Search Results (364)

Background/Objectives: This study investigates the potential of Anisakis sp. as a novel source of α-Gal (Galα1-3Galβ1-4GlcNAc-R) epitopes capable of inducing allergic sensitization in humans. While α-Gal is classically associated with delayed IgE-mediated hypersensitivity following tick bites, emerging evidence suggests that parasitic helminths such as Anisakis sp. may also express α-Gal-containing glycoconjugates, offering an alternative sensitization pathway. Methods: Protein extracts from Anisakis sp. third-stage larvae and mammalian tissues (beef, pork) were analyzed by SDS-PAGE and Western blot using a monoclonal anti-α-Gal antibody (clone M86), and α-Gal epitopes were detected by ELISA. Sera from urticaria patients, stratified by Anisakis sp. sensitization status, were evaluated for anti-α-Gal IgG, IgE, and IgG4 antibodies. Inhibition assays assessed cross-reactivity. Results: Results confirmed the presence of α-Gal epitopes on Anisakis sp. proteins, with prominent bands at ~250 kDa and 65 kDa. Urticaria patients sensitized to Anisakis sp. exhibited significantly elevated anti-α-Gal antibody levels compared to controls. Inhibition ELISA demonstrated substantial reduction in antibody binding with Anisakis sp. extracts, indicating shared antigenic determinants with mammalian α-Gal. Conclusions: These findings establish Anisakis sp. as a source of α-Gal-containing glycoproteins capable of eliciting specific antibody responses in humans, highlighting a potential parasitic route for α-Gal sensitization. Full article

Multivalent lectin–glycan interactions (MLGIs) are vital for viral infection, cell-cell communication and regulation of immune responses. Their structural and biophysical data are thus important, not only for providing insights into their underlying mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information remains to be limited for some important MLGIs, significantly restricting the research progress. We have recently demonstrated that functional nanoparticles, including ∼4 nm quantum dots and varying sized gold nanoparticles (GNPs), densely glycosylated with various natural mono- and oligo- saccharides, are powerful biophysical probes for MLGIs. Using two important viral receptors, DC-SIGN and DC-SIGNR (together denoted as DC-SIGN/R hereafter), as model multimeric lectins, we have shown that α-mannose and α-manno-α-1,2-biose (abbreviated as Man and DiMan, respectively) coated GNPs not only can provide sensitive measurement of MLGI affinities but also reveal critical structural information (e.g., binding site orientation and mode) which are important for MLGI targeting. In this study, we produced mannuronic acid (ManA) coated GNPs (GNP-ManA) of two different sizes to probe the effect of glycan modification on their MLGI affinity and antiviral property. Using our recently developed GNP fluorescence quenching assay, we find that GNP-ManA binds effectively to both DC-SIGN/R and increasing the size of GNP significantly enhances their MLGI affinity. Consistent with this, increasing the GNP size also significantly enhances their ability to block DC-SIGN/R-augmented virus entry into host cells. Particularly, ManA coated 13 nm GNP potently block Ebola virus glycoprotein-driven entry into DC-SIGN/R-expressing cells with sub-nM levels of EC₅₀. Our findings suggest that GNP-ManA probes can act as a useful tool to quantify the characteristics of MLGIs, where increasing the GNP scaffold size substantially enhances their MLGI affinity and antiviral potency. Full article

In the present study, for the first time, a biodegradable and non-toxic polyphosphoramidate glycohydrogel (PPAGHGel) was prepared by crosslinking a polyphosphoramidate glycoconjugate (PPAG) with hexamethylene diisocyanate (HMDI) under mild conditions. Poly(oxyethylene H-phosphonate) (POEHP) was used as a precursor and was converted into PPAG via the Staudinger reaction with glucose-containing azide (2-p-azidobenzamide-2-deoxy-1,3,4,6-tetra-O-trimethylsilyl-α-D-glucopyranose). Then, crosslinking of PPAG was performed to yield PPAGHGel, which was thoroughly characterized. The gel showed a gel fraction of 83%, a swelling degree of 1426 ± 98%, and G″ = 1560 ± 65 Pa. The gel was fully degraded by alkaline phosphatase (400 U/L, pH 9) in 19 days, while hydrolytically, up to 52% degradation was observed under similar conditions. Multivalent studies of the obtained hydrogel with lectin–Concanavalin A were performed. PPAGHGel binds 92% of Concanavalin A within 24 h and the complex remains stable until the amount of glucose reaches 0.3 mM. PPAGHGel acts as a stabilizer for silver nanoparticles (12 nm). SEM shows pores measuring 10 µm (surface) and 0.1 mm (interior) with capillary channels, confirming the gel’s suitability for biosensors, drug delivery, or wound dressings. The cytotoxic (IC50) and cell-adhesive properties of the obtained hydrogel were investigated on human cell lines (HeLa). Antibacterial activity tests were also performed with gel containing silver nanoparticles against skin-associated pathogenic bacteria. The results show that PPAGHGel possesses excellent biocompatibility, non-adhesive properties and antibacterial activity. Full article

Infections caused by Acinetobacter baumannii are increasing worldwide. We evaluated the antibiotic resistance profile, biofilm production, and the frequency of 12 genes encoding carbapenemases and 13 virulence factors in 90 isolates from patients of three hospitals in various regions of Poland. Antibiotic resistance survey was performed using the disc-diffusion method, genes encoding resistance to carbapenems and virulence factors were detected with PCR, and biofilm formation was tested using microtiter plates. A total of 52.2% of isolates were resistant to all tested antibiotic groups (penicillins with β-lactamase inhibitors, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and trimethoprim plus sulfamethoxazole). Among the genes encoding carbapenem resistance, the bla_OXA-23 (68.9%), bla_OXA-40 (83.3%), and ISAba-bla_OXA-51 (18.9%) were detected. The ompA, ata, and recA genes responsible for biofilm formation, adhesion, and stress response, respectively, occurred in all isolates. Genes responsible for the production of other adhesins (bap—94.4%, espA—4.4%, chop—37.7%), biofilm formation (pbpG—90.0%), production of siderophore (basD—97.7%), toxins (lipA—92.2%, cpaA—1.1%), glycoconjugates (bfmR—84.4%), and inducing host cell death (fhaB—71.1%, abeD—93.3%) were also found. A total of 68.8% of isolates produced biofilm. The isolates from Masovia had more virulence genes than isolates from the other regions; moreover, all isolates from Masovia and West Pomerania were multidrug-resistant (MDR), including resistance to carbapenems. Full article

Rhamnose is a natural sugar found in glycoproteins and structural polysaccharides of plants, fungi, and bacteria. Its incorporation into glycoconjugates is mediated by rhamnosyltransferases (RHTs), key enzymes for biomolecular stability and function. While rhamnose biosynthesis has been studied in certain fungal genera, the evolutionary history and distribution of RHTs across the fungal kingdom remain largely unknown. In this study, 351 fungal species were found to encode putative RHTs. Phylogenetic and structural analyses revealed conserved patterns and similarities with previously characterized RHTs. Molecular docking predicted a high affinity of these proteins for UDP-L-rhamnose, and in silico mutagenesis identified key residues potentially involved in substrate binding. Carbohydrate profiling confirmed the presence of rhamnose in the cell walls of multiple fungi, including Aspergillus, Madurella, Metarhizium, and Trichoderma species. Enzymatic assays further supported rhamnose transfer activity. These findings provide the first comprehensive in silico characterization of fungal RHTs, uncovering conserved sequence motifs despite overall diversity, which may be linked to functional adaptation in different fungal lineages. Full article

Extracellular polymeric substances (EPS) are multifunctional biopolymers that have significant biotechnological potential. In this study, forty-seven strains of Rhodococcus actinomycetes were screened for EPS production and the content of its main components: carbohydrates, lipids, proteins, and nucleic acids. The Rhodococcus strains produced lipid-rich EPS (15.6 mg·L⁻¹ to 71.7 mg·L⁻¹) with carbohydrate concentrations varying from 0.6 mg·L⁻¹ to 58.2 mg·L⁻¹ and low amounts of proteins and nucleic acids. Biofilms of R. ruber IEGM 231 were grown on nitrocellulose filters in the presence of n-hexane, n-hexadecane, or diesel fuel. The distribution of β-polysaccharides, glycoconjugates, and proteins between cells and the extracellular matrix was examined using fluorescence microscopy. The observed release of β-polysaccharides into the biofilm matrix in the presence of n-hexane and diesel fuel was regarded as an adaptation to the assimilation of these toxic hydrocarbons by Rhodococcus cells. Atomic force microscopy of the dried EPS film revealed adhesion forces between 1.0 and 20.0 nN, while some sites were highly adhesive (F_a ≥ 20.0 nN). EPS biosynthetic genes were identified, with two glycosyltransferases correlating with an increase in carbohydrate production. The production of EPS by Rhodococcus cells exhibited strain-specific rather than species-specific patterns, reflecting a high genetic diversity of these bacteria. Full article

Glycoconjugate vaccines, consisting of a protein component covalently linked to a glycan antigen, have led to a significant reduction in the global occurrence of bacterial meningitis and pneumonia. They provide robust, lasting immunity in all age groups. However, their production by traditional chemical conjugation approaches has drawbacks in terms of complexity, cost, and lack of flexibility in design, which explains their limited application to a few pathogenic bacteria in the past four decades. Protein glycan coupling technology (PGCT) or bioconjugation, where glycoconjugates are produced in purpose-engineered bacterial cells, is a useful alternative to chemical conjugation and promises an array of low-cost custom-made glycoconjugate vaccines with vast protein glycan combinations. The technology has undergone significant development since its inception, and new advances and refinements continually drive the field forward. Several bioconjugate vaccines are currently in clinical trials, demonstrating the potential of the technology. We will review the wide applicability of bioconjugation and recent developments in each of the components of the technology, namely, glycan expression, protein selection, and the coupling of selected glycan with proteins, all within custom-designed E. coli cells. These advances promise to deliver effective glycoconjugate vaccines for multiple unmet medical needs. Full article

Maternal vaginal and rectal colonization by Streptococcus agalactiae (group B streptococcus, GBS) is the main risk factor for the development of newborn early-onset GBS disease (GBS-EOD). Much effort is in place for its prevention, including the development of vaccines. Currently, both a hexavalent glycoconjugate GBS vaccine against the most prevalent serotypes and a protein subunit vaccine have completed phase two clinical trials. GBS surveillance in both maternal carriage and neonatal disease is therefore important in establishing the coverage of the potential vaccines and in setting up the basis for pre- and post-marketing surveillance. A single-site study was conducted in the years 2020–2021 on the characteristics of 325 GBS strains (serotype distribution; identification of the alpha-like protein family member; and resistance to macrolides, tetracycline, and high-level gentamicin) isolated from the vaginal/rectal site in women in late pregnancy as well as in seven cases of GBS-EOD and one case of GBS-related stillbirth occurring in the same location and time period. The study indicated that the coverage of the developing vaccines was excellent (97.2% for the hexavalent glycoconjugate vaccine and 98.7% for the alpha-like protein subunit vaccine). However, the detection of the serotypes VI, VII, and IX—not covered by current vaccine formulations—accounting for 3.0% of isolates, as well as of negative alpha-like GBS strains from maternal carriage (1.2%), should be closely monitored over time. The high rates of GBS resistance to erythromycin (33.5%) and to clindamycin (29.5% in maternal carriage and 57.1% in GBS-EOD) was mostly due to the ever-increasing spread of the multidrug-resistant ST-17 subclone of serotype III. This finding, along with the newly emerging high-level gentamicin resistance in carriers (4.0%), mainly in serotype IV strains, poses a threat for the continued effectiveness of antibiotic therapy in invasive disease. Full article

Background/Objectives: This study explores the ocular anatomy and glandular components of domestic dogs compared to their ancestor, the wolf, with the aim of identifying evolutionary changes due to domestication and their implications for ocular pathologies. Methods: Utilizing histological and histochemical techniques, including hematoxylin–eosin, Periodic Acid–Schiff, Alcian Blue, and lectins, this research conducts a detailed analysis of the canine and wolf ocular systems, focusing on the eyelids, tarsal glands, and conjunctival tissues. Results: There are marked histological differences between the two species, particularly in the thickness and secretion levels of the conjunctival epithelia and the structure of the tarsal glands. Dogs exhibit a thicker epithelium with greater Periodic Acid–Schiff and Alcian Blue positive secretion, suggesting enhanced ocular protection and lubrication adapted to domestic environments. Conversely, wolves display more concentrated glandular secretions and a predominance of acidic mucopolysaccharides, aligning with their adaptation to natural habitats. Conclusions: Although this study is constrained by the limited number of samples, the use of mixed dog breeds, and the focus on the Iberian wolf, it nonetheless suggests histological and evolutionary differences between domestic dogs and wolves, particularly in structures related to ocular surface protection and lubrication. These differences likely reflect adaptive responses to domestication in dogs and environmental demands in wolves. Importantly, the findings emphasize the clinical and translational potential of using dogs as comparative models for human ocular surface disorders, given their anatomical proximity to humans. Full article

We have previously reported on a novel monoclonal antibody (mAb) we designated F5, which was raised against a glycopeptide derived from the tandem repeat (TR) region of Mucin-4 (MUC4), a heavily O-glycosylated protein that is overexpressed in many pancreatic cancer cells. This mAb was highly specific for the MUC4 glycopeptide antigen in glycan microarrays, ELISA and SPR assays, selectively stained tissue derived from advanced-stage tumors, and bound MUC4⁺ tumor cells in flow cytometry assays. The mAb was also unique in that it did not cross-react with other commercial anti-MUC4 mAbs that were raised in a similar but non-glycosylated TR sequence. Here we describe the selective conjugation of a novel near-infrared dye to this mAb and in vivo biodistribution of this labeled mAb to various MUC4-expressing tumors in mice. The labeled mAb were selectively distributed to both cell-derived xenograft (CDX) flank tumors and patient-derived xenograft (PDX) tumors that expressed MUC4 compared to those that were MUC4-negative. Organ distribution analysis showed high uptake in MUC4⁺ relative to MUC4⁻ tumors. These results suggest that mAb F5 may be used to develop MUC4-targeted, passive antibody-based immunotherapies against Pancreatic Ductal Adenocarcinomas (PDACs) which are notorious for being refractory to many chemo- and radiotherapies Full article

Non-alcoholic steatohepatitis (NASH), a progressive liver disease characterized by lipid accumulation and chronic inflammation, lacks effective therapies targeting its multifactorial pathogenesis. This study investigates marine-derived chondroitin sulfate (CS) as a multi-organelle modulator capable of regulating lipid metabolism, oxidative stress, and inflammation in NASH. By employing subcellular imaging and organelle-specific labeling techniques, we demonstrate that CS restores lysosomal acidification in a NASH model, enabling the reduction of lipid droplets via lysosomal–lipid droplet fusion. Concurrently, CS upregulates dynamin-related protein 1 (DRP1), driving mitochondrial terminal fission to spatially isolate reactive oxygen species (ROS) segments for mitophagy, thereby reducing ROS levels. Notably, pharmacological inhibition of lysosomal activity using chloroquine or bafilomycin A1 abolished the therapeutic effects of CS, confirming lysosomal acidification as an essential prerequisite. Collectively, these findings reveal the potential of CS as a therapeutic agent for NASH and provide critical insights into the subcellular mechanisms underlying its protective effects, thus offering a foundation for future research and therapeutic development. Full article

Despite broad anti-cancer efficacy as Toll-Like Receptor (TLR) 7/8 agonists, imidazoquinolines remain limited in use via systemic administration or in situ vaccination therapies due to inflammatory toxicity. One approach to address this challenge involves better targeting the action of imidazoquinolines by caging them as glycoconjugate prodrugs. Within cancer cells, imidazoquinoline glycoconjugates are activated by hydrolases prior to efflux by ABC transport proteins, where they then elicit tumoricidal effects from the assistance of bystander immune cells, such as tumor-infiltrating lymphocytes and associated macrophages, in local proximity. While this concept of Bystander-Assisted ImmunoTherapy (BAIT) has been established at a molecular level in vitro, tolerability or efficacy of BAIT has not been reported in vivo. Here, we evaluate the MTD and tumor growth delay efficacy of a lead BAIT prodrug (BAIT628) in a male C57BL/6 mouse TRAMP-C2 prostate cancer model to further establish this methodology. Overall, we find that systemic BAIT628 is well tolerated at over 5-fold the dose-limiting inflammatory toxicity of the parent imidazoquinoline (up to 5 mg/mouse/day I.P. for 10 days). Analyzing serum cytokines reveals that IL-10 production, elicited by the mannoside caging group, likely contributes to the enhanced MTD. Using BAIT628 as an in situ vaccination immunotherapy (seven times over 3 weeks) resulted in significant tumor growth delay and increased survival, both alone and in combination with a murinized α-PD-L1 checkpoint blockade. The tumor histology of tumor-infiltrating immune cell subsets (CD4⁺, CD8⁺, CD11c⁺) reveals significant increases in CD11c⁺ populations, consistent with TLR7/8 agonism. Overall, BAIT628 is well tolerated and exhibits significant efficacy in the TRAMP-C2 model. These results demonstrate how the BAIT approach can optimize imidazoquinolines for in vivo tolerability and subsequent efficacy as cancer immunotherapeutics. Full article

Background: 3-Lup-20(29)-ene-3β,28-diol (betulin, BN) is a natural bioactive compound with significant synthetic and pharmacological potential. A growing body of research highlights the increasing interest in BN and its derivatives, driven by their broad biological activities (anticancer, antibacterial, anti-inflammatory, antiretroviral). However, poor bioavailability and low intracellular accumulation limit its pharmaceutical application. Methods: A promising strategy to enhance BN’s therapeutic potential is glycoconjugation. This approach improves drug bioavailability, solubility, and selectivity, particularly in cancer therapy, by leveraging cancer cells’ heightened glucose demand and overexpression of glucose transporters. Incorporating an N-heterocyclic linker, such as a 1,2,3-triazole ring, further enhances biological activity. Results: We developed an efficient method for modifying the betulin backbone at position C28 with sugar units via a (CO)CH₂CH₂COOH linker, based on CuAAC, yielding ten new betulin glycoconjugates with good yields and purity confirmed by spectroscopic analysis (NMR, HRMS). The potential for inhibition of cancer cell proliferation (HCT-116 human colorectal carcinoma cell line and MCF-7 human breast cancer cell line) and cytotoxicity toward normal human dermal fibroblasts (NHDF-Neo) was assessed. Conclusions: The obtained glycoconjugates exhibited higher activity against MCF-7, indicating the selectivity of their action. The development of glycoconjugates based on increased glucose demand and overexpression of its transporters could be an interesting strategy for acquiring anticancer agents, combining innovative chemical solutions with biological complexity. Such an approach may be crucial in the effective fight against cancer diseases. Full article

The storage duration and hatchability of eggs largely depend on the lysozyme content in egg whites; therefore, determining the lysozyme status is important for characterizing their quality. For the first time, a fast and accurate method for determining the active lysozyme in egg whites has been proposed to establish the lysozyme status of eggs using the fluorescence polarization assay and synthetic chitooligosaccharide conjugates with a fluorescent label without sample preparation. The egg whites of hens, black hens, chukars, quails, ducks, geese, turkeys, peacocks, and ostriches were studied. Samples of egg whites from hens, black hens, chukars, and quails demonstrate the possibility of measuring the lysozyme activity. Samples of hen and black hen eggs from a farm showed approximately the same enzymatic activity of lysozyme. A relatively higher enzymatic activity was demonstrated by the samples from quail egg whites; however, a wide range of data was observed among the eggs. Chitooligosaccharide conjugates demonstrate that they bind only to C-type lysozyme, and no interaction with G-type lysozyme has been shown. Lysozyme activity in the egg whites of duck, goose, turkey, peacock, and ostrich eggs has not been detected by using the obtained chitooligosaccharide tracers, which may be related to the structural features of lysozyme in different bird species. Thus, the method of fluorescence polarization (FP), using fluorescently labeled chitopentaoside to determine the lysozyme status, can be used to characterize hen, black hen, chukar, and quail eggs, which will allow for the selection of a batch of eggs with a high content of active lysozyme, for example, for long-term storage. Full article

Oligosaccharides and glycoconjugates play essential roles in various biological processes such as cellular recognition and signaling, and thus have attracted tremendous attention in the synthetic and biological communities over the past few decades. Contributing to this field, we have achieved the synthesis of the aminoxyglycoside pentasaccharide subunit of Pseudomonas aeruginosa polysaccharide synthesis locus (Psl) exopolysaccharide through an efficient 23 step process. This pentasaccharide was designed with an aminooxy derivative at the reducing end, which was used in a 2-step oxime-based bioconjugation to the protein carrier CRM197, with an epitope ratio of 1:4. The conjugate vaccine could generate anti-Psl antibodies that could recognize P. aeruginosa PAO1 bacteria and initiate opsonophagocytic killing of the bacteria. In addition, the aminoxyglycoside could be conveniently conjugated to a bifunctional aldehyde-biotin reagent, which can be used for quantifying antibody titers in vaccination studies. Full article