Search Results (1,773)

Virus-like particles (VLPs) based on the capsid protein (CP) of the ssRNA bacteriophage Beihai32 represent a promising nanoscale platform for the presentation of heterologous peptides. Previous studies have shown that the C-terminus of the CP tolerates long insertions without compromising VLP assembly. Here, we demonstrate that the N-terminus is similarly permissive to extended insertions. Hybrid CPs with one to four copies of the influenza A virus M2e peptide fused to the N-terminus were expressed in Escherichia coli. Fusion proteins containing four copies of M2e self-assembled into spherical VLPs, displaying the inserted peptides on the surface. Subcutaneous immunization of mice with chimeric VLPs induced high titers of M2e-specific antibodies. Unlike C-terminal fusions, the N-terminal insertion prevented the induction of anti-carrier antibody response indicting masking of the carrier protein in the chimeric VLP. To evaluate the capacity of the N-terminus for larger inserts, green fluorescent protein (GFP, 238 a.a.) was attached to the N-terminus of CP. The hybrid protein was expressed in Escherichia coli and formed VLPs. GFP was displayed on the particle surface and retained fluorescent activity. Overall, the phage Beihai32 CP is a versatile platform for the presentation of peptide antigens, supporting its potential application in VLP-based vaccine design. Full article

N-linked glycans (N-glycans) are complex carbohydrate structures covalently attached to the asparagine residues of glycoproteins and play a substantial role in protein folding, stability and biological activity. One of the major challenges in glycomics is the enzymatic release of intact N-glycans from native glycoproteins, as conventional deglycosylation approaches often require prior protein denaturation and exhibit limited activity toward structurally diverse substrates. In this study, we recombinantly produced EndoBI-1, an endo-β-N-acetylglucosaminidase derived from Bifidobacterium longum subsp. infantis ATCC 15697, and investigated its N-glycan release activity on two structurally distinct glycoprotein substrates: bovine lactoferrin (bLF), an animal-derived glycoprotein, and soy protein, a plant-derived glycoprotein. EndoBI-1 was produced using an in vivo cloning approach and purified to homogeneity, as validated by SDS-PAGE analysis. The released N-glycan profiles of each substrate were characterized by HILIC-FLD-QTOF-MS/MS analysis, which showed different glycan compositions depending on the origin: complex-type and sialylated N-glycans were the major structures in the bLF derived fractions, while the fractions derived from soy protein mainly contained high-mannose-type structures. Together, these results demonstrate the capacity of EndoBI-1 to release structurally intact N-glycans from both animal- and plant-derived glycoproteins under native, non-denaturing conditions, establishing this enzyme as a biocatalytic tool for the preparative-scale production of bioactive N-glycans from diverse protein sources. Full article

The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels suggested a strong correlation between exposure to atmospheric particulate matter and an increased risk of developing severe forms of disease. This correlation has been highlighted by several epidemiological studies, indicating the existence of shared molecular mechanisms. Emerging evidence has highlighted the important role of lipid rafts in facilitating viral entry into cells. Specifically, the receptor binding domain of the SARS-CoV-2 spike protein interacts with sialylated glycans of the monosialic ganglioside GM1 and GM2 that are particularly enriched in lipid rafts. This interaction has been proposed to facilitate ACE2 recognition by the spike protein and may contribute to early events involved in viral attachment and entry. Here, we reveal that A549 alveolar lung cells, after DEP exposure, exhibit a significant shift in ACE2 into lipid rafts, accompanied by an increase in the immature form of ADAM17, the sheddase responsible for ACE2 cleavage. Additionally, DEP exposure results in a significant increase in IL-6 release, while no changes were observed in IL-8 and sACE2 release. This treatment does not cause significant alterations in protein levels or membrane redistribution of COX-2 and HO-1, proteins involved in the inflammatory response and oxidative stress following exposure to air pollution, and linked to COVID-19 pathogenesis. Finally, lipidomic analysis by UHPLC-MS revealed that DEP exposure induces a significant increase in GM2 levels, and a concomitant decrease in GM1 and GM3 levels. Together, these results indicate that DEP exposure remodels lipid raft-associated molecular features in A549 cells, including ACE2 membrane redistribution, altered ganglioside composition, and increased IL-6 release. Although these changes may be relevant to cellular mechanisms associated with SARS-CoV-2 susceptibility, the present study does not directly assess viral binding, viral entry, or infection, and further functional studies are required. Full article

Cell surface markers are essential for lineage tracing and for understanding cellular mechanisms of regeneration, yet they remain largely unavailable for echinoderms. Using the starfish Asterias rubens as a model for physiological coelomocyte renewal, we combined Percoll density-gradient fractionation with label-free LC-MALDI proteomics to identify candidate surface proteins that distinguish subpopulations of coelomocytes and coelomic epithelium (CE) cells. However, LC-MALDI is prone to signal variability, sample depletion and nonlinear response. To address the quantitative limitations of LC-MALDI, we implemented a chi-square test with an empirically derived probability density function, which we validated on control mixtures, achieving high precision. Four fractions enriched in distinct cell types were analyzed: coelomocyte fractions 1 (roundish cells) and 4 (large agranulocytes that form cell nets) and CE fractions 1 (type 2 small CE cells) and 4 (ciliated cells), together with a weakly attached CE subpopulation enriched in small putative progenitor cells. LC-MALDI identified 714 protein groups, of which 160 showed significant differential abundance across fractions. Functional enrichment revealed actin cytoskeleton and integrin signaling in coelomocytes, and catenin-related terms in CE-W. Among the 101 predicted membrane proteins, 31 candidate cell surface markers were selected. These include integrins α8β1 and α9β1, three-finger proteins, scavenger receptors, and low-density lipoprotein receptor-related proteins, as well as metallopeptidases. This work provides the first proteome-scale set of candidate surface markers for starfish cell subpopulations, enabling future lineage-tracing studies to resolve the origin of coelomocytes from the coelomic epithelium. Full article

The Powassan virus (POWV) is a neurovirulent tick-borne virus that causes age-associated lethality and long-term neurologic sequelae in 50% of survivors. In aged mice the POWV strain LI9 mirrors human lethality and neuropathology; however, an avirulent POWV mutant, LI9-D308N, fails to enter the CNS or cause lethal disease. The D308N mutation is present in an envelope protein domain associated with cell attachment, yet the role of D308N mutations in cell tropism and neuroinvasion remains to be resolved. Here, we engineered fluorescent mScarlet3 and mNeonGreen reporter genes into WT LI9, and avirulent LI9-D308N viruses and assessed their ability to infect CNS cells in vitro. In addition, we generated replication-defective reporter POWVs that only replicate in NS1-expressing cells by replacing NS1 with fluorescent genes. Similar to WT LI9, fluorescent reporter POWVs spread focally and nonlytically, are stable following passage and reach high titers 2–5 dpi. In NS1-expressing VeroE6 cells, LI9-ΔNS1-FL reporters exhibited robust fluorescence 24 h post-infection (hpi), while fluorescence from LI9-reporter infections was first observed ~32 hpi. Comparing LI9-mScarlet3 and avirulent LI9-D308N-mScarlet3 viruses revealed no difference in their ability to infect human brain microvascular endothelial cells, pericytes, astrocytes, microglia or neuronal cells in vitro. Notably, LI9-mScarlet3 viruses productively and persistently infected differentiated, neuron-like, SH-SY5Y cells without apparent cytotoxicity. These findings indicate that LI9-D308N is capable of infecting blood–brain-barrier and CNS cells, and suggest that neuroinvasion is restricted prior to LI9-D308N engaging CNS cells. These results are consistent with clearance of LI9-D308N from the blood, or the D308N mutation interfering with potential routes of POWV neuroinvasion. Collectively, fluorescent POWV reporter viruses provide insight into the mechanism of POWV neuroinvasion, permit analysis of replication-defective POWVs as vaccines and provide a means of analyzing POWV cell tropism, antivirals and cell-to-cell spread in BSL2 and BSL3 settings.: Full article

Nipah virus (NiV) is a highly lethal zoonotic pathogen with significant pandemic potential, for which no approved antiviral therapies are currently available. Viral entry is mediated by the interaction between the NiV attachment glycoprotein (NiV-G) and host ephrin receptors, particularly ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3), making this interface an attractive therapeutic target. In this study, we evaluated a set of structurally related flavonoids, apigenin, cynaroside, and lonicerin, as potential modulators of the EFNB2–NiV-G and EFNB3–NiV-G interactions. These compounds were selected based on their structural similarity, reported antiviral activity, chemical diversity, and favorable drug-like properties. Apigenin was employed as a reference scaffold due to its well-characterized pharmacological profile and its suitability for guiding analog-based compound selection. Apigenin served as a reference scaffold for selecting structurally related flavonoids, which were analyzed through density functional theory optimization, molecular docking, pharmacokinetic and toxicity predictions, molecular dynamics simulations, and binding free energy calculations. These flavonoids demonstrated high predicted affinity for both the EFNB2–NiV-G and EFNB3–NiV-G interfaces. According to results, these compounds consistently interacted with residues known to play a critical role in receptor recognition, with special emphasis on leucine and tryptophan residues within the G–H loop. These residues are well established as key determinants in the entry process of Nipah virus (NiV) into host cells, highlighting the potential relevance of these flavonoid–protein interactions. Molecular dynamics analyses indicated that flavonoid binding reduced the affinity and the conformational flexibility at the receptor–glycoprotein interfaces and decreased the stability of the complexes. Pharmacokinetic and toxicity predictions suggested favorable drug-like properties for the flavonoids, with apigenin displaying the most balanced profile. Collectively, these results support the potential of selected flavonoids as modulators of EFNB2–NiV-G and EFNB3–NiV-G interactions and provide a rationale for their prioritization in experimental studies aimed at developing scaffolds for the modulation of viral entry against Nipah virus. Full article

Background/Objectives: Resveratrol, a natural polyphenol with anti-inflammatory and antioxidant properties, has emerged as a promising adjunctive agent in periodontal therapy. This systematic review and meta-analysis evaluated evidence from in vitro, in vivo, and human clinical studies regarding the effects of resveratrol on periodontal disease, with the clinical component focusing on systemically healthy non-smoking patients. Methods: Electronic searches of MEDLINE/PubMed, Scopus, Embase, and Web of Science were conducted for studies published between 2000 and 2025. Eligible studies included periodontal-related in vitro cell models, in vivo experimental periodontitis models, and randomized clinical trials assessing resveratrol as an adjunctive therapy. Data extraction, risk-of-bias assessment, and meta-analyses were conducted in accordance with PRISMA guidelines. Results: Fifteen studies were included (five in vitro, six in vivo, and five human randomized controlled trials). Meta-analysis showed the inhibitory effect of resveratrol on the lipopolysaccharide-induced protein expression of IL-1β, IL-6, TNF-α, and IL-8 in vitro, with low to moderate heterogeneity. In animal studies, resveratrol significantly attenuated ligature-induced dental alveolar bone loss, IL-1β protein, and relative mRNA expression. However, reductions in relative mRNA expressions of TNF-α and IL-6 were inconsistent and highly heterogeneous; in contrast, nanoparticle- and liposomal-modified resveratrol consistently and significantly reduced these mRNA levels. In human trials, adjunctive resveratrol was associated with improvements in probing pocket depth and clinical attachment level compared with root planing alone in patients with periodontitis, as well as reductions in bleeding and plaque indices in patients with periodontal diseases, including gingivitis and periodontitis. Conclusions: Resveratrol suppresses pro-inflammatory cytokine expression in vitro and attenuates alveolar bone loss in vivo, with enhanced and more consistent molecular effects observed using modified formulations. Preliminary clinical evidence suggests that resveratrol may be associated with modest adjunctive effects on periodontal outcomes in systemically healthy non-smokers. However, given the limited number of clinical trials, small sample sizes, heterogeneity among studies, short follow-up periods, and limited certainty of the evidence, these findings should be interpreted cautiously. Further well-designed RCTs with longer follow-up periods are required to determine their clinical relevance. Full article

Ubiquitination is a post-translational modification that governs various facets of eukaryotic biology, including protein stability, signaling, and immune regulation. The modification process is mediated by a coordinated enzymatic cascade, in which E3 ubiquitin ligases confer substrate specificity and determine the functional outcome of ubiquitin attachment. In the case of a virus infection, host cellular signaling networks undergo major ubiquitin-dependent changes to protect the host cell, including remodeling of cellular organelles, coordination of innate immunity, and reprogramming of metabolic pathways to prevent virus replication. African swine fever virus (ASFV) has evolved numerous strategies to counteract or evade these responses, thereby manipulating host defenses and promoting its replication. By modulating ubiquitination-dependent host cellular functions, the virus can regulate key immune signaling factors, suppress interferon production, and interfere with inflammatory pathways. These actions not only antagonize antiviral defenses but also remodel cellular homeostasis to favor infection. The important interplay between host defense and viral manipulation underscores the versatility of the ubiquitin system as a battleground in ASFV infection. In this review, we discussed mechanistic insights into how ASFV subverts ubiquitin pathways during host–virus interactions. This comprehensive knowledge might be beneficial for pharmaceutical exploration of host E3 ligase-dependent anti-ASFV treatment. Full article

Glycosylation is the most common post-translational modification in the human proteome, with over half of all human proteins bearing covalently attached glycans. These glycan structures direct protein folding through ER quality control machinery, shield polypeptides from proteolytic degradation, regulate circulatory half-life via the asialoglycoprotein receptor, and serve as molecular signals for immune recognition and intracellular trafficking. For biopharmaceuticals, which constitute a rapidly growing share of approved drugs, glycan profiles are critical quality attributes that directly determine clinical efficacy and safety. Yet achieving the correct glycosylation on a therapeutic protein remains one of the field’s central challenges, as glycan biosynthesis is non-template-driven and highly sensitive to expression system and manufacturing conditions. This review connects the biological functions of glycosylation to the practical strategies of glycoengineering, examining how sequence design, expression system selection, and downstream enzymatic remodeling are used to optimize therapeutic glycoproteins. Clinical case studies spanning monoclonal antibodies, cytokines, and enzyme replacement therapies illustrate how glycan engineering translates into improved patient outcomes. We conclude by surveying emerging technologies poised to make precisely glycosylated therapeutics more accessible. Full article

Eosinophils are highly granulated white blood and tissue cells that play complex roles in the immune system including host protection against helminthic parasites, viruses, fungi, and bacteria. These bone marrow-derived cells cause tissue damage in a range of diseases and disorders, particularly in allergy, asthma, and chronic rhinosinusitis with nasal polyps. Eosinophils are recruited to tissues in response to chemotactic signals, and during inflammation, they release a plethora of mediators, including immunoregulatory cytokines, through multiple pathways involving degranulation, respiratory burst, lipid mediator release, exosome release, and extracellular trap formation. Degranulation from eosinophils has been implicated as a major effector mechanism in airway diseases, particularly late phase asthma responses and in nasal polyps from patients with chronic rhinosinusitis. In degranulation responses, eosinophils release numerous granule proteins by classical exocytosis, compound exocytosis, piecemeal degranulation, and cytolysis, which refers to cell lysis through membrane rupture and cell destruction. Cytolysis can lead to suicidal extracellular trap formation, which is a regulated form of cell death involving the release of extracellular DNA traps and granule proteins. Granule release from eosinophils is dependent on activation of specific and tightly regulated intracellular signaling pathways, including Rac and Rab guanosine triphosphatases, soluble NSF attachment protein (SNAP) receptors (SNAREs), Cdk5 kinase, and actin dynamics. These observations have shown selective and nonredundant roles for signaling in degranulation responses. In this review, we explore findings from the literature on the mechanisms controlling granule-derived mediator release from eosinophils. Full article

Glycosphingolipids (GSLs) are glycoconjugates in which a short and heterogeneous saccharide chain is attached to a lipid moiety called ceramide. Based on their sugar backbone, mammalian GSLs are primarily grouped into the ganglio-, lacto-/neolacto-, and globo-series. Sialic acid—containing GSLs are known as gangliosides. Complex ganglio-series gangliosides are particularly abundant in the brain, whereas simple ganglio-series gangliosides, as well as those belonging to other series or neutral GSLs, are less abundant and typical of non-neural tissues. Congenital disorders in the biosynthesis of the lipid moiety of sphingolipids (SLs) result from defects in enzymes and proteins involved in ceramide biosynthesis and transport. Congenital disorders in the biosynthesis of the sugar chain of GSLs specifically affect ganglio-series ganglioside biosynthesis and are caused by pathogenic variants in GM3 synthase (ST3GAL5) or GM2/GD2/asialo-GM2 synthase (B4GALNT1). Defective variants of the sialyltransferase ST3GAL3 and the galactosyltransferase B4GALT5 have been reported and proposed to impair GSL biosynthesis. The occurrence of these syndromes has provided new insights into the physiological and pathological roles of GSLs. Most of these disorders are associated with completely inactive enzyme variants, leading to severe neurological syndromes. Only a few cases highlighted variants that retained partial activity, resulting in milder phenotypes, which included non-syndromic intellectual disability. It is therefore conceivable that many undiagnosed patients, with mild neurological symptoms, may carry variants retaining residual enzyme activity, insufficient to ensure normal levels of brain GSLs. The purpose of this article is to encourage clinicians to look for additional GLS hereditary disorders associated with a milder phenotype. We also hope to boost future investigations by highlighting the most critical issues emerging from recent literature on SL and GSL biosynthesis and their related defects. Full article

Cartilage engineering is a new therapeutic approach in regenerative medicine. This study explored the chondrogenic potential of human dental pulp stem/stromal cells (hDPSCs) and adipose-derived stem/stromal cells (hAD-MSCs) grown on a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold in relation to the phenotype of primary chondrocytes on PLCL. The effects of PLCL scaffold on the biological features of hDPSC, hAD-MSC, and their chondrogenic differentiation and chondrocytes biology were evaluated via flow cytometry, immunochemistry, biochemistry, and RT–PCR. The results demonstrated that PLCL supported hDPSC, hAD-MSC, and chondrocyte viability and cellular attachment. The chondrogenic potential of hDPSCs and hAD-MSCs on PLCL scaffold was evidenced by the mRNA expression of the cartilage-specific genes. Collagen type II (Col II) and aggrecan (Acan) gene expression and their proteins significantly increased in chondrogenically differentiated hDPSCs and hAD-MSCs on PLCL compared with undifferentiated stem/stromal cells on PLCL. The phenotype of differentiated hDPSCs and hAD-MSCs was comparable to primary chondrocytes grown on PLCL. The results of this study showed that PLCL scaffold promoted chondrogenic differentiation of hAD-MSCs and hDPSCs toward chondrocytes with phenotypic similarities to native chondrocytes. The PLCL scaffold composition has a positive effect on hDPSC, hAD-MSC, and chondrocyte behavior, chondrogenic gene expression, and matrix protein synthesis. Full article

Background/Objectives: The corpus luteum (CL) in the dog is the sole source of progesterone (P4) during diestrus and pregnancy, making it a key regulator of reproductive function. However, robust and functionally validated in vitro models of canine luteal cells remain limited. This study aimed to establish and characterize a reproducible primary culture system of canine luteal cells with preserved steroidogenic activity and regulatory responsiveness. Methods: Ovaries containing CLs were collected from five clinically healthy bitches undergoing routine ovariohysterectomy (OHE). Luteal tissue was mechanically fragmented and enzymatically digested using collagenase type II. Primary cultures were established using an explant-based approach and maintained in Dulbecco’s Modified Eagle Medium/Ham’s F-12 nutrient mixture (DMEM/F12) or Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 20% fetal bovine serum (FBS). Cellular morphology, proliferation, expression of steroidogenic markers—steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), P4 secretion, and responsiveness to forskolin stimulation were evaluated. Results: Cultured luteal cells exhibited stable attachment, proliferation, and a predominantly spindle-shaped morphology. Both media supported maintenance of a steroidogenic phenotype, while RPMI 1640 enabled enhanced proliferation, allowing expansion up to passage three and efficient cryobanking. Cells remained functionally active, secreting progesterone for up to 28 days in vitro. Forskolin stimulation increased progesterone secretion up to 2.7-fold, confirming preserved cyclic AMP-dependent steroidogenic responsiveness. Conclusions: The canine CL is a reliable source of functionally competent luteal cells, and the established culture system represents a physiologically relevant in vitro model. To our knowledge, this is the first functionally validated in vitro model of the canine CL. This platform enables controlled investigations of luteal function, endocrine regulation, and mechanisms of P4 synthesis, supporting its application in mechanistic and translational reproductive research. Full article

Hepatocellular carcinoma is challenging to detect at an early stage, and its severity increases over time. Recently, the incidence of hepatocellular carcinoma has increased, partly due to lifestyle-related factors such as excessive alcohol intake, sedentary behavior, and diets high in fat, which contribute to the growing prevalence of fatty liver and hepatitis. Various therapeutic strategies are being explored for hepatocellular carcinoma, among which therapies targeting deubiquitinating enzymes (DUBs) have attracted growing attention. Ubiquitination acts as a crucial modulator in the regulation of intracellular signaling across many diseases. E3 ligase recognizes the target protein and transfers ubiquitin, received from the E2 enzyme, to the lysine residues of the substrate, thereby conferring specificity to the ubiquitination process. Once a ubiquitin chain is attached to a target protein by an E3 ligase, the protein is directed to the ubiquitin–proteasome system (UPS) for degradation. In this process, the 26S proteasome complex recognizes the ubiquitin chain and degrades the target protein, thereby serving as a major mechanism for maintaining protein homeostasis. Through this pathway, cells regulate signal transduction, eliminate abnormal proteins, and perform various essential functions. On the other hand, deubiquitinating enzymes (DUBs) recognize the ubiquitin chains on target proteins and remove them by hydrolyzing the isopeptide bonds of ubiquitin, thereby enabling the target proteins to evade degradation by the proteasome system. Furthermore, deubiquitinating enzymes independently remove ubiquitin from proteins and can serve as central regulators in signaling pathways related to hepatocellular carcinoma. Full article

Recent studies have highlighted the potential of urinary bladder matrix (UBM) derived from decellularized porcine urinary bladder as a bioactive hydrogel. Despite its complex composition of over 100 proteins, Type I collagen is the primary constituent of UBM. Caco-2 cells are widely used as an in vitro model of the intestinal epithelium; however, to date, no published study has evaluated the effects of UBM on Caco-2 cells. In this study, Electric Cell–Substrate Impedance Sensing (ECIS) was used to measure Caco-2 cell attachment and wound-healing migration on UBM-coated microelectrodes. Our results demonstrate that UBM hydrogel coating at 0.2 mg/mL significantly accelerates cell attachment and enhances migration rates compared to uncoated controls. These stimulatory effects were comparable to those observed with 0.2 mg/mL Type I collagen, suggesting that UBM can function as effectively as Type I collagen. We further monitored barrier formation in Caco-2 cells cultured on UBM-coated transwell membrane inserts using TEER measurements and scanning electron microscopy. The TEER values reached 300 Ω·cm² within three days, indicating the rapid establishment of mature tight junctions. Overall, these results show that UBM hydrogel coatings are effective substrates for Caco-2 cells, performing as well as Type I collagen in all our tests. Full article