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Objective: Hepatic ischemia-reperfusion injury (HIRI) is a common pathological condition in liver surgery and transplantation, and cellular pyroptosis plays a key role in its pathogenesis. However, the clinical application of curcumin is limited by its poor water solubility and low bioavailability. This study aims to develop mesenchymal stem cell (MSC)-derived exosomes loaded with curcumin (Exo-Cur). It also investigates the role and mechanism of Exo-Cur in inhibiting HIRI-related cellular pyroptosis. Methods: The preparation of Exo-Cur was optimized using orthogonal experimental design. Its solubility, stability, particle size distribution, and zeta potential were then evaluated. The morphology of Exo-Cur and its uptake in hepatocytes were observed using laser scanning confocal microscopy. The effect of Exo-Cur on HIRI was assessed through hematoxylin and eosin (HE) staining, ALT and AST measurements, TUNEL assay, CCK-8 assay, and lactate dehydrogenase (LDH) assay. Inflammatory cytokine protein levels were quantified by ELISA, and their mRNA expression was assessed by qRT-PCR. Pyroptosis was assessed by Western blot, immunohistochemistry, and flow cytometry. Additionally, protein expression changes in the PI3K/Akt/mTOR signaling pathway were analyzed using Western blot. Results: Orthogonal experiments determined that the optimal preparation method for Exo-Cur involves cell density at 95%, a curcumin concentration of 30 μg/mL, and a co-cultivation time of 12 h. Characterization results showed that Exo-Cur maintained its typical cup-shaped structure as well as stable particle size and zeta potential. Additionally, its water solubility and its stability in vitro were significantly improved compared to free curcumin. Further mechanistic studies indicated that Exo-Cur could ameliorate the abnormal morphology resulting from HIRI-induced hepatocyte pyroptosis, reduce the proportion of pyroptotic cells, and significantly downregulate the expression of NLRP3 inflammasome and downstream pyroptosis-related proteins (ASC, C-Caspase-1, GSDMD-N). Pathway analysis revealed that Exo-Cur activates the PI3K/Akt/mTOR axis, a pathway inhibited by HIRI. Moreover, rapamycin, an inhibitor of this pathway, reverses Exo-Cur's anti-pyroptosis effect. Conclusions: This study develops an efficient and stable Exo-Cur delivery system, confirming its protective effect against HIRI by activating the PI3K/Akt/mTOR axis and inhibiting NLRP3-mediated cellular pyroptosis. This innovative combination of MSC-derived exosomes combined with curcumin overcomes the limitations in clinical application of curcumin, such as poor bioavailability and stability, and offers a novel nanotherapeutic strategy to treat HIRI clinically.

Background: Lyases are used in a wide scope of applications, making them invaluable tools in both industrial biotechnology and molecular biology. Many examples of lyases belong to the extensive family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, which catalyze numerous reactions involved in amino acid metabolism, like tryptophan indole-lyase (Trpase or Tnase), tyrosine phenol-lyase (TPL), and methionine γ-lyase (MGL). Beyond their role in physiological processes, these lyases can also facilitate the synthesis of other biologically active products from non-canonical substrates. Objectives: Up till now there were only two C-S lyases known for the thiosulfinates' biosynthesis from S-substituted L-cysteine sulfoxides-alliinase and MGL. Our study reveals for the first time that C-C lyases are capable of C-S lyase activity in reactions with S-alkyl, S-allyl and S-benzyl cysteine sulfoxides. Methods: We have compared the kinetic profiles of S-substituted L-cysteine sulfoxide degradation mediated by carbon-sulfur lyase MGL versus carbon-carbon lyases TPL and Trpase. Results: Among other S-alkyl-L-cysteine sulfoxides, petiveriin (S-benzyl-L-cysteine sulfoxide) was proven to be a substrate for all three enzymes. The potential utility of these enzymes in thiosulfinate production was supported by in vitro testing of enzyme-generated thiosulfinates against clinically relevant pathogens such as Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. Conclusions: Both C-S and C-C lyases-MGL, TPL, and Trpase-can be implemented for practical application in thiosulfinate synthesis.

Background/Objectives: Guang Chenpi, the aged pericarp of Citrus reticulata 'Chachi', is a traditional Chinese medicinal food with documented health benefits. This study aimed to systematically evaluate the multifaceted bioactivity of a standardized Guang Chenpi extract (GCE), both alone and in combination with ergothioneine (EGT) and polydeoxyribonucleotide (PDRN), using in vitro and in vivo models. Methods: GCE quality was characterized by LC-MS/MS. Combination regimens of GCE with EGT or PDRN were assessed in UVB-irradiated 3D MelaKutis^® skin tissue for ROS levels, antioxidation defense markers (NNT, GSH-PX1), and melanocyte protein (Pmel17). In zebrafish, GCE was evaluated for toxicity, antioxidant activity, tail fin regeneration, skin barrier protection, melanogenesis inhibition, and expression of collagen (col1a1a, col1a1b, and col1a2) and elastin (elna) genes. Results: In 3D skin models, GCE combined with EGT or PDRN significantly enhanced antioxidant defenses (NNT increased by 113-186%; GSH-PX1 by 173-231%), reduced ROS by 46.27-57.76%, and decreased melanocyte protein (Pmel17) by 23.44-44.27%. In zebrafish, GCE showed low toxicity (≤0.63 mg/mL) and exhibited dose-dependent antioxidant activity (ROS reduction: 27.57-61.85%), enhanced tail fin regeneration (11.35-27.84%), and strengthened skin barrier function (65.20-89.32% protection). GCE also upregulated collagen and elastin gene expression, improved blood circulation, and suppressed melanogenesis. Conclusions: GCE is a promising multifunctional natural ingredient with significant antioxidant, regenerative, and skin-protective properties. Its combination with EGT or PDRN results in enhanced protective effects against UVB-induced skin damage, supporting its potential use in advanced pharmaceutical and cosmeceutical formulations.

Background/objectives: Kidney transplant recipients require lifelong immunosuppression and monitoring to prevent rejection, infection, and graft dysfunction. Current surveillance relies on tacrolimus therapeutic drug monitoring and, when needed, invasive biopsies. Dried blood spot (DBS) sampling provides a minimally invasive, patient-friendly option for remote follow-up. This study aims to develop and evaluate a DBS-based method for CXCL-10 quantification that, in combination with tacrolimus exposure monitoring, could help identify kidney recipients at risk of rejection and cytomegalovirus (CMV) infection and guide immunosuppression adjustment. Methods: The study included 81 selected kidney recipients for CXCL-10-DBS analysis by ELISA (12 T-cell mediated rejection; 10 antibody-mediated rejection; 6 CMV infection and 53 clinical event-free) and 10 healthy volunteers. A Tacrolimus-DBS LC-MS/MS method was developed and validated, and it was compared with the reference method on venous whole blood (WB) LC-MS/MS in a validation cohort (n = 160) and a clinical cohort (n = 36) using linear regression, Passing-Bablok and Bland-Altman analyses. Results: CXCL-10-DBS concentrations were significantly higher in rejectors (p < 0.001), with intermediate increases in CMV infection in comparison with event-free patients and healthy volunteers. ROC analysis demonstrated excellent diagnostic accuracy for rejection (AUC: 0.952; cutoff: 216.2 pg/mL; sensitivity: 100%; specificity: 79%; PPV: 88%; NPV: 100%). In contrast, tacrolimus trough concentrations did not differ significantly among the three clinical groups but showed strong correlation and agreement between DBS and venous WB with no systematic or proportional bias. Conclusions: This pilot study demonstrates the feasibility and diagnostic potential of DBS-based CXCL-10 measurement in adult kidney recipients. Integration of DBS-tacrolimus monitoring supports a minimally invasive pharmacokinetic-pharmacodynamic approach for personalized immunosuppression management.

Background: The SARS-CoV-2 3CLpro is essential for viral replication and an attractive target for antiviral intervention. While most strategies target the catalytic site, recent studies suggest that the dimerization interface and cryptic allosteric pockets offer alternative mechanisms for inhibition. Objective: This study investigated lipid metabolites from the marine sediment-derived Streptomyces sp. DSD454^T as potential multi-site 3CLpro inhibitors. Methods: Metabolites were extracted from cultured biomass and characterized using LCMS-QTOF, MS/MS (LCMS-TQ), and ¹H NMR, with identities confirmed against authentic standards. 3CLpro inhibition was assessed using a FRET-based assay, and ligand-protein interactions were evaluated through molecular docking and MM/GBSA calculations. Lipid content and comparative lipidomic signatures were examined across bioactive Streptomyces strains through LCMS-TQ and BODIPY^TM 493/503 staining. Results: Palmitoleic and linoleic acids were identified as major constituents and inhibited SARS-CoV-2 3CLpro with IC₅₀ values of 1.59 µg/mL (6.25 µM) and 5.29 µg/mL (18.88 µM). Molecular docking predicted that both fatty acids bind not only to the catalytic site but also to the dimerization interface and cryptic allosteric pocket. Additional lipids, including 9-heptadecenoic acid, linolenic acid, 9-HODE, and monoacylglycerols such as aggrecerides A-C and glyceryl-based lipids, showed similarly favorable multi-site binding profiles. Streptomyces sp. DSD454^T also exhibited substantial lipid accumulation (~63% of crude extract). Across bioactive Streptomyces strains, a conserved lipid signature correlated strongly with 3CLpro inhibition. Conclusions: This study highlights the potential of microbial lipids as promising scaffolds for developing catalytic and allosteric SARS-CoV-2 3CLpro inhibitors and underscore marine Streptomyces as a valuable source of structurally simple yet mechanistically versatile antiviral metabolites.

Background/Objectives: Nature has evolved millions of venom-derived peptides with diverse biological functions, a substantial fraction of which target complex membrane proteins such as G-protein-coupled receptors and ion channels. Many of these peptides are stabilized by multiple disulfide bonds, endowing them with exceptional structural stability and favorable pharmacological properties. Methods: Leveraging this natural diversity, we developed a robust venom peptide therapeutics discovery system built on phage display technology and constructed a library using approximately 482 venom-derived scaffolds. The library design was guided by a machine learning (ML) model capable of predicting mutation-tolerant residues that preserve peptide foldability, maximizing structural integrity and sequence diversity. Results: The resulting VCX library was evaluated through screening against four diverse targets (CD47, DLL3, IL33, and P2X7R), yielding strong binders for all four, a success rate of 100%. Furthermore, by integrating high-throughput recombinant expression of thioredoxin-venom fusion proteins along with ML-assisted affinity maturation, we rapidly identified potential leads for DLL3 binders. Conclusions: This venom-based discovery platform offers significant advantages in both functionality and developability compared with conventional peptide discovery approaches. By combining natural structural diversity, ML-guided design, and recombinant expression, it enables efficient identification of "antibody-like" binders with molecular weights much smaller than those of antibodies. Consequently, it provides a powerful strategy for developing next-generation peptide therapeutics targeting challenging protein-protein interactions and complex membrane proteins.

Background/Objectives: Astragalus root is a classical qi-tonifying traditional Chinese medicine that has demonstrated potential therapeutic efficacy in type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). However, the precise mechanisms underlying its effects on the comorbidity of these two disorders remain unclear. This study investigated the molecular mechanisms by which Astragalus root ameliorated T2DM-NAFLD comorbidity. Methods: Network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experiments were employed to elucidate the potential roles and mechanisms of Astragalus root in the management of T2DM-NAFLD comorbidity. Results: A total of 25 bioactive constituents and 152 corresponding targets associated with Astragalus root were identified. PPI network analysis revealed the top ten core candidate targets, among which six possessed suitable crystal structures for molecular docking, including interleukin-6 (IL-6), threonine-protein kinase 1(AKT1), transcription factor AP-1(JUN), tumor necrosis factor (TNF), cysteine-dependent aspartate-specific protease 3 (CASP3), and estrogen Receptor 1(ESR1). Kyoto encyclopedia of genes and genomes (KEGG) analysis further identified the phosphatidylinositol 3-kinase (PI3K)-AKT as the most significantly enriched pathway. Molecular docking validated the potential binding modes of formononetin to the six core targets, a finding that was further confirmed by molecular dynamics simulations, which proved the stability of the resulting complexes. In vitro experiments demonstrated that formononetin obviously decreased lipid droplet accumulation, downregulated total cholesterol (TC) and triglyceride (TG) levels, suppressed the expression of TNF-α, IL-6, and interleukin-1β (IL-1β), decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and enhanced glutathione (GSH) content and superoxide dismutase (SOD) activity. These therapeutic effects were achieved through inhibition of protein expression within the PI3K/AKT/mechanistic target of rapamycin (mTOR) signaling pathway. Conclusions: This study determined the potential therapeutic targets and underlying mechanisms of formononetin derived from Astragalus root in the T2DM-NAFLD management, thereby providing a scientific basis for its clinical application.

Background: Carbonic anhydrases (CAs) are known to play important roles in several physiological and pathological processes; among them, CAs IX and XII are of particular relevance in cancer therapy due to their involvement in tumor growth and progression. Methods: In this study, a novel series of benzenesulfonamides incorporating a hydrazinocarbonyl-ureido linker alongside a 6-arylpyridine tail was synthesized and evaluated for inhibitory activity through a stopped-flow CO₂ hydrase assay on four hCA isoforms. Results: Some of the new compounds exhibited great activity and selectivity toward the tumor-expressed CA XII isoform over the off-target isoforms CA I and CA II. Based on these results, they were selected for ADME prediction studies, showing favorable drug-like properties. To further investigate their binding mode, these compounds were docked into the four hCA isoforms. Conclusions: Overall, the results underscore the potential of compounds bearing a 6-arylpyridine tail along with a hydrazinocarbonyl-ureido linker as a foundation for further inhibitor development.

Background: Herpes simplex keratitis (HSK), caused by herpes simplex virus type 1 (HSV-1), is a major cause of infectious blindness. Macrophages are key antiviral effector cells, yet the metabolic mechanisms driving their protective responses remain poorly defined. This study aimed to determine whether interleukin-33 (IL-33) modulates macrophage metabolism and function to enhance antiviral protection in HSK. Methods: Bone marrow-derived macrophages (BMDMs) were stimulated with IL-33, followed by phenotypic and functional characterization using qRT-PCR, flow cytometry, and immunofluorescence. Integrated transcriptomic and non-targeted LC-MS metabolomic profiling was performed to uncover regulatory pathways. For in vivo validation, differently treated BMDMs were adoptively transferred subconjunctivally into a mouse HSK model. Clinical scoring, fluorescein staining, TCID₅₀ quantification of tear samples, and corneal viral gene detection were used to evaluate disease severity and viral burden. Results: IL-33 stimulation increased CD169 and MHC-II expression, expanded the CD169⁺ macrophage subset, and suppressed HSV-1 replication in vitro. Multi-omics integration identified 616 differentially expressed genes and 417 differentially expressed metabolites, revealing substantial remodeling of lipid and amino acid metabolism and suggesting a critical IL-33-lipoprotein lipase (LPL)-palmitoylcarnitine (L-PC) metabolic axis. In vivo, prophylactic adoptive transfer of IL-33-treated BMDMs significantly reduced corneal opacity, epithelial injury, tear viral titers, and virogene expression. LPL inhibition eliminated these benefits, whereas L-PC supplementation partially restored antiviral and clinical improvements. Conclusions: IL-33 reprograms macrophages toward a CD169⁺ antiviral phenotype through an LPL-dependent metabolic pathway, establishing an LPL-L-PC axis essential for enhanced antiviral function and protection against HSK. These findings highlight metabolic tuning of macrophages as a potential preventive immunomodulatory approach for HSV-1-induced ocular disease.

Background/Objectives: Infections caused by Candida albicans, Candidozyma auris, and Candida parapsilosis increasingly challenge current treatment options as resistance to currently used antifungals is continuously developing. Neutralizing antimicrobial peptides (nAMPs), which modulate pathogenic behavior rather than inducing cell death, represent a promising approach to fighting against fungal infections. Methods: This study established a whole-cell phage display workflow to identify novel nAMPs, and therefore three independent biopanning processes with the Ph.D.-12 phage display library against C. albicans, C. auris, and C. parapsilosis cells were conducted. Results: Phage display produced species-selective, high-affinity peptides that were non-cytotoxic to human cells and did not affect planktonic Candida viability. These peptides inhibited early biofilm formation, and several also slowed early biofilm maturation down. Conclusions: These findings demonstrate that whole-cell phage display as a powerful and adaptable discovery tool is suitable for identifying nAMPs that neutralize biofilm development without toxicity towards human cells. Beyond the peptides described here, this approach expands the methodological toolbox for antifungal research and provides a sustainable approach for generating targeted peptides.