Search Results (564)

SLC35A2-CDG is a congenital disorder of glycosylation caused by mutations in the SLC35A2 gene encoding a Golgi-localized UDP-galactose transporter. This transporter plays an essential role in glycan synthesis by transporting UDP-galactose from the cytoplasm into the Golgi lumen. Its dysfunction leads to impaired galactose-containing glycans and various neurological symptoms, although the underlying mechanisms remain largely unknown. We identified a novel SLC35A2-CDG patient carrying a pathogenic variant (c.617_620del, p.(Gln206ArgfsTer45)) who exhibited neurological abnormalities including bilateral ventriculomegaly. To investigate the disease mechanism, we established the first Drosophila model of SLC35A2-CDG. Knockout of Ugalt, the fly ortholog of SLC35A2, resulted in embryonic lethality, indicating its essential role. Knockdown of Ugalt reduced mucin-type O-glycans on muscles and neuromuscular junctions (NMJs), without affecting N-glycans. Ugalt knockdown larvae exhibited mislocalized NMJ boutons accompanied by a deficiency in basement membrane components on muscles. This phenotype resembles that of mutants of dC1GalT1 and dGlcAT-P, both involved in mucin-type O-glycosylation. Genetic interaction between Ugalt and dC1GalT1 was confirmed through double knockdown and double heterozygous analyses. Given that Drosophila NMJs are widely used as a model for mammalian central synapses, our findings suggest that Ugalt regulates NMJ architecture via mucin-type O-glycosylation and provide insights into the molecular basis of neurological abnormalities in SLC35A2-CDG. Full article

Glycosylation, the enzymatic addition of glycans to proteins and lipids, is a critical post-translational modification that influences protein folding, stability, trafficking, immune modulation, and cell signaling. The vast structural diversity of glycans arising from differences in monosaccharide composition, branching, and terminal modifications such as sialylation, fucosylation, and sulfation underpins their functional specificity and regulatory capacity. This review provides a comprehensive overview of glycan biosynthesis, with a focus on N-glycans, O-glycans, glycosaminoglycans (GAGs), and glycolipids. It explores their essential roles in maintaining cellular homeostasis, development, and immune surveillance. In health, glycans mediate cell–cell communication, protein interactions, and immune responses. In disease, however, aberrant glycosylation is increasingly recognized as a hallmark of numerous pathological conditions, including cancer, neurodegenerative disorders, autoimmune diseases, and a wide range of infectious diseases. Glycomic alterations contribute to tumor progression, immune evasion, therapy resistance, neuroinflammation, and synaptic dysfunction. Tumor-associated carbohydrate antigens (TACAs) and disease-specific glycoforms present novel opportunities for biomarker discovery and therapeutic targeting. Moreover, glycan-mediated host–pathogen interactions are central to microbial adhesion, immune escape, and virulence. This review highlights current advances in glycomics technologies, including mass spectrometry, lectin microarrays, and glycoengineering, which have enabled the high-resolution profiling of the glycome. It also highlights the emerging potential of single-cell glycomics and multi-omics integration in precision medicine. Understanding glycome and its dynamic regulation is essential for uncovering the molecular mechanisms of disease and translating glycomic insights into innovative diagnostic and therapeutic strategies. Full article

The Sultanate of Oman boasts remarkable biodiversity, exemplified by such species as the Arabian leopard (Panthera pardus nimr) and the Arabian oryx (Oryx leucoryx), national symbols that highlight the extensive conservation efforts required to protect the country’s natural heritage. During decades, Omani authorities have taken significant measures to safeguard wildlife and preserve the natural environment. A sanctuary dedicated to the reintroduction of the Arabian Oryx, after extinction in nature in 1972, was established in 1980 in the Al Wusta Governorate under the patronage of the Royal Diwan and currently administrated by the recently established Environment Authority. During the almost 40 years since the reintroduction and the creation of the sanctuary, the oryx population has grown slowly but constantly. In 2021, the sanctuary hosted 738 oryx, allowing the start of the reintroduction of the species into the natural environment. Small groups of animals were released into the wild in selected areas. No animal health adverse events were recorded, and mortality was generally due to injuries received as a consequence of fighting, in particular during mating season. Standard veterinary care, including control of internal and external parasites, was regularly provided. In some occasions, immunization against certain diseases, such as clostridial infections, pasteurellosis, or mycoplasmosis, was also applied. In 2023, an FMD outbreak in cattle reported in Dhofar, about 500 km from the Al Wusta sanctuary, motivated specific prophylactic actions to prevent the risk of diffusion to oryx. From December 2023 to January 2024, an immunization program was undertaken using an FMD vaccine against serotypes A, O, and SAT 1, mostly in male oryx, while pregnant oryx were avoided for abortion risk due to handling. The following year, in January 2025, a severe outbreak occurred in oryx herds held in the sanctuary. The rapid onset and the spread of clinical symptoms among animals (100% morbidity in the second day after the first appearance of signs in some individuals) were suggestive of a highly contagious disease. The animals suffered from severe depression and inappetence, rapidly followed by abundant salivation, erosions of the oral mucosa and tongue, and diarrhea, with a short course characterized by prostration and death of the animal in the most severe cases. Therapeutical attempts (administration of antibiotics and rehydration) were mostly ineffective. Laboratory investigations (ELISA and PCR) ruled out contagious bovine pleuropneumonia (CBPP), Johne’s disease and Peste des petits ruminants (PPR). Both serology and antigen detection showed positiveness to foot-and-mouth disease (FMD). Out of a total population of 669 present in the sanctuary at the beginning of the outbreak, 226 (33.78%) oryx died. Despite the vaccinal status, the 38.49% of dead animals resulted being vaccinated against FMD. Taking into account the incalculable value of the species, the outbreak represented a very dangerous event that risked wiping out the decades of conservation efforts. Therefore, all the available means, such as accrued biosecurity and adequate prophylaxis, should be implemented to prevent the recurrence of such health risks. The delicate equilibrium of wild fauna in Oman requires study and support for an effective protection, in line with the national plan “Vision 2040”, targeting the inclusion of the Sultanate within the 20 best virtuous countries for wildlife protection. Full article

Background: While most Escherichia coli strains are harmless members of the gastrointestinal microbiota, certain pathogenic variants can cause severe intestinal and extraintestinal diseases. A notable outbreak of E. coli O104:H4, involving both enteroaggregative (EAEC) and enterohemorrhagic (EHEC) strains, occurred in Europe, resulting in symptoms ranging from bloody diarrhea to life-threatening colitis and hemolytic uremic syndrome (HUS). Since treatment options remain limited and have changed little over the past 40 years, there is an urgent need for an effective vaccine. Such a vaccine would offer major public health and economic benefits by preventing severe infections and reducing outbreak-related costs. A multiepitope vaccine approach, enabled by advances in immunoinformatics, offers a promising strategy for targeting HUS-causing E. coli (O104:H4 and O157:H7 serotypes) with minimal disruption to normal microbiota. This study aimed to design an immunogenic multiepitope vaccine (MEV) construct using bioinformatics and immunoinformatic tools. Methods and Results: Comparative proteomic analysis identified 672 proteins unique to E. coli O104:H4, excluding proteins shared with the nonpathogenic E. coli K-12-MG1655 strain and those shorter than 100 amino acids. Subcellular localization (P-SORTb) identified 17 extracellular or outer membrane proteins. Four proteins were selected as vaccine candidates based on transmembrane domains (TMHMM), antigenicity (VaxiJen), and conservation among EHEC strains. Epitope prediction revealed ten B-cell, four cytotoxic T-cell, and three helper T-cell epitopes. Four MEVs with different adjuvants were designed and assessed for solubility, stability, and antigenicity. Structural refinement (GALAXY) and docking studies confirmed strong interaction with Toll-Like Receptor 4 (TLR4). In silico immune simulations (C-ImmSim) indicated robust humoral and cellular immune responses. In Conclusions, the proposed MEV construct demonstrated promising immunogenicity and warrants further validation in experimental models. Full article

Mastitis is a common disease for dairy cows that exerts tremendously detrimental impacts on the productivity of cows and economic viability of pasture. Dihydromyricetin (DMY) is a flavonoid monomeric compound that possesses anti-inflammatory and antioxidant activity. This study aimed at dissecting the effects of DMY on the lactation performance, blood parameters, gut microbiota, and metabolite profiles of dairy cows with subclinical mastitis (SM). The results showed that dietary supplementation with DMY resulted in a reduction in milk somatic cell count, an increase in serum T-AOC and CAT activity, as well as a decrease in serum MDA content. DMY significantly enhanced the prevalence of Coprococcus and Roseburia and reduced the proportion of Cyanobacteria, Proteobacteria, and Dehalobacterium. The amino acid degradation, antibiotic resistance, and O-antigen building blocks biosynthesis (E. coli) capacity of gut microbes were notably diminished by DMY supplementation in cows with SM. Moreover, fecal and plasma metabolomic analysis revealed that DMY intervention reduced the abundance of pro-inflammatory metabolites including arachidonic acid analogues, ω-6 PUFA, and structural components of bacteria. Nevertheless, the levels of anti-inflammatory and antioxidant metabolites involving secondary bile acids, antioxidant vitamins, specific amino acid analogues, etc. were elevated by DMY administration. Overall, DMY might ameliorate SM via enhancing antioxidant capacity and improving the structure of the hindgut microbial community and metabolite profiles in dairy cows. These findings underscore the potential of DMY as a valuable dietary supplement for the improvement of mammary inflammatory diseases in dairy cows. Full article

Background: Brucellosis is a zoonotic bacterial disease primarily controlled through quarantine, culling, and vaccination. Live attenuated vaccines remain the most effective countermeasure, yet their application is limited by residual virulence and diagnostic interference. This study developed three rough-type attenuated Brucella melitensis mutants (G7, G8, G16) and evaluated their potential as DIVA (Differentiating Infected from Vaccinated Animals) vaccine candidates. Methods: Rough phenotypes were characterized through heat agglutination, acridine orange staining, and immunoblotting. Macrophage cytotoxicity was assessed via LDH release assays, while RT-qPCR analyzed macrophage activation capacity. Mouse infection and immunization-challenge experiments, complemented by histopathology, evaluated residual virulence and protective immunity. Antibody profiles were determined by ELISA, and DIVA capability was verified using LPS-coated ELISA. Results: G7 and G8 exhibited complete rough phenotypes, whereas G16 retained partial O-antigen (semi-rough). All rough mutants induced macrophage cytotoxicity and activation. The strains showed attenuated virulence with no viable bacteria recovered from spleens at 4 weeks post-inoculation. Histopathology revealed no liver lesions at 6 weeks post-inoculation. Immunized mice predominantly produced IgG2a-dominated Th1-type responses. The immune protection levels of G7 and G16 matched the reference vaccine M5–90Δ26, while G8 showed slightly lower efficacy. LPS-ELISA effectively differentiated vaccinated from infected animals via concurrent IgM/IgG detection. Conclusions: This study demonstrates that the rough-type B. melitensis mutants G7 and G16 serve as promising DIVA vaccine candidates, offering strong protection with low residual virulence while enabling serological differentiation between vaccinated and infected animals, highlighting their potential as effective vaccines for brucellosis control. Full article

Background/Objectives: Among Enterobacterales, OXA-48-like-producing Proteus mirabilis strains have been scarcely detected. Herein, we characterized a bla_OXA-48-harbouring P. mirabilis strain recovered from Greece (Pm GR-1), while phylogenomics and comparative genomics analyses with previously published bla_OXA-48 carriers were also assessed. Methods: Characterization of Pm GR-1 was performed by the Vitek^® Compact and Mass Spectrometry systems, antimicrobial susceptibility testing, detection of beta-lactamases, multilocus-sequence typing (MLST), and whole-genome sequencing (WGS). In silico prediction of mobile genetic elements (MGEs), genomic islands (GIs), antimicrobial resistance genes (ARGs) and virulence factors (VFs), and phylogenetic, core-genome SNP and comparative genomics analyses were executed using bioinformatic tools. Results: Pm GR-1 was isolated from a urine sample of an outpatient in a Greek hospital. It exhibited a multidrug-resistant phenotype, being susceptible only to amikacin and ceftazidime/avibactam. It co-carried several beta-lactamase genes on the chromosome (bla_OXA-48, bla_CTX-M-14, bla_TEM-1) and a plasmid (bla_TEM-2) and several other ARGs, but also mutations associated with quinolone resistance in the DNA gyrase and topoisomerase IV subunits. It belonged to the international clone ST135 that has also been detected among OXA-48-producing P. mirabilis strains from Germany and the USA. Pm GR-1 was genetically related to those from Germany, sharing highly similar MGEs, GIs, ARGs and VFs, including the chromosomal bla_OXA-48 genetic structure, the O-antigen locus, the flagella locus, the MR/P fimbriae operon, and the urease gene cluster. Conclusions: To our knowledge, this is the first report from Greece of a bla_OXA-48-possessing P. mirabilis strain. The emergence of bla_OXA-48 among P. mirabilis strains of the international clone ST135 in different geographical regions is worrying. Close monitoring of these strains is required in One Health settings. Full article

Avian pathogenic Escherichia coli (APEC) poses a global threat to poultry health and public safety due to its high lethality, limited treatment options, and potential for zoonotic transmission via the food chain. However, long-term genomic surveillance remains limited, especially in countries like China where poultry farming is highly intensive. This study aimed to characterize the population structure, virulence traits, and antimicrobial resistance of 81 APEC isolates from diseased chickens collected over 16 years from Shandong and neighboring provinces in eastern China. The isolates were grouped into seven Clermont phylogroups, with A and B1 being dominant. MLST revealed 27 STs, and serotyping identified 29 O and 16 H antigens, showing high genetic diversity. The minor phylogroups (B2, C, D, E, G) encoded more virulence genes and had higher virulence-plasmid ColV carriage, with enrichment for iron-uptake, protectins, and extraintestinal toxins. In contrast, the dominant phylogroups A and B1 primarily carried adhesin and enterotoxin genes. Antimicrobial resistance was widespread: 76.5% of isolates were multidrug-resistant. The minor phylogroups exhibited higher tetracycline resistance (mediated by tet(A)), whereas the major phylogroups showed increased resistance to third- and fourth-generation cephalosporins (due to bla_CTX-M-type ESBL genes). These findings offer crucial data for APEC prevention and control, safeguarding the poultry industry and public health. Full article

Brucellosis continues to be a significant global zoonotic infection, with diagnosis largely relying on the detection of antibodies against the Brucella O-polysaccharide (O-PS) A and M antigens. In this study, computational methods, including homology modeling, molecular docking, and molecular dynamics simulations, were applied to investigate the interaction of the four murine monoclonal antibodies (mAbs) YsT9.1, YsT9.2, Bm10, and Bm28 with hexasaccharide fragments of the A and M epitopes. Through stringent stability criteria, based on interaction energies and mobility of the antigens, high-affinity binding of A antigen with YsT9.1 antibody and M antigen with Bm10 antibody was predicted. In both the complexes hydrophobic interactions dominate the antigen–antibody binding. These findings align well with experimental epitope mapping, indicating YsT9.1’s preference for internal sequences of the A epitope and Bm10’s preference for internal elements of the M epitope. Interestingly, no stable complexes were identified for YsT9.2 or Bm28 interacting with A or M antigen. This study provides valuable insights into the mechanism of molecular recognition of Brucella O-antigens that can be applied for the development of improved diagnostics, synthetic glycomimetics, and improved vaccine strategies. Full article

Celiac disease (CD), a human leukocyte antigen-associated disorder, is caused by gluten sensitivity and is characterized by mucosal alterations in the small intestine. Currently, its diagnosis involves the determination of serological markers. The traditional method for clinically determining these markers is the enzyme-linked immunosorbent assay. However, immunosensors offer sensitivity and facilitate the development of miniaturized and portable analytical systems. This work focuses on developing an amperometric immunosensor for the quantification of IgA antibodies against tissue transglutaminase (IgA anti-TGA) in human serum samples, providing information on a critical biomarker for CD diagnosis. The electrochemical device was designed on a polyimide substrate using a novel solid ink of wax and carbon nanofibers (CNFs). The working electrode microzone was defined by incorporating aminofunctionalized TiO₂ nanoparticles (TiO₂NPs). The interactions and morphology of CNFs/wax and TiO₂NPs/CNFs/wax electrodes were assessed through different characterization techniques. Furthermore, the device was electrochemically characterized, demonstrating that the incorporation of CNFs into the wax matrix significantly enhanced its conductivity and increased the active surface area of the electrode, while TiO₂NPs contributed to the immunoreaction area. The developed device exhibited remarkable sensitivity, selectivity, and reproducibility. These results indicate that the fabricated device is a robust and reliable tool for the precise serological diagnosis of CD. Full article

Background/Objectives: An accurate quantification of the effective antigens from different serotypes is essential for the quality control of multivalent vaccines, but it remains challenging. Herein, we developed a simple and high-throughput method using differential scanning fluorimetry (DSF) for quantifying foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent vaccines. Methods: Purified serotypes A and O FMDV were used to establish and validate the method. The DSF parameters, including the dye concentration, thermal scanning velocity, and PCR tube material, were optimized at different FMDV concentrations. The established DSF method was validated for the quantification of monovalent and A/O bivalent FMDV, and was compared with the ultracentrifugation of 86 samples from different processing stages and serotypes. Results: The DSF showed that the melting temperature (T_m) of type A (56.2 °C) was significantly higher than that of type O FMDV (50.5 °C), indicating that their T_m can be distinguished in bivalent antigens. After optimizing the DSF parameters, a strong correlation (R² > 0.998) was observed between the 146S concentration and the maximum of the first derivative of the DSF fluorescence (d(RFU)/dT) for both serotypes A and O FMDV. The method demonstrated good reproducibility (RSD < 10%) and high sensitivity (limit of detection: 0.7 μg/mL). Using a multiple linear regression analysis, the simultaneous quantification of A and O FMDV in the bivalent mixtures achieved recovery rates of 82.4–105.5%, with an RSD < 10% for most of the samples. Additionally, the DSF results correlated well with the ultracentrifugation data (Pearson ρ = 0.9789), validating its accuracy and broad applicability. Conclusions: In summary, DSF represents a simple, rapid, and high-throughput tool for the quality control of monovalent and bivalent FMDV vaccines. Full article

Background: Since the massive outbreak of foot-and-mouth disease (FMD) in South Korea in 2010–2011, cloven-hoofed livestock have been immunized with serotype O and A vaccines across the country. Other serotypes of FMD vaccines were stockpiled in overseas FMD vaccine factories as antigen banks. Once a manufacturing facility has been established in South Korea, the overseas antigen banks will be replaced by domestic one. Therefore, this study aimed to evaluate the commercial potential of the previously developed SAT2 vaccine candidate (SAT2 ZIM-R). Methods: The optimal condition was determined at various virus concentrations, infection times, and pH levels, resulting in 0.01 MOI for SAT2 ZIM-R for 24 h infection at a pH of 7.5. Results: When the SAT2 ZIM-R virus was produced in flasks from 40 to 1000 mL in fivefold increments, all scales of production yielded > 7.0 µg/mL of antigens. Using a bioreactor, 5.6 µg/mL of antigens was recovered from a 1 L viral culture. The optimal conditions of viral inactivation kinetics were determined to be 1 mM of binary ethyleneimine (BEI) treatment at 26 °C for 24 h, with approximately 91% of the antigen being retained after virus inactivation. When the SAT2 ZIM-R experimental vaccine was administered twice to pigs, the neutralizing antibody titer increased approximately 500-fold after booster immunization. Conclusions: To the best of our knowledge, this is the first study to evaluate the antigen productivity, viral inactivation kinetics, and immunogenicity of the SAT vaccine strain in pigs. In the future, the SAT2 ZIM-R vaccine may be a useful candidate vaccine for a domestic antigen bank. 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

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

Listeria monocytogenes is a foodborne pathogen frequently exposed to oxidative stress in diverse environmental conditions. Cyclic di-AMP (c-di-AMP) is a second messenger that plays a key role in stress resistance. This study investigates the role of pdeA (degrades c-di-AMP) and how c-di-AMP accumulation affects catalase activity and oxidative stress response and gene expression. Survival and catalase activity assays were conducted under oxidative stress, and c-di-AMP levels were quantified in L. monocytogenes 10403S under aerobic, anaerobic, and L-cysteine-supplemented conditions. ΔpdeA, which accumulates c-di-AMP, exhibited greater sensitivity to oxidative stress (4.6 log reduction for the wild type (WT) vs 7.34 log reduction for ΔpdeA at 10 h) and lower catalase activity than the WT in the early stationary phase. However, in the late stationary phase, while the catalase activity levels of ΔpdeA remained stable (~6.33 cm foam height), it became resistant to oxidative stress (5.85 log reduction). These findings indicate that pdeA contributes to catalase activity in L. monocytogenes. Transcriptomic analysis revealed differential expression of pathways mainly including pentose phosphate pathway, carbon metabolism, O-antigen nucleotide sugar biosynthesis and ABC transporters in ΔpdeA compared to WT. Our transcriptomic data provided promising insights into the molecular mechanisms underlying c-di-AMP regulation, which may enhance stress resistance. Moreover, oxidative stress led to increased intracellular c-di-AMP levels. Under L-cysteine supplementation, catalase activity levels in WT were similar to ΔpdeA (~1.86 cm foam height for both), but the latter showed enhanced oxidative stress resistance and c-di-AMP levels. Anaerobic conditions also elevated c-di-AMP levels in WT and ΔpdeA but resulted in greater oxidative stress sensitivity. Understanding these regulatory mechanisms provides valuable insights into oxidative stress resistance, with potential implications for food safety and pathogen control. Full article