic acid A (1) and the xanthene barceloneic acid C2 (2), together with two new compounds derived from 2, barceloneic acids D and E (3 and 4). Compound 3 is a cyclopentanone adduct of the xanthene 2, whereas 4 is a polyketide-indole hybrid, highlighting the chemical reactivity of the xanthene 2 scaffold. Through combinatorial heterologous expression and feeding assays, we identified a predicted glucose-methanol-choline oxidoreductase (BarcL) as the enzyme responsible for converting diphenyl ether 1 to xanthene 2. This defines a previously uncharacterised oxidative scaffold-remodelling step in barceloneic acid biosynthesis, revealing how an emodin-derived pathway can access a xanthene scaffold. Deletion of the backbone gene barcB in M. anisopliae did not significantly alter virulence towards Tenebrio molitor (mealworm) under the conditions tested, suggesting that barceloneic acids are not essential for pathogenicity in this insect model.

Cinnamate 4-hydroxylase (C4H) is a key cytochrome P450 enzyme in the phenylpropanoid pathway, providing essential precursors for lignin and flavonoid biosynthesis. While well characterized in seed plants, the functional and regulatory features of C4H in lycophytes remain less well understood. In this study, a C4H gene (SmC4H) from the lycophyte Selaginella moellendorffii was cloned and functionally characterized. Sequence and comparative analyses showed that SmC4H retains the characteristic CYP73 framework, including a non-canonical PSRF sequence, and falls within the supported land-plant C4H/CYP73 cluster in a maximum-likelihood phylogeny. SmC4H also displayed the complete 10-motif architecture observed in the sampled land-plant C4Hs and a 3-exon/2-intron organization shared by many of these genes. Heterologous expression in yeast confirmed its cinnamate 4-hydroxylase activity, and kinetic analysis showed that SmC4H exhibited its highest apparent catalytic efficiency toward trans-cinnamic acid. In addition, SmC4H was capable of accepting the bulky cinnamate analog 2-naphthoic acid as a substrate, indicating a degree of substrate permissiveness. Subcellular localization analysis demonstrated that SmC4H is localized to the endoplasmic reticulum. Expression profiling revealed that SmC4H transcripts were most abundant in stems, which also exhibited the highest mean relative lignin content among the tissues examined. In contrast, flavonoid content was highly enriched in leaves, suggesting distinct tissue-level accumulation patterns of phenylpropanoid metabolites. Finally, promoter activity assays demonstrated that the SmC4H promoter (pSmC4H) is capable of driving gene expression in both vascular (Nicotiana benthamiana) and non-vascular (Marchantia polymorpha) plants. These findings provide a comparative reference for plant C4H biochemistry and promoter activity.

Although the biosynthesis of active ingredients in medicinal plants is co-regulated by diverse environmental stimuli, the endocrine mechanism by which brassinosteroids (BRs) coordinate physiological resilience with the in situ spatial distribution of saikosaponins in Bupleurum chinense DC. under combined drought and salinity remains largely elusive. We hypothesized that exogenous brassinosteroids (BRs) application alleviates oxidative impairment provoked by single and combined osmotic/ionic stresses while orchestrating the tissue-level topological routing of saikosaponin biosynthesis. Here, B. chinense seedlings were exposed to drought (15% PEG6000), salinity (150 mM NaCl), and combined stress with or without root-applied BRs (0.2 mg/L). Plant growth, foliar cytomembrane integrity, antioxidant enzyme kinetics, and key transcriptional profiles were evaluated and integrated with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)-based spatial metabolomics. Stress-induced morphological inhibition and membrane lesion ranked salinity > combined stress > drought. Exogenous BRs markedly counteracted stress-evoked membrane lipid peroxidation by sustaining CAT, SOD, and POD activities and curtailing H2O2 and superoxide accumulation, as corroborated by in situ DAB and NBT cytochemical visualization. Under drought and salinity, BRs further amplified the stress-elicited elevation of SS-a, SS-c, SS-d, and total saikosaponins through coordinated transcriptional activation of MVA/MEP pathway genes and pentacyclic triterpene synthases (HMGR, FPS, SS, SE, and β-AS). In sharp contrast, under combined drought and salinity, BRs application failed to upregulate these biosynthetic genes, revealing an antagonistic metabolic arrest driven by acute carbon allocation trade-offs. Spatial metabolomics demonstrated that common precursors (IPP, FPP, and 2,3-oxidosqualene) displayed an ubiquitous distribution across the whole root cross-section (cork, cortex, phloem, and xylem), whereas bioactive saikosaponins (SS-a, SS-c, SS-e, SS-f, and acetyl-SS-a) were preferentially compartmentalized in the secondary phloem and periderm. Exogenous BRs dynamically intensified precursor flux toward the outer cortical/phloem sink under single stress regimes. In summary, this work systematically characterizes BRs-regulated spatial patterns of saikosaponins under combined stress. It offers theoretical evidence for decoding stress adaptation mechanisms of B. chinense and provides practical support for optimizing its ecological cultivation techniques.

Glycogen phosphorylase (GP) catalyzes the rate-limiting step of glycogenolysis and supplies precursors for chitin biosynthesis in insects. Acyl urea compounds structurally related to benzoylphenylurea (BPU) insecticides are potent inhibitors of mammalian GP, raising the question of whether insect GP could serve as an independent insecticidal target. Here, we show that a mammalian GP inhibitor (GPI) potently inhibits recombinant Plutella xylostella GP (PxGP; IC50=2.96 nM), while the BPU insecticide diflubenzuron does not. Molecular docking and MM/GBSA analysis predict that this selectivity reflects differential side-chain engagement. Despite potent in vitro inhibition, GPI exhibits no insecticidal activity, and RNAi-mediated knockdown of PxGP does not impair development or survival. We provide evidence that insects compensate through upregulation of gluconeogenic enzymes (PEPCK, G-6-Pase) and protein catabolism, with enzyme activity measurements confirming ~29% reduction in GP-a activity following RNAi. Fitness assessment reveals transient larval weight loss with complete recovery of pupal weight, adult morphology, and fecundity. These findings demonstrate that GP is functionally non-essential under the tested conditions, likely through gluconeogenic compensation and alternative carbon sources, and highlight the importance of metabolic network redundancy in target-based insecticide design.

Furofuran lignans are the primary insecticidal and medicinal bioactive compounds in Phryma leptostachya, yet their species-specific biosynthetic pathways remain incompletely deciphered. We established a Tobacco rattle virus-based virus-induced gene silencing system, identifying vacuum-assisted infiltration of Agrobacterium tumefaciens strain GV3101 at the four-leaf stage as the optimal inoculation condition. Silencing the PlCYP81Q38 gene via this system markedly decreased root accumulation of sesamin and 6-demethoxyleptostachyol acetate. Combined transcriptomic and metabolomic analyses revealed that PlCYP81Q38-silenced plants exhibited upregulated phenylpropanoid pathway genes, including phenylalanine ammonia lyase, cinnamoyl-CoA reductase (CCR), and dirigent protein 1, alongside elevated levels of lignan precursors (coniferyl alcohol, (+)-pinoresinol). Within this pathway, CCR enzymes catalyze hydroxycinnamoyl-CoA substrates across multiple branches. We characterized the PlCCR family and confirmed that PlCCR4 acts as a dedicated catalytic enzyme for lignan biosynthesis. It exhibited the highest in vitro catalytic efficiency for feruloyl-CoA (Kcat/Km = 244.90 min-1/nM) and was essential for lignan biosynthesis in vivo. Its homolog PlCCR5 exhibited significantly lower efficiency; homology modeling and molecular docking indicated that an Asp64-to-Glu64 substitution in the NADPH-binding region may alter the cofactor microenvironment, plausibly reducing its activity. This study identifies PlCCR4 as an important upstream enzyme, providing a valuable genetic target for engineering sustainable production of bioactive lignan insecticides.

Prolonged glucocorticoid (GC) therapy is frequently complicated by secondary vitamin D3 deficiency, which acts as a potential pathogenetic modulator of GC-induced neurotoxicity. This study investigated the role of disturbed calcium homeostasis and neuroinflammation in GC-induced brain damage and evaluated the protective potential of vitamin D3 (VD3, cholecalciferol). Female Wistar rats (n = 54) were divided into control, GC-treated (prednisolone, 5 mg/kg/day, 30 days, per os), and VD3-correction (1000 IU/kg/day, concurrent with GC) groups. Analysis of calcium-binding protein levels (calbindin-D28K, S100B, osteocalcin) and neurospecific markers (NF-L, MBP, GFAP) was performed using ELISA, Western blot, and confocal microscopy. Prolonged prednisolone administration caused significant systemic and local 25(OH)D3 deficiency, accompanied by a twofold reduction in osteocalcin protein content and elevated calbindin-D28K levels. Reactive astrogliosis, characterized by increased GFAP[+] cell counts and S100B content, occurred alongside decline in NG2[+] oligodendrocyte precursors. Significant neuroinflammation was recorded: TNF-α levels rose 1.62-fold in the cortex and 1.65-fold in the hippocampus, while IL-1β increased 2.66-fold and 2.40-fold, respectively. Furthermore, markedly increased serum levels of NF-L, S100B, GFAP, and MBP indicated alterations consistent with blood-brain barrier dysfunction and neuro-glial disintegration. VD3 therapy effectively restored serum markers (osteocalcin, S100B, MBP), suppressed astrocyte reactivity, and normalized cortical cytokine levels while partially replenishing NG2[+] cell pool. Although its impact on hippocampal inflammation was limited, these findings demonstrate that maintaining adequate VD3 levels is paramount for preserving CNS integrity by stabilizing calcium buffering and suppressing neuroinflammation. Our results emphasize clinical importance of monitoring VD3 status to mitigate risk of glucocorticoid-induced neurotoxicity and associated neurological complications.

The processes of clot formation and clot dissolution with fibrinolysis underpin the pathophysiological mechanisms of acute ischaemic stroke (AIS) and its treatment. Detailed understanding of the modulation of this system is required to develop effective and safe stroke therapeutics. Clot formation occurs with the interaction of the coagulation cascade, along with platelets and fibrin meshwork. Conversely, fibrinolysis involves the serine protease plasmin, its zymogen, plasminogen and its enzymatic conversion by tissue plasminogen activator. The regulators and inhibitors that modulate the interactions in clot formation and fibrinolysis can be potential targets for new and emerging stroke therapies. The aim of this narrative review is to help inform stroke clinicians and researchers about the role of the fibrinolytic system and its inhibitors in AIS and the potential for new therapeutic targets. We outline the processes involved in clot formation and dissolution, the role of endogenous inhibitors of the fibrinolytic system, followed by potential therapeutic targets including tissue plasminogen activators as well as novel therapies and adjuncts. Better understanding of these processes will provide insight into the variable response to current stroke fibrinolytics amongst AIS patients and help to develop new and improved AIS therapeutics in future.

The number of oxygenated derivatives of cholesterol, its precursors, and other biologically relevant sterols that exist in biological systems is unknown and may extend substantially beyond the oxysterols currently characterized. Successive oxidation, reduction, hydrolysis, sulfation, glucuronidation, esterification, and conjugation reactions can generate a large chemical space that we refer to as the oxysterolome. However, chemical possibility does not establish endogenous existence, and endogenous existence does not establish biological function. Defining this metabolic space requires a progression from analytical annotation to structural authentication, metabolic reconstruction, and biological validation. Iterative metabolism broadens physicochemical diversity, making analytical coverage method-dependent and requiring complementary approaches for different metabolite classes. Authenticated metabolites should subsequently be connected through precursor-product relationships, enzyme dependence, isotope tracing, metabolic flux, and spatial organization. Biological relevance requires separate evidence linking endogenous exposure and pathway perturbation to molecular mechanisms and phenotypes. Further metabolism can generate, terminate, or redirect biological activity, but these outcomes cannot be predicted from structural complexity. The oxysterolome should therefore be approached not as an expanding catalogue of oxygenated sterols, but as an experimentally reconstructed metabolic network integrating molecular identity, pathway connectivity, flux, and biological causality.

The prevalence of proline residues in intrinsically disordered proteins, functional hinges of enzymes, and protein interaction sites highlights the importance of studying their local dynamics and interactions at atomic resolution. While nuclear magnetic resonance (NMR) spectroscopy is, in principle, ideally suited for such investigations, the absence of an amide proton precludes the use of conventional 1H-15N amide-detected experiments. 1H-13C-based approaches, though possible, remain challenging in large proteins. In this work, we present a selective labeling strategy that introduces an isolated 1H2δ-13Cδ-15N spin system in proline residues in otherwise deuterated proteins. Our method leverages the enzymatic conversion of ornithine, synthesized from cost-effective isotopic precursors. We demonstrate the utility of this approach by applying it to a 134 kDa enzyme using magic-angle spinning NMR, highlighting its potential to efficiently probe functionally relevant sites in proteins.

Ether glycerophospholipids (ether lipids) are a subclass of glycerophospholipids that are synthesized at the peroxisome and constitute nearly 20% of the total phospholipid content in metazoans. Biochemically, ether lipids have an alkyl chain attached by an ether bond at the sn-1 position of the glycerol backbone (alkyl-ether) primarily containing choline or ethanolamine head groups. Some ether lipids contain a cis bond adjacent to the ether linkage (alkenyl-ether) and are commonly referred to as plasmalogens. While the importance of ether lipids in humans is clear, highlighted by the severity of inherited peroxisomal disorders caused by ether lipid deficiency, the molecular roles of these lipids remain poorly understood. Here, we identify a link between ether lipid metabolism, peroxisome degradation, and oxygen availability. We find Fatty acyl-CoA-reductase 1 (FAR1), the rate-limiting enzyme in peroxisomal ether lipid metabolism, acts as a peroxisome receptor necessary for peroxisome degradation when oxygen is limiting. Hypoxia increases choline ether lipid abundance, promotes autophagy activation, and peroxisome degradation. Mechanistically, FAR1 binds ATG8-family protein LC3B and is necessary and sufficient to control peroxisome degradation under hypoxia. FAR1's enzymatic function as well as its capacity to bind LC3B is necessary to degrade peroxisomes under hypoxia. Similarly, loss of multiple components of peroxisomal ether lipid metabolism blocks peroxisome degradation under hypoxia in cells and in vivo. In mouse models of chronic HIF activation, Far1 is a direct HIF target gene and is necessary for peroxisome degradation in this setting. Increasing ether lipids using ether lipid precursors activates autophagy and promotes autophagy-dependent cardioprotection against ischemic injury. This work reveals FAR1 as a critical regulator of peroxisome homeostasis in the setting of oxygen limitation.

Urokinase is often regarded as an older thrombolytic displaced by fibrin-selective agents in reperfusion medicine. Yet its decline has been uneven. This mini-review reframes its contemporary role through three determinants: route, compartment, and access. The systemic role of native urokinase has receded, particularly in stroke. Adjunctive thrombolysis after endovascular reperfusion remains unsettled: POST-UK and POST-TNK were neutral, whereas PEARL and ANGEL-TNK reported higher 90-day excellent-outcome rates. Trial differences preclude a uniform conclusion or generalization to primary intra-arterial thrombolysis and local fibrinolysis. Native urokinase retains a niche when delivered directly into clot-containing compartments or established catheters, including intracranial blood collections, chronic subdural hematoma, selected venous thromboembolism, loculated pleural disease, and catheter-related thrombotic dysfunction. In these settings, it functions as a low-cost, familiar, locally delivered fibrinolytic that facilitates clot clearance or drainage. Supporting evidence remains heterogeneous and often derives from retrospective cohorts, small studies, and variable protocols. Recombinant human prourokinase should be distinguished pharmacologically and clinically from native urokinase. As a zymogen precursor preferentially activated at the fibrin-rich thrombus surface, it represents a renewed systemic reperfusion pathway. Following the historical proof of concept provided by PROACT II, PROST and PROST-2 showed intravenous recombinant human prourokinase to be non-inferior to alteplase for excellent 90-day functional outcomes; PROST-2 additionally reported lower rates of symptomatic intracranial hemorrhage and major bleeding. External generalizability and direct comparison with tenecteplase remain unresolved. The contemporary urokinase pathway therefore reflects a dual evolution: native urokinase retains focused local and access-driven roles, whereas recombinant prourokinase has re-entered systemic reperfusion therapy.

Genetically encoded reporters are indispensable tools for monitoring gene expression, transformation efficiency, and diverse cellular processes in plants. The betalain-based RUBY reporter has recently emerged as a convenient visual marker requiring no substrates or specialized equipment, and it has been widely used for a variety of applications. However, the developmental and metabolic consequences of its expression remain unexplored. Here, we show that independent transgenic lines of Arabidopsis thaliana expressing RUBY exhibit profound alterations in growth and reproductive development. RUBY-expressing plants were significantly smaller, produced fewer leaves, flowered later, and frequently developed malformed siliques with reduced seed yield. In addition, root architecture was severely impaired, with a marked reduction in the number of lateral roots. This phenotype was not rescued by indole-3-acetic acid (IAA). Targeted metabolomic profiling revealed widespread alterations in amino acid and hormone pools, including reduced indole-3-acetic acid and gibberellin, alongside accumulation of auxin precursors. Disruption of RUBY expression by suppressing the third enzyme (glucosyltransferase) restored the RUBY-associated phenotypes to wild-type. Together, our findings show that RUBY expression in Arabidopsis thaliana is not metabolically neutral; rather, it perturbs both development and metabolism by disrupting hormone and amino acid homeostasis. These results highlight reporter-associated trade-offs and underscore the necessity of regulated expression systems, such as inducible or tissue-specific expression, to expand the utility of RUBY in plant research and biotechnology.

Cryoconite granule formation underpins the physical structure and biogeochemical functioning of glacier cryoconite holes. Central to this process is the extracellular polymeric substance (EPS) matrix, produced by microorganisms, which binds microbial cells and mineral particles into stable aggregates while retaining organic matter and nutrients. However, the physiological traits governing the EPS matrix construction and degradation remain poorly understood. Here, we investigated microbial capacities for biofilm formation, EPS-related substrate utilization, and extracellular hydrolysis in Antarctic cryoconite communities over a 40-day melt season. After five weeks of incubation, 49 of 56 (88%) cryoconite debris samples formed detectable biofilms, with biofilm-forming capacity peaking in samples collected after 30 days and significantly exceeding that at the beginning and after 10 days of the melt season (p < 0.05). Across the melt season, the proportion of C-, N-, P- and S-containing substrates utilized increased from 33% to 90% of the 260 compounds tested, with significantly greater utilization of organic nitrogenous compounds than other substrate groups (p < 0.05). Biofilm-forming capacity correlated positively with utilization of EPS-backbone precursors, EPS-associated glycosides, and compounds associated with EPS modification (Spearman's ρ = 0.6, p < 0.05). Forty bacterial genera were recovered, dominated by Cryobacterium, Polaromonas, Arthrobacter, and Chryseobacterium, with representative isolates exhibiting biofilm-forming phenotypes and diverse extracellular enzyme activities. Together, these findings demonstrate complementary microbial capacities for biofilm formation and EPS matrix-associated organic matter transformation that may influence carbon and nutrient cycling in Antarctic cryoconite ecosystems.