With the global ban on antibiotic growth promoters, identifying safe, effective alternatives for poultry production is urgent. Probiotics, particularly Bacillus amyloliquefaciens (BA), have shown promise in improving animal health and performance, but research in geese remains limited. This study aimed to evaluate the effects of dietary BA supplementation on growth performance, immune function, antioxidant capacity, intestinal morphology, microbiota, and transcriptome in growing Zi geese. A total of 180 50-day-old male Zi geese were randomly assigned to a control group (basal diet) or an experimental group (basal diet supplemented with 1 × 108 CFU/g BA) for 40 days. Growth performance, serum immune indices, intestinal antioxidant parameters, morphology, jejunal microbiota, and transcriptomic profiles were assessed. BA supplementation significantly increased average daily gain and decreased feed-to-gain ratio (p < 0.01). Serum IgA, IgG, IgM, and IL-4 increased, while IL-2, IL-1β, and TNF-α decreased (p < 0.01). Intestinal CAT, GSH-Px, and T-AOC increased (CAT in duodenum and jejunum, p < 0.01; CAT in ileum, p < 0.05; GSH-Px in duodenum and ileum, p < 0.01; GSH-Px in jejunum, p < 0.05; T-AOC in all segments, p < 0.01), and jejunal MDA decreased (p < 0.01). Villus height and V/C ratio improved. Jejunal microbial diversity increased, with higher Proteobacteria and lower Pseudomonas and Escherichia-Shigella. Transcriptomic analysis revealed DEGs enriched in calcium signaling, cell junction, and intestinal barrier pathways. Dietary BA at 1 × 108 CFU/g enhances growth, immunity, and intestinal health in Zi geese by improving gut function, remodeling microbiota, and modulating gene expression, supporting its use as a green feed additive.

Wound-induced cellular reprogramming is a fundamental process for plant regeneration, yet the molecular mechanisms of this process in cotton remain poorly understood. In this study, we identified and systematically characterized the GhWINDs gene family in upland cotton (Gossypium hirsutum), revealing its plant-specific conserved features. Among these genes, GhWIND1-A, which encodes a nucleus-localized AP2/ERF transcription factor, significantly promoted callus enlargement when overexpressed in both Arabidopsis thaliana and upland cotton. Further analyses demonstrated that GhMYC2, a central regulator of jasmonic acid (JA) signaling, directly binds to and activates the GhWIND1-A promoter and also promotes callus enlargement in cotton hairy roots. Transcriptomic and molecular biology analyses showed that GhWIND1-A acts as a key regulatory hub coordinating the reprogramming of multiple phytohormone pathways and directly activates GhSWEET4, which encodes a sugar transporter, thereby providing the metabolic energy required for rapid callus enlargement. Consistently, treatment with JA inhibitors reduced endogenous JA levels, suppressed GhSWEET4 expression, and significantly inhibited callus enlargement, whereas exogenous MeJA application elevated JA content, induced GhSWEET4 expression, and promoted callus enlargement. Collectively, these findings reveal a JA-GhMYC2-GhWIND1-A-GhSWEET4 regulatory cascade that links wound-induced hormonal signaling with metabolic energy regulation to promote callus enlargement. Given that cotton is a major industrial fiber crop and that callus enlargement is a critical trait for tissue culture and genetic transformation, this study provides new insights into the molecular basis of callus enlargement in cotton and identifies potential regulatory targets for improving cotton tissue culture systems.

The industrial cultivation of the medicinal mushroom Cordyceps militaris relies heavily on expensive animalderived supplements such as silkworm pupae powder, creating economic and sustainability challenges. This study evaluated spent mushroom substrates (SMSs) from seven saprophytic species as alternative cultivation inputs. Among them, Hypsizygus marmoreus-SMS proved the most effective, increasing fresh fruiting body weight by 35.33% and matching the yield-promoting performance of silkworm pupae powder. To identify the key growth-enhancing factor, we integrated targeted metabolomic profiling and exogenous validation assays. Malic acid, highly enriched in H. marmoreus-SMS, was identified as a growth-associated organic acid; exogenous malic acid supplementation increased fresh weight by 20.97%. Spatiotemporal transcriptomic analysis further revealed that malic acid induced a stage-specific transcriptional response, prominently at the primordium stage, marked by coordinated remodeling of central carbon metabolism, including the TCA cycle, gluconeogenesis, the pentose phosphate pathway, and one-carbon metabolism. These metabolic shifts suggest an enhanced supply of precursors for fruiting body development. Our findings establish H. marmoreus-SMS not merely as an agro-industrial by-product repurposed as a nutrient source, but as a dual-function input that combines basal nutrition with malic acid-mediated developmental reprogramming. This work provides a mechanistic basis for replacing animalderived supplements with valorized SMS in circular mushroom production.

We report the first collective biomimetic synthesis of xanchryones A (1), B (2), and I-N (3-8, respectively), all featuring a benzoxazole scaffold, by employing a synthetic strategy based on biogenetic building blocks (BBBs). These compounds were efficiently assembled in six steps from commercially available 3,4,5-trimethoxyphenol. Notably, the benzoxazole ring was constructed for the first time using rationally designed p-quinones and amino acids as substrates, enabling precise control of the nitrogen and oxygen positions in the benzoxazole ring. This BBB-based biomimetic strategy provides a concise route to structurally diverse natural benzoxazoles and offers insight into their biogenetic origin.

Seven undescribed diarylheptanoid derivatives, alpinins A - G (1-7), together with nine known ones (8-16), were isolated and identified from the rhizomes of Alpinia officinarum Hance. Architecturally, compounds 1-4 represent four unusual dimeric diarylheptanoids featuring distinct linkage patterns, while compounds 5 and 6 are newly identified adducts consisting of diarylheptanoid motifs with a sesquiterpene and a monoterpene unit, respectively. The structures and absolute configurations of these compounds were elucidated by comprehensive spectroscopic data, DFT-based DP4+ analysis, as well as electronic circular dichroism (ECD) calculations. Additionally, the α-synuclein aggregation inhibitory activities of these isolates were evaluated using native mass spectrometry and fluorescence-based assays. Among them, compounds 7, 10, 12, and 13 exhibited significant inhibitory effects on α-synuclein aggregation and showed potent neuroprotective activities against α-synuclein aggregate-induced cytotoxicity in SH-SY5Y cells.

Abstract C 18 H 16 O 5 , monoclinic, P 2 1 / n (no. 14), a = 13.4677(4) Å, b = 4.0887(2) Å, c = 26.1789(8) Å, β = 93.996(3)°, V = 1,438.05(9) Å 3 , Z = 4, R gt ( F ) = 0.0356, w R ref ( F 2 ) = 0.0356, T = 293.75(10) K.

Huangqin Qingre Chubi Capsules(HQC) are commonly used in clinical practice to treat rheumatoid arthritis(RA). It is composed of Scutellariae Radix, Gardeniae Fructus, Coicis Semen, Clematidis Radix et Rhizoma, and the stir-fried Persicae Semen. However, the pharmacological substance basis and mechanism are not yet clear. This study systematically elucidated the pharmacological substance basis and mechanism of HQC by the research strategy of &quot;identification of target tissue migration components-network mechanism prediction-multidimensional experimental verification&quot;. An adjuvant-induced arthritis(AA) rat model was established, and the serum and synovium migration components of HQC in normal and AA model rats were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS). These serum and synovium migration components were integrated for network pharmacology prediction, which was followed by multi-dimensional validation using molecular docking, RT-qPCR, and pharmacodynamic evaluation. The results showed that 46 serum and 13 synovium migration components were identified in normal rats, and 59 serum and 15 synovium migration components were identified in AA rats, indicating that the RA disease environment affects the entry of HQC into the bloodstream and migration to the synovium. Comparative analysis of components common and unique to normal and model groups identified geniposide, baicalin, daucosterol, coptisine, acteoside, luteolin, and crocin as migration components reaching the target site, reflecting the target tissue enrichment characteristics of HQC. Network pharmacology of the migration components screened 7 core targets: GAPDH, TNF-α, AKT1, PTGS2, NF-κB1, MAPK1, and SphK1, which were enriched in key RA signaling pathways including PI3K-AKT, HIF-1, VEGF, and TNF. Molecular docking and RT-qPCR confirmed good binding activity and regulatory capacity between key components and core targets. In vivo validation further demonstrated that HQC significantly improved joint inflammation and synovial hyperplasia in AA rats. This study confirmed that HQC exerted an anti-RA effect by targeting chronic inflammation and metabolic disorders through multi-target regulation of PI3K-Akt and other signaling pathways via serum and synovium migration components. The research provides a scientific basis for the clinical application of HQC and offers a new strategy for studying the mechanisms of TCM.

Pumpkin (Cucurbita moschata) seed protein isolate (PSPI) is a promising plant protein, yet its fermented gel exhibits poor quality for practical applications. To overcome the limitations of PSPI and enhance its applicability in gelation systems, a synergistic strategy combining protein-glutaminase (PG) co-fermentation with ultrasound (US) pretreatment was developed for fermented pumpkin seed protein isolate (PSPFG). The results showed that compared with native PSPFG, PG co-fermentation effectively accelerated the fermentation process, shortening the fermentation endpoint time by 4 h (from 10 h to 6 h). US pretreatment induced protein unfolding and exposed more hydrophobic groups, thus enhancing gel binding strength and reducing the risk of syneresis and gel collapse. The combined treatment of US+PG significantly reduced gel particle size from 30.82 ± 2.97 μm to 8.91 ± 0.80 μm with a more uniform distribution, improved gel cohesiveness, and formed gels with enhanced water-holding capacity (WHC) by 25.54% compared with native PSPFG, thereby generating a finer fermented gel structure suitable for alternative yogurt products. LF-NMR analysis further confirmed that the synergistic treatment eliminated free water content and alleviated syneresis in the gels. Overall, the synergistic combination of ultrasonication and PG deamidation provided a reliable strategy for constructing plant protein gels with faster fermentation kinetics and higher structural stability.

Bacillus subtilis is a widely used agricultural probiotic for plant disease control. We previously reported the novel B. subtilis HF1, which produces abundant fengycin and surfactin lipopeptide homologues. This study investigates fengycin's functions in HF1. Surprisingly, the fengycin-deficient mutant Pfen-1 exhibited enhanced antagonism against Sclerotinia sclerotiorum. Using Pfen-1 as an ideal tool to probe fengycin function, we tested lipopeptide extracts against 20 plant pathogenic fungi. Results indicated that fengycin is the primary antifungal lipopeptide in HF1 against most fungi but not against S. sclerotiorum. We elucidated the specific, efficient biocontrol mechanism of Pfen-1 against S. sclerotiorum in rapeseed. Pfen-1 consistently showed higher activity than HF1 in some key enzymes including cellulase, proteinase, chitinase, amylase, and β-1,3-glucanase - across different culture stages, a finding corroborated by transcriptome data. Additionally, Pfen-1 significantly boosted defensive enzyme activity in rapeseed, contributing to its excellent biocontrol efficacy. This work provides new insights into fengycin's functions and offers a promising mutant strain for developing more effective agents against S. sclerotiorum in rapeseed.

Arabinoxylan (AX) from wheat bran is an effective antifreeze polysaccharide, but its structure–activity relationship in ionic environments remains poorly understood. In this study, representative Hofmeister salts (Na2SO4, NaCl, NaNO3, KCl, and MgCl2) were introduced into AX solutions to elucidate the ion-specific regulation of AX antifreeze activity. Heterogeneous ice nucleation, single ice-crystal growth, and ice recrystallization inhibition analyses demonstrated that ions significantly affect AX activity, with anions playing a more pronounced role than cations. At 0.05 M, Na2SO4 markedly enhanced the antifreeze activity of AX, whereas NaCl and NaNO3 showed limited or inhibitory effects. Structural analyses revealed that AX adopts a flexible random-coil conformation in solution, while sulfate ions promoted a more compact molecular organization. Integrated mechanistic analyses suggested that ion-induced conformational rearrangement and hydration regulation collectively modulate AX–associated hydration environments and enhance its ice-affinity. Specifically, SO42-–induced AX compaction was accompanied by altered interfacial hydrogen-bonding states, stabilized local hydration interactions, and modulation of long-range hydrogen-bond network dynamics. These findings establish an ion-specific conformation–hydration coupling mechanism for understanding and regulating polysaccharide antifreeze activity.

The rational design of antifreeze peptides remains a significant challenge. In this study, we developed a series of short peptides with antifreeze activity: G-1 (GPACCCGPA), G-2 (GPACCCGPA)2, and G-3 (GPACCCGPA)3, by mimicking the repetitive sequences and structural motifs of natural antifreeze proteins (AFPs). Notably, antifreeze activity was enhanced with increasing peptide length and molecular weight, with G-3 exhibiting the most pronounced ice recrystallization inhibition. Systematic assays, including splat cooling, sucrose sandwich, and nanoliter osmometry, revealed that peptide elongation markedly suppressed ice growth rates without inducing thermal hysteresis (TH). Structural analyses demonstrated that increased peptide length enhanced α-helical content and promoted the formation of ordered nanostructures with moderate aggregation, enabling multiple ice-binding motifs to interact directly with the ice-water interface. The combined results of water spectral analysis and molecular dynamics simulations further revealed that peptides modulated the organization of surrounding water clusters by increasing local hydrogen-bond ordering while disrupting long-range hydrogen-bond dynamics. This dual modulation weakened the integrity of the extended hydrogen-bond network in bulk water and delayed the transition into a highly ordered crystalline ice phase. Mechanistically, G-3 achieves dual functionality by coupling efficient initial ice templating with strong inhibition of crystal growth, providing a clear rationale for its superior activity. Furthermore, the peptides exhibited excellent biocompatibility and significantly improved post-thaw cell viability. Overall, the design strategy proposed in this study highlights the potential of modular peptide engineering for developing biocompatible, tunable cryoprotectants, offering mechanistic insights into peptide-water interactions and clarifying the molecular basis of peptide-mediated antifreeze activity.

BACKGROUND:Tuberculosis is the leading cause of death among chronic infectious diseases caused by a single pathogen. Macrophages serve as the first line of immune defense, and pulmonary epithelial cells are essential for lung structural integrity and function. However, these cells are highly susceptible to death following Mycobacterium tuberculosis (Mtb) infection. Preserving cell viability has been shown to significantly enhance host anti-infective capacity. PURPOSE:This study aimed to screen an in-house natural product library derived from Lingnan-region herbal medicines in China for cell-protective activity in H37Rv-infected cells and investigate the underlying mechanisms. METHODS:Cell viability in macrophages and epithelial cells was assessed using CCK-8 and LDH assays. Pathways involved in cell death inhibition were explored via RNA-seq. Natural product target proteins were identified using DARTS and LC-MS. Gene and protein expression were validated by qRT-PCR and western blotting. Intracellular H37Rv viability was determined by CFU counting. RESULTS:A novel natural compound, cleistoperlone J (CJ), was identified as a cell viability-protective agent. CJ inhibited ferroptosis by modulating cellular antioxidant balance. Glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in glutathione metabolism, acted as a critical mediator of CJ. By binding to G6PD, CJ enhanced its level and activity via inhibition of ubiquitination-mediated degradation, thereby suppressing ferroptosis in Mtb-infected cells. Notably, both CJ and the G6PD activator AG1 potentiated the antibacterial activity of isoniazid in Mtb-infected macrophages. CONCLUSION:These findings suggest that CJ holds promise as an adjunctive therapeutic agent for tuberculosis chemotherapy by inhibiting ferroptosis in Mtb-infected cells.

Securinine (1) and its three stereoisomers, allosecurinine (2), virosecurinine (3) and viroallosecurinine (4), can be produced in the medicinal plant Flueggea suffruticosa. Their stereochemistry critically impacts biological activity: only securinine acts as a potent GABAA receptor antagonist and was once clinically used as a central nervous system stimulant. However, the mechanism underlying their stereochemical control has remained elusive. Here, through isotope labeling, we elucidated that (±)-menisdaurilide and (±)-aquilegiolide serve as key intermediates. They can non-enzymatically couple with Δ1-piperideine to form neosecurinane alkaloids, subsequently undergoing intramolecular rearrangement to generate 1–4. Notably, the C6–OH configuration of (±)-menisdaurilide and (±)-aquilegiolide serves as a gatekeeper for controlling selective production of levorotatory products 1/2 and dextrorotatory products 3/4. To identify enzymes governing this stereoselective formation of C6–OH, we employed an isotope-guided spatial transcriptomic strategy, which revealed a critical spatial discrepancy: biosynthesis of securinine and its stereoisomers occurs in stems, whereas they accumulate in roots. Leveraging this spatial localization insight, we ultimately identified three stem-preferentially expressed ketoreductases that mediate stereoselective biosynthesis of these alkaloids. This study comprehensively delineates the mechanistic basis for stereochemical control of securinine and highlights the utility of the isotope-guided spatial transcriptomic strategy in efficiently identifying biosynthetic genes of plant natural products.

Oxidative stress drives skin aging, barrier impairment, and inflammatory amplification, making dietary antioxidants potential systemic contributors to cutaneous redox homeostasis. Edible fungi are distinctive sources of redox-active metabolites, particularly ergothioneine, a stable sulfur-containing antioxidant whose cellular uptake is mediated by the ergothioneine transporter OCTN1 (SLC22A4). This review evaluates ergothioneine, polysaccharides and β-glucans, cordycepin, phenolics, and Ganoderma triterpenoids as processing-sensitive dietary bioactives with redox relevance rather than topical cosmetic ingredients. We examine how cultivation, drying, cooking, extraction, fermentation, and microbial biomanufacturing determine antioxidant formation, retention, oral bioaccessibility, and dose realism, and how antioxidant response, inflammatory, mitochondrial, and gut microbiota-mediated pathways connect intake to skin endpoints. The strongest oral evidence concerns biomarker-linked ergothioneine-rich Pleurotus. Smaller Flammulina velutipes and Sparassis crispa trials report hydration or transepidermal water loss signals without comparable exposure biomarkers, whereas purified ergothioneine provides provisional non-mushroom food evidence. Compared with better-established oral ingredients, edible fungi offer distinctive food technology advantages but a narrower human evidence base. Priorities include processing-aware quality and safety markers, contaminant control, standardized digestion models, dose-realistic exposure estimates, and biomarker-anchored randomized human trials.

Crystallographic analysis has become the most reliable method for elucidating the chemical structures and absolute configurations of natural products. In this study, we present an in situ acetylation-tagging approach to enable the cocrystallization of difficult-to-crystallize molecules bearing hydroxyl or amino groups with cyclic trinuclear complexes (Ag3Pz3). Single crystals are readily obtained upon slow evaporation after adding acetylating reagents and then Ag3Pz3 into an NMR tube. This acetylation tagging procedure introduces electron-rich acetyl groups with conformational flexibility that serve as beacons for electron-deficient Ag3Pz3, leading to effective π-acid···base supramolecular interactions. Using this NMR tube protocol, we have successfully determined the structures of over 40 difficult-to-crystallize molecules, including flexible long-chain alcohols, macrocyclic alcohols, and aliphatic amines. These molecules, if not acetylated, would not cocrystallize with Ag3Pz3. Further applications demonstrate the effectiveness of this protocol in determining the chemical structures and absolute configurations of highly complex natural glycosides and pharmaceutical molecules, showcasing broad substrate compatibility and its potential for analyzing difficult-to-crystallize molecules containing hydroxyl or amino groups.

Avian metapneumovirus subtype B (aMPV/B) infections significantly affect the global poultry industry. However, the virulence determinants and attenuation mechanism remain unknown. Here, a series of chimeric and mutant viruses was constructed, and their pathogenicity was evaluated in a specific-pathogen-free (SPF) chicken model. First, substitutions in different genes (N, P, F, SH, G, and L) and amino acid sites (323rd, 396th, and 522nd residues in F protein) in virulent (LN16-V) and attenuated (LN16-A) viruses revealed that the residue 396th in F protein is closely related to replication ability in vitro and in vivo and is a critical determinant of aMPV/B virulence in chickens. Further studies revealed that the K396R mutation in the F protein decreases the adsorption of aMPV/B to DF-1 cells, which reduces the binding of the F protein with αVβ1 integrin. Structural and surface plasmon resonance analysis indicated that the K396R mutation primarily reduced the electrostatic potential of the RDD motif of the F protein that binds with αVβ1 and decreased the binding between the F protein and αVβ1, which is a critical determinant of the replication ability of aMPV/B. Collectively, these findings not only contribute to a better understanding of the aMPV attenuation mechanism but also offer novel strategies for developing Metapneumovirus members live vaccines.IMPORTANCEBoth aMPV and hMPV belong to the Metapneumovirus family and cause the most acute respiratory diseases in poultry and humans, respectively. Recently, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the USA, largely attributed to aMPV/B infections. Live-attenuated vaccines developed by the blind passage of virulent strains in tissue culture have been widely used to prevent aMPV/B infection. However, the mechanism of aMPV/B attenuation remains unclear. Here, we identified the F gene as a key determinant of the virulence of aMPV/B and confirmed that residue 396 in the F protein plays an important role in attenuating the virulence of aMPV/B. Importantly, we found that the K396R mutation decreased the binding affinity between the F protein and αVβ1 and reduced the replication ability of aMPV/B. This is the first study to identify the key virulence genes and amino acid residues of aMPV/B and elucidate the molecular mechanisms underlying the attenuation of virulence. Our work provides fundamental insights into aMPV/B pathogenicity and offers direction for guiding the rational design of novel and more effective vaccines against aMPV/B and, by extension, related pathogens, such as hMPV.

Six novel monoterpenoid indole alkaloid (MIA) heterodimers, hunterlanines A-F (1-6), were isolated from Hunteria zeylanica. Compounds 1 and 2 possess unprecedented carbon skeletons featuring cage-like 6/5/6/6/6/5/6 heptacyclic and 6/5/6/6/6/6 hexacyclic cores, respectively. Compounds 3 and 4 represent the first MIA-phenylpropanoid heterodimers characterized by an unusual C-C and N-C connectivity, forming two different 6/5/5/8/6 pentacyclic scaffolds. Compounds 5 and 6 exemplify novel heterodimers in which the MIA moiety was coupled with indole derivative or indole via C-C linkages. Their structures were established by spectroscopic analyses, X-ray crystallography, and quantum chemical calculations. Notably, compounds 1, 4, and 6 exhibited significant neuroprotective activity against l-glutamate-induced neural injury in HT-22 cells.

Cold-active cellulases are highly desirable for temperature-sensitive biomass valorization and food processing, yet they remain scarce in conventional industrial fungal platforms. In this study, a novel cold-induced cellobiohydrolase, VvCBHI-II, was mined from the mushroom Volvariella volvacea and successfully engineered into the industrial workhorse Trichoderma reesei via site-specific homologous replacement. Structural homology modeling revealed that the substitution of the flexible B3 loop with a β-sheet creates a more open substrate-binding cleft in VvCBHI-II. Consequently, the purified VvCBHI-II exhibited robust endoglucanase-like characteristics with superior catalytic efficiency on amorphous cellulose. At 10 °C, the engineered cellulase complex demonstrated an 8.1-fold increase in filter paper activity compared to the wild-type strain. Mechanistic structural analyses indicated that the open cleft architecture elongates and weakens the hydrogen-bonding network with the cellobiose product, facilitating rapid product dissociation and alleviating severe cold-induced product inhibition. In practical applications, the engineered cold-active enzyme complex exhibited an exceptional saccharification capacity on natural pear pomace at 10 °C. Furthermore, when applied to simulated fruit juice processing, it significantly maximized the extraction yield, elevated the sweetness response, and substantially mitigated undesirable bitterness and astringency. This study elucidates the structural-functional paradigm of cold-adapted cellobiohydrolases and provides a promising strategy for formulating highly efficient, energy-saving biocatalysts for the food and biorefinery industries.