Search Results (185)

Aberrant glycosylation is a recognized hallmark of cancer, establishing Golgi α-mannosidase II (GMII) as strategic therapeutic target. While the natural alkaloid swainsonine demonstrated potent anticancer activity, its clinical use is hampered by toxicity from off-target inhibition of the lysosomal α-mannosidase (LMan). This review surveys computational methodologies advancing inhibitor development from empirical observations to precision structural optimization. We examine the evolution from Molecular Docking to advanced Quantum Mechanics (QM) and Molecular Dynamics (MD), highlighting their combined role in modeling metalloenzyme flexibility and energetics. Analysis reveals that selectivity relies on exploiting peripheral structural divergences, organelle-specific pH gradients, and distinct substrate conformational itineraries. In this context, electronic structure calculations and pKa predictions prove critical for designing “electrostatic switches”, inhibitors binding neutrally at Golgi pH while incurring lysosomal repulsion. Structurally, targeting the non-conserved “anchor site”, mimicking specific transition-state ring distortions and utilizing conformationally restricted scaffolds represent the most effective strategies. Integrating dynamic sampling with rigorous energetic profiling is therefore crucial for developing the next generation of safe, selective GMII inhibitors. Full article

The Svx protein is an established virulence factor in the phytopathogenic pectolytic bacterium Pectobacterium atrosepticum and is secreted into the host plant apoplast. However, its particular role has long remained enigmatic. In our recent studies, we showed that Svx proteins from pectolytic bacteria are metallopeptidases composed of two domains: peptidase and acyltransferase-like domains. Structural organization of the peptidase domain active site led us to hypothesize that its preferred substrates are extensins—hydroxyproline-rich glycoproteins of the plant cell wall. Nevertheless, direct experimental confirmation of extensin hydrolysis by Svx was lacking, and the precise role of the acyltransferase-like domain remained unclear. The present study aimed to address these issues. We showed that Svx indeed cleaves extensins while not degrading some other glycosylated and non-glycosylated proteins. The acyltransferase-like domain was shown to be critical for recognition of arabinan substituents in extensins, thereby providing optimal enzyme–substrate complementarity. Deletion of the acyltransferase-like domain abolished extensin hydrolysis by the truncated variant of Svx. Our study provides the first example of an apoplast-secreted protease from a phytopathogenic bacterium whose specificity toward specific target proteins (extensins) is achieved, at least in part, through structural elements that specifically recognize the distinctive glycosylation pattern of the target proteins. Full article

Yeast selection plays a strategic role in winemaking, influencing not only the quality and style of the final product but also the expression of the cultivar. This study evaluated the impact of selected Saccharomyces cerevisiae strains on the fermentation of three white grape cultivars grown in Western Sicily: Grillo, Catarratto, and Moscato Giallo (Vitis vinifera L.). A standardized vinification protocol was applied to assess the fermentative performance and effects on the chemical composition, aromatic profile, and sensory profile. Alcoholic fermentation kinetics, major analytical parameters, free and glycosylated volatile compounds, and sensory attributes were monitored. Significant differences were observed among the yeast strains in their fermentation dynamics and production of secondary metabolites. Notably, certain strains enhanced the aromatic expressions of the cultivars, particularly in Moscato Giallo, modulating the free and glycosylated terpene profiles. This approach to fermentation highlights the potential to optimize wine quality through yeast selection, aligning the strain performance with the specific needs of each cultivar. Furthermore, the use of efficient yeast strains may reduce reliance on additives, contributing to more sustainable and economically viable winemaking. Full article

Wilson’s disease (WD) is an autosomal recessive disorder of copper metabolism caused by ATP7B mutations, characterized by hepatic copper accumulation and multisystem involvement. Several rare inherited and acquired conditions can closely mimic WD, posing diagnostic challenges and the risk of inappropriate therapy. By examining neuroimaging patterns and distinguishing between diagnostic criteria, this narrative review provides a comprehensive synthesis of WD-mimicking disorders, emphasizing their molecular mechanisms, clinical phenotypes, and biochemical features. WD-mimicking disorders encompass ATP7A-related neurodegenerations (Menkes disease, occipital horn syndrome, X-linked distal hereditary motor neuropathy), MEDNIK syndrome, Huppke–Brendel syndrome, aceruloplasminemia, congenital disorders of glycosylation, primary familial intrahepatic cholestasis type 3, and acquired copper deficiency syndromes. Mechanisms include systemic copper deficiency, impaired intracellular trafficking, defective ceruloplasmin biosynthesis, secondary hepatic copper accumulation, and abnormal glycosylation. Clinical features range from neurodevelopmental delay, movement disorders, and hepatic dysfunction to dermatologic, hematologic, and connective-tissue abnormalities. Biochemical profiles may overlap with WD, particularly low serum ceruloplasmin and total copper, altered urinary copper excretion, and elevated hepatic copper in some disorders. Neuroimaging and genetic testing provide critical discriminative value. Management is largely supportive, with disease-specific therapies available in selected conditions, such as subcutaneous copper in Menkes disease or monosaccharide supplementation in certain congenital disorders of glycosylation subtypes. Accurate differentiation between WD and WD-mimicking disorders requires careful integration of clinical, biochemical, imaging, and molecular data. Recognition of distinctive features and understanding underlying pathophysiology are essential to avoid misdiagnosis and inappropriate anti-copper therapy, optimize management, and improve patient outcomes. Full article

Blueberries are widely consumed due to their richness in nutrients, yet they are also prone to quality deterioration after being harvested, even at cold temperatures. Non-thermal physical technology is an important auxiliary method worth considering for maintaining the quality of this fruit while refrigerated. In this study, a static magnetic field (SMF) was applied as a complementary pretreatment strategy prior to cold storage of blueberries. The optimal SMF parameters were identified as 5 mT exposure for 12 h, as this significantly retarded decay and softening. The contents of ascorbic acid, total polyphenols, flavonoids and proanthocyanidins were elevated by 20.0%, 17.7%, 23.9%, and 9.1%, respectively. Concurrently, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging capacity, catalase (CAT), and superoxide dismutase (SOD) activity markedly improved. Targeted metabolomic analysis revealed that SMF pretreatment significantly regulated polyphenol metabolic pathways and redirected polyphenol biosynthesis toward more stable and functional compounds, including three hydroxycinnamic acids, quercetin, dihydromyricetin, glycosylated hesperetin, and acylated delphinidin derivates. The synergistic effect of these SMF-elevated phenolics and the reinforced antioxidant system preserved the overall cold-storage quality of blueberries. These findings underscore the potential of SMF pretreatment as an effective physical technique for reducing postharvest blueberry losses. Full article

Coumarins represent a distinctive class of non-classical carbonic anhydrase inhibitors that interact with the entrance region of the catalytic pocket rather than directly coordinating the catalytic Zn²⁺ ion. In this study, a series of glycosylated coumarins was synthesized through a copper-catalyzed multicomponent reaction involving propargyl glycosides, salicylaldehyde, and tosyl azide, providing efficient access to iminocoumarin-based glycosides derived from natural carbohydrates. The inhibitory activity of the synthesized compounds was evaluated against human carbonic anhydrase isoforms I, II, IX, and XII using a stopped-flow CO₂ hydrase assay. The compounds showed negligible inhibition of the cytosolic isoforms hCA I and hCA II, while displaying moderate activity toward the tumor-associated isoforms hCA IX and hCA XII, with Ki values ranging from 12.9 to 41.8 μM. Among the series, 6-O-(2H-chromene-2-one-3-yl-methyl)-D-galactopyranose (10a) emerged as the most potent inhibitor of hCA IX and XII. Structure–activity relationship analysis indicated that deprotected glycosyl derivatives exhibit improved inhibitory activity compared to protected analogues. To rationalize these observations, molecular docking followed by molecular dynamics simulations and MM-GBSA binding free energy calculations were performed for both anomeric forms of compound 10a. The computational results revealed a clear preference for the β-anomer, particularly in hCA IX and XII, where favorable interactions with catalytic threonine residues and isoform-specific aromatic residues stabilize the ligand within the active-site entrance. These findings provide a molecular explanation for the experimentally observed selectivity and highlight glycosyl coumarins as potential starting points for further optimization toward selective inhibitors of tumor-associated carbonic anhydrases. Full article

Astaxanthin (AST), despite its high bioactivity, exhibits poor stability and low bioavailability due to its strong lipophilicity and inherent degradation susceptibility. To overcome such a challenge, we developed a food-grade oleogel delivery system using a soy protein–arabinoxylan (SA) glycosylated complex modulated by different concentrations (0.5–3%) of sucrose ester (SE) or soy lecithin. We show that the emulsifier concentration has a non-linear effect on the oleogel microstructure: an optimal level of 1% had a significant impact on the interfacial compactness and network density, giving rise to improved thermal stability, rheological strength and AST encapsulation efficiency (81.27%). During in vitro digestion, the SA matrix in combination with emulsifiers allowed gastric protection and intestinal-targeted release of AST with a bioaccessibility of up to 88.84% (SAO-SE-AST). This controlled release profile directly translated into enhanced in vivo antioxidant efficacy in wild-type Bristol N2 Caenorhabditis elegans, as evidenced by reduced lipofuscin accumulation, elevated thermotolerance (survival rate: 64.44–73.33%), suppressed reactive oxygen species levels and activation of endogenous antioxidant enzymes (superoxide dismutase as well as glutathione peroxidase). Collectively, this research has uncovered that food-grade emulsifiers are not only stabilizers, but also key regulators of oleogel architecture and bioactive functionality. These results provide a structure–digestion–bioactivity correlation for protein–polysaccharide oleogels, representing a rational design strategy for high-performance delivery systems of lipid-soluble nutraceuticals. Full article

Phyllanthus emblica (amla) exhibits anticancer activity, but its extracts often suffer from poor stability and bioavailability. This study developed amla extract-loaded niosomes to enhance delivery and evaluate their anticancer activity against MCF-7 and HCT116 cell lines, supported by in silico analyses. Methodology: Amla extract was prepared using a 50% aqueous–alcoholic solvent system and lyophilized. Niosomes were prepared by the thin-film hydration method and characterized for physicochemical properties. Anticancer activity was evaluated through in vitro cytotoxicity studies, supported by molecular docking and in silico pharmacokinetic analyses. Results: Optimized niosomes exhibited spherical morphology, good homogeneity (PDI < 0.30), anionic surface charge, high entrapment efficiency (70.5 ± 5.9%), and sustained diffusion-controlled release. In vitro cytotoxicity demonstrated a strong concentration-dependent anticancer activity of amla-loaded niosomes across a range of concentrations (31.25–1000 µg/mL) against both MCF-7 and HCT116 cell lines. At 1000 µg/mL, cell viability decreased to 7.0% and 5.4% in MCF-7 and HCT116 cells, respectively, with calculated IC₅₀ values of 245 µg/mL and 158 µg/mL. Molecular docking and pharmacokinetic predictions supported the potential multi-target anticancer relevance of major phytochemicals, including hydrolyzable tannins, phenolic acids, flavonoid aglycones and glycosides, and highlighted bioavailability limitations for certain high-affinity glycosylated flavonoids, reinforcing the rationale for vesicular encapsulation. Conclusions: Amla extract-loaded niosomes represent a promising vesicular system for enhanced, sustained delivery of anticancer activity in vitro, with complementary in silico findings supporting mechanistic plausibility and translational rationale. Further studies are warranted to evaluate their performance in vivo. Full article

Background: Rabies is a highly fatal zoonotic disease that causes approximately 59,000 human deaths worldwide each year. Current inactivated rabies vaccines require multiple doses and are associated with high costs. The full-length rabies virus glycoprotein (RVG), a membrane protein, exhibits substantial instability in its trimeric structure during recombinant expression. This instability makes it difficult to obtain high-purity, correctly folded antigens. Objectives: This study focuses on the preparation of a full-length recombinant RVG subunit vaccine candidate expressed in a glycoengineered Pichia pastoris system with mammalian-like glycosylation. Methods: The full-length RVG gene (including the transmembrane domain and cytoplasmic tail) from the Challenge Virus Standard-11 (CVS-11) strain was codon-optimized and inserted into the pPICZαA vector to construct the recombinant expression plasmid pPICZαA-RVG. The plasmid was transformed into glycoengineered Pichia pastoris X33-7 (low-mannose type) by electroporation for inducible expression. The target protein was purified by nickel affinity chromatography, anion-exchange chromatography, and Superdex-200 size-exclusion chromatography. The structural characteristics of the purified protein were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The purified antigen was formulated with the adjuvants AS03 or MF59. BALB/c mice (n = 5 per group) were immunized intramuscularly following a four-dose schedule (days 0, 7, 14, and 28). Antigen-specific IgG antibody titers were measured by ELISA, and neutralizing antibody titers were determined using the rapid fluorescent focus inhibition test (RFFIT). Results: Glycoengineered Pichia pastoris yeast strains expressing wild-type RVG (RVG-WT) or a mutant variant (RVG-M6: R84S, R199S, H270P, R279S, K300S, and R463S) were successfully constructed. The purified RVG antigen formed nanoparticles with an average particle size of approximately 75 nm. Immunized mice generated robust RVG-specific IgG responses, with titers reaching approximately 6.31 × 10⁵ for RVG-WT after the fourth immunization, compared to 3.16 × 10³ for RVG-M6 and 5.62 × 10³ for the RVG-WT-PEG control. Two weeks after the fourth immunization, RVG-WT formulated with AS03 or MF59 induced significant neutralizing antibody responses compared with the control group (p < 0.0001 and p < 0.01, respectively). The neutralizing antibody titers reached 1:79.43 in the AS03 group and 1:33.11 in the MF59 group, whereas the WT-PEG + AS03 control group showed a low titer of 1:3.72. In contrast, RVG-M6 formulated with MF59 failed to induce detectable neutralizing antibodies (1:3.02). Furthermore, RVG-WT + AS03 induced significantly higher neutralizing antibody responses than the WT-PEG + AS03 control group (p < 0.0001), and a significant difference was also observed between the RVG-WT + MF59 and RVG-M6 + MF59 groups (p < 0.01). Conclusions: The glycoengineered Pichia pastoris expression system successfully produced uniform full-length rabies virus glycoprotein nanoparticles with high purity. When formulated with the AS03 adjuvant, RVG-WT induced high-titer neutralizing antibodies in mice, suggesting a promising strategy for the development of recombinant subunit vaccines against rabies. However, this study is limited by the absence of challenge studies and validation in target animal species, which will be further investigated in future work. Full article

Aberrant protein glycosylation is a key driver of colorectal cancer (CRC) progression, contributing to tumour growth, metastasis, and immune evasion. In this study, computational approaches were employed to explore the potential of Brefeldin A as an inhibitor of two glycosylation-associated regulatory proteins: Protein Kinase C alpha (PrKCα) and Mitogen-Activated Protein Kinase 1 (MAPK1). Using computational docking and structural analyses, Brefeldin A was predicted to bind effectively to both targets, thereby inhibiting their enzymatic activities. Detailed investigations revealed that Brefeldin A interacts favourably within the active sites of MAPK1 and PrKCα, forming stable complexes by optimal binding interactions. Key residues contributing to binding stabilisation were identified in both MAPK1 and PrKCα. For MAPK1, residues such as Lys114 and Ser153 played a significant role in hydrogen bonding interactions, while for PrKCα, Gln105, Asn154, and Asp167 were notably involved. These interactions included both hydrogen bonds and hydrophobic contacts, which collectively contributed to the strength and specificity of ligand binding. The identification of these residues provides insight into the molecular mechanisms underlying the stabilisation of the Brefeldin A-kinase complexes. Binding affinity estimations showed that Brefeldin A bound to MAPK1 exhibited a binding energy of −22.18 ± 4.50 kcal/mol. In contrast, the Brefeldin A bound to PrKCα demonstrated a slightly stronger binding energy of −23.90 ± 5.36 kcal/mol. Collectively, these findings underscore Brefeldin A’s potential as a novel inhibitor targeting glycosylation-related proteins in CRC, offering a promising therapeutic strategy to impede CRC progression. This work not only proposes Brefeldin A as a promising therapeutic lead but also supports glycosylation inhibition as a valuable approach for CRC control, with broader implications for drug discovery in glycan-related oncogenic pathways. Full article

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a serious threat to the swine industry in China. As a major pig-producing province, Shandong requires continuous epidemiological monitoring of PRRSV. To elucidate the molecular epidemiology of the virus, 1621 clinical samples were collected from suspected cases across different regions of Shandong Province between 2023 and 2025, primarily from Tai’an, Linyi, Jining, and Liaocheng. RT-qPCR detection showed that the positive rate for PRRSV-2 was 20.05% (325/1621). Genetic analysis based on ORF5 and NSP2 genes indicated that Sublineage L1C (NADC30-like) was the dominant strain for 38.38% of ORF5 gene and 72.73% of NSP2 sequencing results. This was followed by Sublineage L8E and L1A and L5A strains. Key virulence-related mutations were identified at residues R13 and R151 in the GP5 protein, which are associated with enhanced pathogenicity. Additionally, variations in neutralizing epitope and the number of N-glycosylation sites (ranging from 2 to 5 per strain) suggested potential immune evasion. Notably, 26.79% (15/56) of sequenced samples showed discordant ORF5 and NSP2 genotyping results, indicating widespread recombination among PRRSV strains in Shandong Province. These finding demonstrated that the genetic diversity, high recombination frequency, and key amino acid variations in circulating PRRSV strains collectively undermine vaccine effectiveness. This study highlights the need to optimize vaccination strategies, enhance biosecurity measures, and implement effective disease control and elimination programs to reduce the impact of PRRSV in Shandong Province. Full article

Background/Objectives: Dihydroartemisinin (DHA), a bioactive metabolite of Artemisia annua, displays potent antitumor activity in multiple cancers. However, its dose-dependent transcriptional regulatory networks in colorectal cancer (CRC) remain insufficiently understood. This study aimed to clarify the molecular mechanisms of low- and high-dose DHA in human CRC cells and reveal the dose-dependent crosstalk among related biological processes. Methods: We integrated RNA-seq transcriptomic profiling and functional validation in HCT116 cells treated with 20 μM (low-dose) or 50 μM (high-dose) DHA. Differentially expressed genes (DEGs) were screened at FDR ≤ 0.05 and |log₂(fold change)| ≥ 1, followed by GO and KEGG enrichment analyses. Results: DHA inhibited cell viability dose-dependently, with an IC₅₀ of 50 μM. We identified 280 and 678 DEGs in low-and high-dose groups, respectively. Low-dose DHA induced apoptosis via GADD45α/β and ATF4/DDIT3-mediated endoplasmic reticulum stress and triggered senescence through G2/M phase arrest. High-dose DHA mainly modulated gene expression signatures associated with ferroptosis by regulating iron homeostasis and lipid peroxidation at the transcriptional level. Both doses suppressed glycolysis, lipid, and folate metabolism; high-dose DHA also inhibited MGAT5B-mediated glycosylation. DHA regulated five core signaling pathways dose-dependently, with high-dose DHA further repressing Wnt3a/16 and BMP4/6. Conclusions: This study first identifies ferroptosis-related gene networks as key transcriptional targets. It reveals dose-dependent crosstalk among cell death, senescence, metabolic reprogramming, and signaling, providing a transcriptomic framework and gene targets for optimizing DHA-based colorectal cancer therapy. Full article

Rapid antigenic drift in the coronavirus spike protein motivates alternative antiviral strategies. We target the conserved nucleocapsid (N) protein—central to RNA binding, genome packaging, and replication—and perform a comparative, cross-species 3D structure-based in silico evaluation. A library of 494 compounds (natural, phytochemical, synthetic) was docked with AutoDock Vina against the MERS-CoV N–terminal RNA–binding domain (NTD; PDB 7DYD) and the C–terminal dimerization domains (CTD) of SARS-CoV (2CJR) and SARS-CoV-2 (8R6E), reflecting the availability of high-resolution, functionally relevant domain structures for each virus. Top-ranked poses underwent ADME profiling and 100 ns GROMACS molecular-dynamics (MD) simulations. Myricetin 3-O-β-D-Galactopyranoside (myricetin) showed the most favorable predicted docking scores across targets (−8.9 kcal/mol, MERS–NTD; −10.1, SARS–CTD; −9.8, SARS-CoV-2 CTD). Curcumin showed moderate predicted affinity (−7.1 to −8.1), while MCC950 achieved consistently favorable docking score (−7.9 to −9.0). ADME results highlighted a trade-off: glycosylated flavonoids offered rich interaction networks but violated oral drug-likeness criteria (e.g., high TPSA), whereas MCC950 met Lipinski/Veber guidelines, supporting translational potential. MD analyses revealed ligand- and target-specific stability: myricetin maintained persistent binding over 100 ns in the SARS-CoV-2 CTD with lower RMSD than comparators; curcumin exhibited transient stability (~30 ns) in MERS- and SARS-bound complexes; MCC950 showed intermittent interactions. Collectively, these findings suggest that the conserved N protein RNA-binding groove represents a resistance-resilient target for broad-spectrum antiviral discovery. Natural flavonoids provide promising scaffolds for optimization, and MCC950 warrants further exploration given its drug-like profile. As this study is purely computational, the results are hypothesis-generating and should be validated via RNA-binding disruption assays, antiviral cell studies, and in vivo models. Full article

Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic metabolic diseases, and inhibition of α-glucosidase activity represents an effective therapeutic strategy. Chitin is the most abundant renewable polysaccharide in the ocean, with its monosaccharide being N-acetylglucosamine (NAG). To evaluate the potential of NAG glycosides as novel α-glucosidase inhibitors, three common phenolic compounds were modified via NAG glycosylation. Their inhibitory activities were assessed at both the enzymatic and cellular levels. In addition, density functional theory (DFT), molecular dynamics (MD) simulations, and molecular docking analyses were employed to systematically investigate the effects of NAG glycosylation on enzyme inhibition and the underlying mechanisms. Compared with the parent phenolic compounds, NAG glycosides exhibited significantly enhanced α-glucosidase inhibitory activity, with NAG introduction markedly improving their binding affinity to α-glucosidase. Among them, glycoside 3a displayed the optimal inhibitory effect, comparable to acarbose, and at the cellular level, its activity at high concentrations was comparable to or slightly higher than that of metformin. Circular dichroism (CD) and MD analyses indicated that glycoside 3a increased the conformational flexibility of key residues and enhanced the structural looseness of the enzyme, thereby inhibiting its activity. NAG glycosides constitute a promising class of marine-derived α-glucosidase inhibitors, warranting further structural optimization and rational design to enhance their activity and selectivity. Full article

The highly chitinolytic marine bacterium Vibrio jasicida KMM 6832, which exhibits potent antifungal activity, possesses an atypical Glycosyl Hydrolase family 19 (GH19) chitinase (ChitVjs). This is the first report of a GH19 gene in V. jasicida, an enzyme generally absent in this species and rare within the Harveyi clade. Phylogenetically, ChitVjs-like enzymes from the genera Vibrio and Aeromonas form a distinct cluster, separate from typical plant and bacterial GH19 counterparts. Despite high sequence identity (80–94%) with characterized homologs from V. parahaemolyticus and V. cholerae, ChitVjs is distinguished by its obligate halophilicity (optimum 0.3–0.4 M NaCl), an acidic isoelectric point (pI 4.72), and a broader cation-activation profile (K⁺, Ni²⁺, Ca²⁺, Cu²⁺, Co²⁺). The recombinant ChitVjs was produced in E. coli as a soluble 63 kDa protein. It functions as a stable, salt-dependent endo-chitinase/chitosanase, exhibiting optimal activity at 40 °C and pH 7.0. The enzyme displays high affinity for colloidal chitin (K_M 0.377 mg·mL⁻¹), is activated by DTT and Tween 80, and shows moderate stability in organic solvents. Furthermore, unlike its primarily catabolic relatives, ChitVjs suppresses conidial germination in marine-derived Aspergillus strains. These findings suggest that ChitVjs significantly contributes to the competitive fitness of V. jasicida KMM 6832 in high-salinity marine environments through both nutrient acquisition and antagonism. Full article