Search Results (5,158)

SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis. Full article

Neospora caninum, as well as Toxoplasma gondii, secrete proteins that facilitate the invasion of host cells and the regulation of host immune response and metabolism. However, the localization of the secretory proteins in infected animal brains has not been studied in detail. Here, we investigate the brain and intracellular distribution of the secretory proteins in experimentally infected mice and naturally infected calves through histopathology and immunohistochemistry (IHC) to detect surface antigen 1 (NcSAG1), cyclophilin (NcCYP), profilin (NcPF), dense granule protein 6 (NcGRA6), and NcGRA7. These methods revealed that numerous tachyzoites positive for NcSAG1, NcCYP, NcPF, NcGRA6, and NcGRA7 were localized in and around the animals’ necrotic lesions, and NcGRA7 was diffusely observed in the necrotic lesions of the infected mice. Moreover, IHC revealed that NcGRA6 and NcGRA7 were distributed in the cytoplasm of infected neurons around the parasites in the infected mice and calves. This suggests that NcGRA6 and NcGRA7 might be directly related to the alteration of neuronal metabolism and activity, and that NcGRA7 might be related to the formation of necrotic lesions. Full article

Objectives: The aim of the present study was to better understand the antibody concentrations in healthcare workers (HCWs) from a hospital in Mexico City with a high density of COVID-19 patients. Methods: Up to 243 HCWs were recruited in 2020 and 2022 and were sorted into three groups: hybrid immunity (HI, natural infection plus vaccination), vaccine-induced immunity (VI), and unvaccinated but RT-qPCR negative at the beginning of the pandemic (UV). Peripheral blood and nasopharyngeal swab samples were obtained; additionally, saliva samples were obtained from the UV group. The titers of IgG, IgM, and IgA against the SARS-CoV-2 receptor-binding domain (RBD) and nucleocapsid (NCP) proteins were assessed using an in-house ELISA, and positivity to the virus was determined via RT-qPCR. Results: Most HI and VI participants were positive for serum anti-RBD IgG (92.8% and 100%, respectively), while 26.6% (for HI) and 19% (for VI) were positive for anti-NCP IgG. Regarding serum anti-RBD IgA, the VI and HI groups had positive rates of 87.3% and 66%, respectively. In contrast, the UV group showed a rate of 5.7% but the positivity for IgA in saliva was higher (52% for RBD and 35% for NCP). In addition, the highest antibody titers were obtained for anti-RBD IgG and IgA in the HI and VI groups, respectively. In saliva, the IgA antibody titer was higher for the RBD antigen (1:1280). Conclusions: These results strengthen our understanding of antibody concentrations in HCWs during two critical years of the pandemic in a general hospital with many COVID-19 patients. Full article

Background/Objectives: Human endogenous retroviruses (ERVs) are genomic sequences integrated into the human genome from ancestral exogenous retroviruses and are epigenetically silenced under normal conditions. Growing evidence has shown that they can be reactivated in human diseases such as cancers and autoimmune diseases. However, their clinical implications in colon cancer are yet to be explored. Methods: RNA-seq data were downloaded from RNA Atlas and TCGA for cell lines and tissue samples, respectively. After alignment, ERV expression was quantified against comprehensively compiled ERVs (3220). ERV expression profiles were compared between sequencing protocols, cancer and normal cells, and matched tumor and normal tissue pairs. Unsupervised clustering was used to identify ERV-defined tumor subtypes and their associations with clinical and other molecular features. ERV association with disease-specific survival (DSS) was performed using the Cox regression model. Results: PolyA and total RNA protocols were comparable in ERV expression detection. Cancer cells had significantly increased ERV expression and reactivation. Upregulated ERVs were significantly enriched in viral protein interactions with cytokine and cytokine receptors. ERV expression-defined tumor classes were significantly associated with tumor mutation burden and immuno-phenotypes such as antigen processing and presenting machinery and tumor immune infiltration score. Survival analysis identified 152 ERVs to be independently associated with DSS. Conclusions: ERV abnormal expression is common in colon cancer. The ERV-defined subtypes are associated with tumor immunity, and some ERVs are independently associated with patient outcomes. Full article

Mannan oligosaccharide (MOS), a prebiotic derived from yeast cell walls, has been shown to enhance growth performance and health status in various aquatic species. As an exogenous antigen adjuvant, MOS modulates T-cell-mediated immune responses, thereby improving immune function and suppressing excessive inflammatory reactions. This study aimed to evaluate the effects of dietary MOS supplementation on growth performance, serum biochemical parameters, muscle composition, digestive enzyme activity, antioxidant and immune status, and the mTOR signaling pathway in juvenile GIFT tilapia (Oreochromis niloticus). Juveniles (initial body weight: 16.17 ± 1.32 g) were randomly assigned to six treatment groups (three replicate tanks per group) and fed diets supplemented with MOS at 0, 0.2%, 0.4%, 0.6%, 0.8%, and 1% (equivalent to 0, 2, 4, 6, 8, and 10 g/kg of diet, respectively) for 60 days. Compared with the control group, fish fed MOS-supplemented diets exhibited significantly higher (p < 0.05) weight gain rates, specific growth rates, and protein efficiency ratios, along with a significantly lower (p < 0.05) feed conversion ratio. Serum albumin, high-density lipoprotein, and lysozyme levels were significantly increased (p < 0.05), whereas triglycerides, low-density lipoprotein, aspartate aminotransferase, and alanine aminotransferase levels were significantly decreased (p < 0.05). In the liver, head kidney, and spleen, the expression of pro-inflammatory genes (tumor necrosis factor α, interleukin 1β, interleukin 6, interleukin 8, and interferon γ) was significantly downregulated (p < 0.05), while the expression of antioxidant and protective genes (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, nuclear factor erythroid 2-related factor 2, lysozyme, alkaline phosphatase, interleukin-10, transforming growth factor β, and heat shock protein 70) as well as mTOR signaling pathway-related genes (mammalian target of rapamycin, akt protein kinase B, phosphatidylinositol 3 kinase, and ribosomal protein S6 kinase polypeptide 1) was significantly upregulated (p < 0.05). Overall, MOS positively affects tilapia’s growth, health, and immunity, with 0.60% identified as the optimal dietary level based on growth performance. 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

Enhancing protein–protein interactions is a key therapeutic strategy to ensure effective protein function in terms of pharmacokinetics and pharmacodynamics and can be accomplished with methods like directed evolution or rationale design. Previously, two papers suggested the possible enhancement of protein–protein binding affinity via destabilizing mutations. This paper reviews the results of the previous literature and adds new data to show the generality of the strategy that destabilizing the unbound protein without significantly changing the free energy of the complex can enhance protein–protein interactions for therapeutic benefit. The first example presented is that of a variant of human growth hormone (hGHv) containing 15 mutations that improve the binding to the hGH binding protein (hGHbp) by 400-fold while retaining full biological activity. The second example is that of the YTE mutations (M252Y/S354T/T256E) in the Fc region of a monoclonal antibody (mAb). The YTE mutations improve the binding of the mAb to FcRn at pH 6.0 10-fold, resulting in elongated serum half-life of the mAb. In both cases, (i) chemical titration or differential scanning calorimetry (DSC) showed the mutations destabilize the unbound mutant proteins, (ii) isothermal titration calorimetry (ITC) showed extremely favorable enthalpy (ΔH) and unfavorable entropy (ΔS) upon binding to their respective target molecule compared with the wildtype, and (iii) hydrogen/deuterium exchange–mass spectrometry (HDX-MS) revealed that these mutations increase the free energy of unbound mutant protein without significantly affecting the free energy of the bound state, resulting in an enhancement to the binding affinities. The third example presented is that of the JAWA mutations (T437R/K248E) also located in the Fc region of a mAb. The JAWA mutations facilitate antibody multimerization upon binding to cell surface antigens, allowing for enhanced agonism and effector functions. Both DSC and HDX-MS showed that the JAWA mutations destabilize the unbound Fc, although the complex was not characterized due to weak binding. Enhancement of protein–protein interactions through incorporation of mutations that increase the free energy of a protein’s unbound state represents an alternative route to decreasing the protein–protein complex free energy through optimization of the binding interface. Full article

Norovirus–bacterial interactions influence viral replication and immune responses, yet the molecular details that mediate binding of these viruses to commensal bacteria are unknown. Studies with other enteric viruses have revealed that LPS and other lipid/carbohydrate structures facilitate virus–bacterial interactions, and it has also been shown that human noroviruses (HuNoVs) can interact with histo-blood group antigen (HBGA)-like compounds on the surface of bacterial cells. Based on these findings, this study hypothesized that carbohydrate-based compounds were the ligands that facilitated binding of both human and murine noroviruses (MNV) to bacteria. Using glycan microarrays, competitive inhibition assays, and a panel of bacterial mutants, the project assessed the influence of specific glycans on viral attachment to bacteria. Protein-based interactions were also examined. The results supported previous work which demonstrated that HuNoVs strongly bind HBGA-like glycans, while MNV displayed distinct binding to other glycans including aminoglycosides and fucosylated structures. Ultimately, this work demonstrates that HuNoVs have more limited binding requirements for bacterial attachment compared to MNV, and the MNV binding to bacteria may involve both specific structures as well as electrostatic interactions. Given the importance of commensal bacteria during viral infection, defining the molecular mechanisms that mediate virus–bacteria interactions is critical for understanding infection dynamics and may be useful in the development of disease therapeutics and novel technologies for viral detection from food and environmental sources. Full article

Background: Fixation influences the quality of staining across species, especially in neuroscience, where accurate visualization of neuronal structures and protein localization is crucial for understanding brain function and pathology. This study compared two commonly used fixatives—9% glyoxal (G-fix) and Davidson’s solution (D-fix)—regarding their effects on autofluorescence, immunolabeling specificity, and histological quality in medaka brain tissue. Methods: Mixed-sex medaka from five strains were fixed with either G-fix or D-fix. Autofluorescence was assessed in posterior bodies and brain tissues, including sections stained with fluorescently conjugated secondary antibodies alone. Tissues were also injected with fluorescent dyes or immunolabeled for neuronal markers (PGP9.5, NeuN, and NCAM) using fluorescent secondary antibodies. Hematoxylin and eosin (H&E) staining and immunohistochemistry were used to evaluate tissue morphology and chromogenic antigen detection. Results: Both fixatives induced autofluorescence: D-fix enhanced blue signals, while G-fix increased green and red fluorescence. These autofluorescence levels were significantly weaker than those from fluorescent dyes or PGP9.5 immunolabeling. Posterior body tissue showed patterns similar to deparaffinized brain sections, supporting its use for pre-screening fixation. G-fix yielded more neuron-specific PGP9.5 staining, whereas D-fix showed broader signal distribution. NeuN and NCAM were not detected, likely due to antibody incompatibility. PGP9.5 was undetectable by immunohistochemistry, while D-fix provided superior H&E staining quality. Conclusions: Although both fixatives induced autofluorescence, their signals were weaker than those of conventional dyes and antibodies. Glyoxal improved specificity for neuronal immunofluorescence, while Davidson enhanced histological detail. These findings provide practical guidance for optimizing fixation strategies in medaka-based neuroscience and histopathological research. Full article

T lymphocytes in human skin play essential roles in immune surveillance and tissue homeostasis, with distinct populations residing in the epidermal and dermal compartments. To characterize the molecular basis of their compartmentalized functional specialization, we performed proteomic analysis of total T cell populations isolated from healthy human skin, combining flow cytometry and liquid chromatography–tandem mass spectrometry. We quantified 5985 proteins across epidermal and dermal T cell populations, identifying 2177 significantly differentially expressed proteins (FDR < 0.05), including 1008 with >2-fold changes. Compared with dermal T cells, epidermal T cells showed elevated intensity of tissueresidency marker CD69, co-stimulatory protein CD27, complement components (C3, C4a, and Factors B and D), and proteins involved in oxidative phosphorylation and cholesterol metabolism. Epidermal T cells also exhibited higher levels of antimicrobial S100 proteins, chemokine receptor CCR6, IL-18, and MHC class I molecules, while, in contrast, dermal T cells showed increased expression of CXCR4, IL-16, and MHC class II-related proteins. While these distinct proteomic signatures suggest compartment-specific adaptations in metabolism, immune surveillance, and antigen presentation, the results should be interpreted as exploratory, given methodological limitations. Nonetheless, this study provides a valuable molecular resource for understanding the specialization of T cells within different skin layers and offers a basis for future investigations into skin immune biology and its potential implications in disease. Full article

Objectives: The construction of subunit vaccines based on antigens that can induce strong cellular immunity is a widely accepted strategy to develop new tuberculosis vaccines. This study screens immunogens with potential for subunit vaccine development from seven candidate antigens and then verifies their vaccine efficacy. Design: C57BL/6 mice were immunized subcutaneously with purified PPE19, PPE50, FadD21, Rv1505c, Rv1506c, Rv2035, and Rv0976c proteins formulated with Freund’s adjuvant to evaluate both the antigen-specific Th1 cellular immune responses and IgG level. After the vaccination of mice with recombined pcDNA3.1 expressing Rv0976c, intravenous or aerosol infection with M. tb were further challenged to assess protective efficacy. Results: Purified PPE19, PPE50, FadD21, and Rv0976c proteins generated strong antigen-specific Th1 cellular immune responses in mice. Compared to Ag85A, Rv0976c also stimulated higher IgG antibody level in mice. In particular, Rv0976c stimulated high and specific IgG antibody levels in serum from TB patients. The vaccination of mice with DNA vaccines expressing Rv0976c, followed by intravenous challenge with Bacillus Calmette–Guerin (BCG) Pasteur or M. tb, resulted in significant levels of protection that are comparable to or better than that afforded by the two leading antigens, Ag85A and PPE18. Conclusions: These results indicated that Rv0976c was a better protective antigen. Future studies to combine Rv0976c with other antigens and evaluate its effectiveness as a booster of BCG or as a therapeutic vaccine are warranted. Full article

Background/Objectives: Human leukocyte antigen B27 (HLA-B27) is a genetic marker strongly associated with various inflammatory rheumatic diseases, particularly those within the spondyloarthritis spectrum. Its presence influences disease onset, clinical severity, and therapeutic strategies. However, comparative data between HLA-B*27-positive and -negative patients, especially in Eastern European populations, remain limited. The study aimed to investigate the clinical, paraclinical, and psychosocial differences between HLA-B*27-positive and -negative individuals diagnosed with rheumatic diseases, in order to better understand the implications of HLA-B27 status on disease expression and patient quality of life. Methods: A cross-sectional, observational study was conducted between June 2023 and December 2024 at the Emergency Clinical Hospital for Children “Sf Ioan” in Galati, Romania, in collaboration with “Dunarea de Jos” University. Fifty adult patients with various rheumatic conditions were enrolled and stratified into HLA-B*27-positive (n = 22) and -negative (n = 28) groups. Data collection included clinical evaluations, laboratory biomarkers (CRP = C-reactive protein; ESR = erythrocyte sedimentation rate), and a structured quality-of-life questionnaire. Statistical analysis was performed using SPSS v27. Results: HLA-B*27-positive patients were significantly younger (mean age 46.00 vs. 55.07 years, p = 0.018) and had higher CRP levels (>1 mg/dL in 53.33% vs. 0%, p = 0.001). Ankylosing spondylitis was more prevalent in this group (22.73% vs. 3.57%, p = 0.039). Magnetic resonance imaging (MRI) was more frequently used (68.18% vs. 39.29%, p = 0.042), indicating greater suspicion of axial involvement. HLA-B27-positive patients also reported higher perceived stress (mean score 2.41 vs. 1.21, p < 0.001). Conclusions: HLA-B*27 positivity is associated with earlier disease onset, increased systemic inflammation, greater axial involvement, and higher psychological stress. These findings emphasise the need for personalised, multidisciplinary care that integrates both medical and psychological support for HLA-B*27-positive patients. Full article

Background: Rice bran proteins and their hydrolysates exhibit anticancer activity. Our previous study demonstrated that Riceberry glutelin and its hydrolysates possessed potent in vitro antioxidant and antimutagenic properties. However, their cancer chemopreventive effects in animals remain unclear. Methods: This study investigated chemopreventive mechanisms in diethylnitrosamine (DEN)- and 1,2-dimethylhydrazine (DMH)-induced preneoplastic lesions, including glutathione S-transferase placental form (GST-P)-positive foci in the liver and aberrant crypt foci (ACF) in the colon of rats. Rats received GTL, GTLH, and total protein hydrolysate (TPH) at 500 mg/kg body weight, five days per week for ten weeks. Results: GTLH significantly reduced GST-P-positive foci in the liver and ACF in the colon, while GTL decreased GST-P-positive foci only in the liver. However, TPH did not affect preneoplastic lesions in both the liver and the colon. GTLH suppressed cell proliferation by reducing proliferating cell nuclear antigen (PCNA)-positive cells and promoted apoptosis, as indicated by an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells in both organs. GTL also decreased PCNA-positive cells in the liver and colon. Moreover, GTLH significantly upregulated BAX and CASP3 in the liver, while only BAX in the colon was observed. Conclusions: This study highlighted the cancer chemopreventive potential of Riceberry GTLH with its underlying mechanism to reduce the number of preneoplastic lesions in the liver and colon through cell proliferation inhibition and apoptosis induction. These findings suggested that this protein hydrolysate might be used as a functional food ingredient or dietary supplement for cancer prevention. Full article

Glycosylation plays a pivotal role in regulating the functions and immunogenicity of antigens. Targeting the receptor-binding domain (RBD) of the spike protein (S protein) of SARS-CoV-2, we examined the impact of different glycoforms on RBD antigen immunogenicity and the underlying mechanisms. IgG-specific antibody titers and pseudovirus neutralization were compared in mice immunized with RBD antigens bearing different glycoforms, which were prepared using glycoengineering-capable Pichia pastoris and mammalian cell expression systems with distinct glycosylation pathways. The glycosylation impacted the surface charges of the RBD antigen, and influenced its adsorption onto alum. This may further lead to variations in the antigen’s immunogenicity. The high-mannose variant of the RBD antigen (H-MAN/RBD) expressed in wild-type Pichia pastoris induced significantly higher IgG-specific antibody titers and pseudovirus neutralization activity compared with the complex RBD variant (Complex/RBD) expressed in mammalian cells (293F) or glycoengineering-capable Pichia pastoris. The rate of H-MAN/RBD adsorption onto aluminum hydroxide (alum) adjuvant was significantly higher than that of Complex/RBD. It was assumed that H-MAN/RBD might carry more negative charges because of its phosphomannose-modified surfaces, leading to a higher rate of adsorption onto the positively charged alum and enhancing the immune response. To assess the impact of phosphomannose modification on antigen immunogenicity, a yeast strain was engineered to prepare a low-mannose RBD antigen (L-MAN/RBD); additionally, a yeast strain was constructed to generate a low-phosphomannose-modified RBD antigen (L-MAN-P/RBD). In conclusion, phosphomannose modification substantially enhanced the immunogenicity of RBD by altering the surface charges of the RBD antigen and facilitating its adsorption onto alum. These findings offer novel insights and strategies for vaccine design and immunotherapeutic approaches. Full article

Background: Toxoplasma gondii (T. gondii) infection causes serious diseases in immunocompromised patients and causes congenital toxoplasmosis in infants. T. gondii microneme protein 8 (MIC8) and apical membrane antigen 1 (AMA1) are essential proteins involved in parasitic invasion. Methods: In this study, we generated virus-like particles (VLPs) and recombinant vaccinia virus (rVV) containing MIC8 or AMA1 proteins. Vaccine efficacy was evaluated in mice (BALB/c) upon challenge infection with T. gondii ME49. Results: Intramuscular immunization with heterologous vaccines (rVV + VLPs; rVV for prime and VLPs for boost) elicited T. gondii-specific IgG antibody responses in mice. Four weeks after the boost, all mice were orally challenged with T. gondii ME49, and protective immunity was assessed. The responses of antibody-secreting cells for IgG2a and IgG2b and those of memory B cells and CD4+ and CD8+ T cells were higher in the rVV + VLP group than in the VLP + VLP group. The rVV + VLP group exhibited a significant reduction in cyst count in the brain. Conclusions: These findings indicate that heterologous vaccination with vaccinia viruses and VLPs improves vaccine efficacy. Full article