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Search Results (1,065)

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13 pages, 2281 KiB  
Article
Amphipathic Alpha-Helical Peptides AH1 and AH3 Facilitate Immunogenicity of Enhanced Green Fluorescence Protein in Rainbow Trout (Oncorhynchus mykiss)
by Kuan Chieh Peng and Ten-Tsao Wong
J. Mar. Sci. Eng. 2025, 13(8), 1497; https://doi.org/10.3390/jmse13081497 - 4 Aug 2025
Abstract
Vaccination is the most effective method to counteract infectious diseases in farmed fish. It secures aquaculture production and safeguards the wild stock and aquatic ecosystem from catastrophic contagious diseases. In vaccine development, recombinant subunit vaccines are favorable candidates since they can be economically [...] Read more.
Vaccination is the most effective method to counteract infectious diseases in farmed fish. It secures aquaculture production and safeguards the wild stock and aquatic ecosystem from catastrophic contagious diseases. In vaccine development, recombinant subunit vaccines are favorable candidates since they can be economically produced in large quantities without growing many pathogens, as in inactivated or attenuated vaccine production. However, recombinant subunit vaccines are often weak or deficient in immunogenicity, resulting in inadequate defenses against infections. Technologies that can increase the immunogenicity of recombinant subunit vaccines are in desperate need. Enhanced green fluorescence protein (EGFP) has a low antigenicity and is susceptible to folding changes and losing fluorescence after fusing with other proteins. Using these valuable features of EGFP, we comprehend two amphipathic alpha-helical peptides, AH1 and AH3, derived from Hepatitis C virus and Influenza A virus, respectively, that can induce high immune responses of their fused EGFP in fish without affecting their folding. AH3-EGFP has the most elevated cell binding, significantly 62% and 36% higher than EGFP and AH1-EGFP, respectively. Immunizations with AH1-EGFP or AH3-EGFP significantly induced higher anti-EGFP antibody levels 300–500-fold higher than EGFP immunization after the boost injection in rainbow trout. Our results suggest that AH1 and AH3 effectively increase the immunogenicity of EGFP without influencing its structure. Further validation of their value in other recombinant proteins is necessary to demonstrate their broader utility in enhancing the immunogenicity of subunit vaccines. We also suggest that EGFP and its variants are promising candidates for initially screening proper immunogenicity-enhancing peptides or proteins to advance recombinant subunit vaccine development. Full article
(This article belongs to the Section Marine Aquaculture)
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26 pages, 4785 KiB  
Article
Osteogenic Differentiation of Mesenchymal Stem Cells Induced by Geometric Mechanotransductive 3D-Printed Poly-(L)-Lactic Acid Matrices
by Harrison P. Ryan, Bruce K. Milthorpe and Jerran Santos
Int. J. Mol. Sci. 2025, 26(15), 7494; https://doi.org/10.3390/ijms26157494 (registering DOI) - 2 Aug 2025
Viewed by 178
Abstract
Bone-related defects present a key challenge in orthopaedics. The current gold standard, autografts, poses significant limitations, such as donor site morbidity, limited supply, and poor morphological adaptability. This study investigates the potential of scaffold geometry to induce osteogenic differentiation of human adipose-derived stem [...] Read more.
Bone-related defects present a key challenge in orthopaedics. The current gold standard, autografts, poses significant limitations, such as donor site morbidity, limited supply, and poor morphological adaptability. This study investigates the potential of scaffold geometry to induce osteogenic differentiation of human adipose-derived stem cells (hADSCs) through mechanotransduction, without the use of chemical inducers. Four distinct poly-(L)-lactic acid (PLA) scaffold architectures—Traditional Cross (Tc), Triangle (T), Diamond (D), and Gyroid (G)—were fabricated using fused filament fabrication (FFF) 3D printing. hADSCs were cultured on these scaffolds, and their response was evaluated utilising an alkaline phosphatase (ALP) assay, immunofluorescence, and extensive proteomic analyses. The results showed the D scaffold to have the highest ALP activity, followed by Tc. Proteomics results showed that more than 1200 proteins were identified in each scaffold with unique proteins expressed in each scaffold, respectively Tc—204, T—194, D—244, and G—216. Bioinformatics analysis revealed structures with complex curvature to have an increased expression of proteins involved in mid- to late-stage osteogenesis signalling and differentiation pathways, while the Tc scaffold induced an increased expression of signalling and differentiation pathways pertaining to angiogenesis and early osteogenesis. Full article
(This article belongs to the Special Issue Novel Approaches for Tissue Repair and Tissue Regeneration)
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18 pages, 3111 KiB  
Article
Ectopic Recruitment of the CTCF N-Terminal Domain with Two Proximal Zinc-Finger Domains as a Tool for 3D Genome Engineering
by Eugenia A. Tiukacheva, Artem V. Luzhin, Natalia Kruglova, Anastasia S. Shtompel, Grigorii Antonov, Anna Tvorogova, Yegor Vassetzky, Sergey V. Ulianov and Sergey V. Razin
Int. J. Mol. Sci. 2025, 26(15), 7446; https://doi.org/10.3390/ijms26157446 - 1 Aug 2025
Viewed by 169
Abstract
Enhancer-promoter interactions occur in the chromatin loci delineated by the CCCTC-binding zinc-finger protein CTCF. CTCF binding is frequently perturbed in genetic disorders and cancer, allowing for misregulation of genes. Here, we developed a panel of chimeric proteins consisting of either full-length or truncated [...] Read more.
Enhancer-promoter interactions occur in the chromatin loci delineated by the CCCTC-binding zinc-finger protein CTCF. CTCF binding is frequently perturbed in genetic disorders and cancer, allowing for misregulation of genes. Here, we developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human HOXD locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region, anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect the epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF-binding site. Together, our data indicate that the ectopic recruitment of the CTCF N-terminal part could be an appropriate tool for 3D genome engineering. Full article
(This article belongs to the Section Molecular Biology)
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41 pages, 7499 KiB  
Article
Development of a Broad-Spectrum Pan-Mpox Vaccine via Immunoinformatic Approaches
by Japigorn Puagsopa, Panuwid Jumpalee, Sittichoke Dechanun, Sukanya Choengchalad, Pana Lohasupthawee, Thanawat Sutjaritvorakul and Bunyarit Meksiriporn
Int. J. Mol. Sci. 2025, 26(15), 7210; https://doi.org/10.3390/ijms26157210 - 25 Jul 2025
Viewed by 890
Abstract
Monkeypox virus (MPXV) has caused 148,892 confirmed cases and 341 deaths from 137 countries worldwide, as reported by the World Health Organization (WHO), highlighting the urgent need for effective vaccines to prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time [...] Read more.
Monkeypox virus (MPXV) has caused 148,892 confirmed cases and 341 deaths from 137 countries worldwide, as reported by the World Health Organization (WHO), highlighting the urgent need for effective vaccines to prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time consuming, and susceptible to reversion to virulence. Alternatively, a reverse vaccinology approach offers a rapid, efficient, and safer alternative for MPXV vaccine design. Here, MPXV proteins associated with viral infection were analyzed for immunogenic epitopes to design multi-epitope vaccines based on B-cell, CD4+, and CD8+ epitopes. Epitopes were selected based on allergenicity, antigenicity, and toxicity parameters. The prioritized epitopes were then combined via peptide linkers and N-terminally fused to various protein adjuvants, including PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and the cholera toxin B subunit (CTB). All vaccine constructs were computationally validated for physicochemical properties, antigenicity, allergenicity, safety, solubility, and structural stability. The three-dimensional structure of the selected construct was also predicted. Moreover, molecular docking and molecular dynamics (MD) simulations between the vaccine and the TLR-4 immune receptor demonstrated a strong and stable interaction. The vaccine construct was codon-optimized for high expression in the E. coli and was finally cloned in silico into the pET21a (+) vector. Collectively, these results could represent innovative tools for vaccine formulation against MPXV and be transformative for other infectious diseases. Full article
(This article belongs to the Section Molecular Informatics)
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12 pages, 2083 KiB  
Article
Modification of Silver-Loaded Biodegradable Polymer Nanoparticles with Bacterial Membrane Vesicles for Treating Intracellular Bacterial Infections
by Wei Xu, Sayo Maruyama and Takuro Niidome
Materials 2025, 18(15), 3470; https://doi.org/10.3390/ma18153470 - 24 Jul 2025
Viewed by 173
Abstract
Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen capable of surviving and replicating within macrophages, which causes foodborne diseases such as gastroenteritis. To develop a strategy against intracellular bacteria in macrophages, we designed silver-loaded biodegradable polymer nanoparticles functionalized [...] Read more.
Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen capable of surviving and replicating within macrophages, which causes foodborne diseases such as gastroenteritis. To develop a strategy against intracellular bacteria in macrophages, we designed silver-loaded biodegradable polymer nanoparticles functionalized with S. Typhimurium membrane vesicles (MVs). Silver nanoparticles (Ag NPs) were initially encapsulated within biodegradable poly(lactic-co-glycolic) nanoparticles (Ag-P NPs), which were then surface-modified with polyethyleneimine to form Ag-PP NPs. These were subsequently fused with S. Typhimurium MVs via a sonication method to generate Ag-PP@MV NPs. The resulting MV-coated nanoparticles displayed a similar protein profile to that of native MVs and exhibited antibacterial activity against intracellular S. Typhimurium. Notably, the enhanced cellular uptake of the MV-modified NPs contributed to their intracellular bactericidal efficacy. This study highlights MV modification as a promising strategy to improve NP delivery to macrophages for treating persistent intracellular infections. Full article
(This article belongs to the Section Biomaterials)
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21 pages, 3415 KiB  
Article
SARS-CoV-2 RBD Scaffolded by AP205 or TIP60 Nanoparticles and Delivered as mRNA Elicits Robust Neutralizing Antibody Responses
by Johnathan D. Guest, Yi Zhang, Daniel Flores, Emily Atkins, Kuishu Ren, Yingyun Cai, Kim Rosenthal, Zimeng Wang, Kihwan Kim, Charles Chen, Richard Roque, Bei Cheng, Marianna Yanez Arteta, Liping Zhou, Jason Laliberte and Joseph R. Francica
Vaccines 2025, 13(8), 778; https://doi.org/10.3390/vaccines13080778 - 22 Jul 2025
Viewed by 1164
Abstract
Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD [...] Read more.
Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article
(This article belongs to the Section COVID-19 Vaccines and Vaccination)
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15 pages, 2357 KiB  
Article
Development of a Novel, Highly Sensitive System for Evaluating Ebola Virus Particle Formation
by Wakako Furuyama, Miako Sakaguchi, Hanako Ariyoshi and Asuka Nanbo
Viruses 2025, 17(7), 1016; https://doi.org/10.3390/v17071016 - 19 Jul 2025
Viewed by 456
Abstract
Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and effective countermeasures remain limited. The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently [...] Read more.
Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and effective countermeasures remain limited. The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently target the viral particle formation process. In this study, we established a simple and highly sensitive screening system to evaluate VP40-mediated virus-like particle (VLP) formation under biosafety level −2 conditions. The system uses the HiBiT luminescence-based reporter fused to VP40, allowing for the detection of VP40 release. Our results demonstrate that the HiBiT sequence fused at the N-terminus [HiBiT-VP40 (N)] retains VP40′s ability to form VLPs, supporting its use as a functional reporter. Furthermore, we validated the system by assessing the role of Rab11-dependent trafficking in VP40-mediated budding and by evaluating the effect of nocodazole, a microtubule depolymerizer, on VLP release. This novel screening system provides a convenient and reliable platform for screening potential inhibitors targeting the late stages of EBOV infection, including viral particle formation and release. Additionally, its potential adaptability to other filoviruses suggests wide applicability in the discovery and development of additional novel therapeutic agents. Full article
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13 pages, 1764 KiB  
Article
Surface Display of Avian H5 and H9 Hemagglutinin Antigens on Non-Genetically Modified Lactobacillus Cells for Bivalent Oral AIV Vaccine Development
by Fuyi Liu, Jingbo Chang, Jingqi Huang, Yuping Liao, Xiaonan Deng, Tingting Guo, Jian Kong and Wentao Kong
Microorganisms 2025, 13(7), 1649; https://doi.org/10.3390/microorganisms13071649 - 11 Jul 2025
Viewed by 348
Abstract
A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus [...] Read more.
A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines. Full article
(This article belongs to the Section Food Microbiology)
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17 pages, 1548 KiB  
Article
CD19-ReTARGTPR: A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies
by Anne Paulien van Wijngaarden, Isabel Britsch, Matthias Peipp, Douwe Freerk Samplonius and Wijnand Helfrich
Cancers 2025, 17(14), 2300; https://doi.org/10.3390/cancers17142300 - 10 Jul 2025
Viewed by 404
Abstract
Background/Objectives: The physiological activation of cytotoxic CD8pos T cells (CTLs) relies on the engagement of the TCR/CD3 complex with cognate peptide-HLA class I (pHLA-I) on target cells, triggering cell lysis with appropriate cytokine release and minimized off-target toxicity. In contrast, current [...] Read more.
Background/Objectives: The physiological activation of cytotoxic CD8pos T cells (CTLs) relies on the engagement of the TCR/CD3 complex with cognate peptide-HLA class I (pHLA-I) on target cells, triggering cell lysis with appropriate cytokine release and minimized off-target toxicity. In contrast, current immunotherapies for CD19-expressing hematological malignancies, such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs), bypass TCR/pHLA interactions, resulting in CTL hyperactivation and excessive cytokine release, which frequently cause severe immune-related adverse events (irAEs). Thus, there is a pressing need for T cell-based therapies that preserve physiological activation while maintaining antitumor efficacy. Methods: To address this, we developed CD19-ReTARGTPR, a novel fusion protein consisting of the immunodominant cytomegalovirus (CMV) pp65-derived peptide TPRVTGGAM (TPR) covalently presented by a soluble HLA-B*07:02/β2-microglobulin complex fused to a high-affinity CD19-targeting Fab antibody fragment. The treatment of CD19-expressing cancer cells with CD19-ReTARGTPR makes them recognizable for pre-existing anti-CMVpp65 CTLs via physiological TCR-pHLA engagement. Results: Our preclinical data demonstrate that CD19-ReTARGTPR efficiently redirects anti-CMV CTLs to eliminate CD19-expressing cancer cells, including both established cell lines and primary chronic lymphocytic leukemia (CLL) cells. Unlike CD19-directed CAR T cells or the CD19/CD3 BiTE blinatumomab, CD19-ReTARGTPR mediated robust cytotoxic activity without triggering supraphysiological cytokine release. Importantly, this approach retained efficacy even against cancer cells with low CD19 expression. Conclusions: In summary, we provide a robust proof-of-concept study and show that CD19-ReTARGTPR offers a promising alternative strategy for T cell redirection, enabling the selective and effective killing of CD19-expressing malignancies while minimizing cytokine-driven toxicities through physiological CTL activation pathways. Full article
(This article belongs to the Special Issue New Insights of Hematology in Cancer)
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17 pages, 3221 KiB  
Article
An mRNA Vaccine Targeting the C-Terminal Region of P1 Protein Induces an Immune Response and Protects Against Mycoplasma pneumoniae
by Fenglian Zhang, Chengwei Li, Yanan Wu, Hongyun Chuan, Shaohui Song, Yun Xie, Qi Zhu, Qianqian Chen, Fei Tong, Runfang Zhang, Guangbo Yuan, Xiaoyan Wu, Jian Zhou and Guoyang Liao
Int. J. Mol. Sci. 2025, 26(13), 6536; https://doi.org/10.3390/ijms26136536 - 7 Jul 2025
Viewed by 517
Abstract
Mycoplasma pneumoniae, a cell wall-deficient pathogen, primarily affects children and adolescents, causing Mycoplasma pneumoniae pneumonia (MPP). Following the relaxation of non-pharmaceutical interventions (NPIs) post COVID-19, there has been a global increase in MPP cases and macrolide-resistant strains. Vaccination against M. pneumoniae is [...] Read more.
Mycoplasma pneumoniae, a cell wall-deficient pathogen, primarily affects children and adolescents, causing Mycoplasma pneumoniae pneumonia (MPP). Following the relaxation of non-pharmaceutical interventions (NPIs) post COVID-19, there has been a global increase in MPP cases and macrolide-resistant strains. Vaccination against M. pneumoniae is being explored as a promising approach to reduce infections, limit antibiotic misuse, and prevent the emergence of drug-resistant variants. We developed an mRNA vaccine, mRNA-SP+P1, incorporating a eukaryotic signal peptide (tissue-type plasminogen activator signal peptide) fused to the C-terminal region of the P1 protein. Targeting amino acids 1288 to 1518 of the P1 protein, the vaccine was administered intramuscularly to BALB/c mice in a three-dose regimen. To evaluate immunogenicity, we quantified anti-P1 IgG antibody titers using enzyme-linked immunosorbent assays (ELISAs) and assessed cellular immune responses by analyzing effector memory T cell populations using flow cytometry. We also tested the functional activity of vaccine-induced sera for their ability to inhibit adhesion of the ATCC M129 strain to KMB17 cells. The vaccine’s protective efficacy was assessed against the ATCC M129 strain and its cross-protection against the ST3-resistant strain. Transcriptomic analysis was conducted to investigate gene expression changes in peripheral blood, aiming to uncover mechanisms of immune modulation. The mRNA-SP+P1 vaccine induces P1 protein-specific IgG antibodies and an effector memory T-cell response in BALB/c mice. Adhesion inhibition assays demonstrated that serum from vaccinated mice attenuatesthe adhesion ability of ATCC M129 to KMB17 cells. Furthermore, three doses of the vaccine confer significant and long-lasting, though partial, protection against the ATCC M129 strain and partial cross-protection against the ST3 drug-resistant strain. Transcriptome analysis revealed significant gene expression changes in peripheral blood, confirming the vaccine’s capacity to elicit an immune response from the molecular level. Our results indicate that the mRNA-SP+P1 vaccine appears to be an effective vaccine candidate against the prevalence of Mycoplasma pneumoniae. Full article
(This article belongs to the Section Molecular Immunology)
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29 pages, 5028 KiB  
Article
Moloney Murine Leukemia Virus-like Nanoparticles Pseudo-Typed with SARS-CoV-2 RBD for Vaccination Against COVID-19
by Bernhard Kratzer, Pia Gattinger, Peter A. Tauber, Mirjam Schaar, Al Nasar Ahmed Sehgal, Armin Kraus, Doris Trapin, Rudolf Valenta and Winfried F. Pickl
Int. J. Mol. Sci. 2025, 26(13), 6462; https://doi.org/10.3390/ijms26136462 - 4 Jul 2025
Viewed by 592
Abstract
Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of [...] Read more.
Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG2a and IgG1 after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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17 pages, 3309 KiB  
Article
Mitochondrial Fragmentation and Long Noncoding RNA MALAT1 in Diabetic Retinopathy
by Renu A. Kowluru and Jay Kumar
Int. J. Mol. Sci. 2025, 26(13), 6429; https://doi.org/10.3390/ijms26136429 - 3 Jul 2025
Viewed by 478
Abstract
Mitochondria are dynamic in nature and depending on the energy demand they fuse and divide. This fusion-fission process is impaired in diabetic retinopathy and the promoter DNA of Mfn2, a fusion gene, is hypermethylated and its expression is downregulated. Long noncoding RNAs [...] Read more.
Mitochondria are dynamic in nature and depending on the energy demand they fuse and divide. This fusion-fission process is impaired in diabetic retinopathy and the promoter DNA of Mfn2, a fusion gene, is hypermethylated and its expression is downregulated. Long noncoding RNAs (RNAs with >200 nucleotides that do not encode proteins) can regulate gene expression by interacting with DNA, RNA, and proteins. Several LncRNAs are aberrantly expressed in diabetes, and among them, MALAT1 is upregulated in the retina, altering the expression of the genes associated with inflammation. Our aim was to investigate MALAT1’s role in mitochondrial dynamics in diabetic retinopathy. Using MALAT1-siRNA-transfected human retinal endothelial cells (HRECs) and human retinal Muller cells (RMCs) incubated in 20 mM D-glucose, Mfn2 expression and activity and its promoter DNA methylation were quantified. Mitochondrial integrity was evaluated by analyzing their fragmentation, ultrastructure, membrane potential, and oxygen consumption rate. Compared to normal glucose, high glucose upregulated MALAT1 expression and downregulated Mfn2 expression and activity in both HRECs and RMCs. MALAT1-siRNA ameliorated the glucose-induced increase in Mfn2 promoter DNA hypermethylation and its activity. MALAT1-siRNA also protected against mitochondrial fragmentation, structural damage, and reductions in the oxygen consumption rate. In conclusion, the upregulation of MALAT1 in diabetes facilitates Mfn2 promoter DNA hypermethylation in retinal vascular and nonvascular cells, leading to its suppression and the accumulation of the fragmented/damaged mitochondria. Thus, the regulation of MALAT1 has the potential to protect mitochondria and provide a possible new target to inhibit/prevent the blinding disease in diabetic patients. Full article
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15 pages, 2342 KiB  
Article
CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy
by Giada Becchi, Michael Whitehead, Joshua P. Harvey, Paul E. Sladen, Mohammed Dushti, J. Paul Chapple, Patrick Yu-Wai-Man and Michael E. Cheetham
Int. J. Mol. Sci. 2025, 26(13), 6364; https://doi.org/10.3390/ijms26136364 - 2 Jul 2025
Viewed by 393
Abstract
Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a [...] Read more.
Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9–VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9–VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA. Full article
(This article belongs to the Special Issue Advanced Research in Mitochondrial Genetics)
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15 pages, 2136 KiB  
Article
POSA-GO: Fusion of Hierarchical Gene Ontology and Protein Language Models for Protein Function Prediction
by Yubao Liu, Benrui Wang, Bocheng Yan, Haiyue Jiang and Yinfei Dai
Int. J. Mol. Sci. 2025, 26(13), 6362; https://doi.org/10.3390/ijms26136362 - 1 Jul 2025
Viewed by 324
Abstract
Protein function prediction plays a crucial role in uncovering the molecular mechanisms underlying life processes in the post-genomic era. However, with the widespread adoption of high-throughput sequencing technologies, the pace of protein function annotation significantly lags behind that of sequence discovery, highlighting the [...] Read more.
Protein function prediction plays a crucial role in uncovering the molecular mechanisms underlying life processes in the post-genomic era. However, with the widespread adoption of high-throughput sequencing technologies, the pace of protein function annotation significantly lags behind that of sequence discovery, highlighting the urgent need for more efficient and reliable predictive methods. To address the problem of existing methods ignoring the hierarchical structure of gene ontology terms and making it challenging to dynamically associate protein features with functional contexts, we propose a novel protein function prediction framework, termed Partial Order-Based Self-Attention for Gene Ontology (POSA-GO). This cross-modal collaborative modelling approach fuses GO terms with protein sequences. The model leverages the pre-trained language model ESM-2 to extract deep semantic features from protein sequences. Meanwhile, it transforms the partial order relationships among Gene Ontology (GO) terms into topological embeddings to capture their biological hierarchical dependencies. Furthermore, a multi-head self-attention mechanism is employed to dynamically model the association weights between proteins and GO terms, thereby enabling context-aware functional annotation. Comparative experiments on the CAFA3 and SwissProt datasets demonstrate that POSA-GO outperforms existing state-of-the-art methods in terms of Fmax and AUPR metrics, offering a promising solution for protein functional studies. Full article
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18 pages, 2850 KiB  
Article
An mRNA Vaccine Expressing Blood-Stage Malaria Antigens Induces Complete Protection Against Lethal Plasmodium yoelii
by Amy C. Ott, Patrick J. Loll and James M. Burns
Vaccines 2025, 13(7), 702; https://doi.org/10.3390/vaccines13070702 - 28 Jun 2025
Viewed by 784
Abstract
Background and Objectives: To evaluate the mRNA vaccine platform for blood-stage Plasmodium parasites, we completed a proof-of-concept study using the P. yoelii mouse model of malaria and two mRNA-based vaccines. Both encoded PyMSP119 fused to PyMSP8 (PyMSP1/8). One [...] Read more.
Background and Objectives: To evaluate the mRNA vaccine platform for blood-stage Plasmodium parasites, we completed a proof-of-concept study using the P. yoelii mouse model of malaria and two mRNA-based vaccines. Both encoded PyMSP119 fused to PyMSP8 (PyMSP1/8). One was designed for secretion of the encoded protein (PyMSP1/8-sec); the other encoded membrane-bound antigen (PyMSP1/8-mem). Methods: Secretion of PyMSP1/8-sec and membrane localization of PyMSP1/8-mem were verified in mRNA-transfected cells. As recombinant PyMSP1/8 (rPyMSP1/8) is known to protect mice against lethal P. yoelii 17XL infection, we first compared immunogenicity and efficacy of the PyMSP1/8-sec mRNA vaccine versus the recombinant formulation in outbred mice. Animals were immunized three times followed by challenge with a lethal dose of P. yoelii 17XL-parasitized RBCs (pRBCs). Similar immunization and challenge experiments were conducted to compare PyMSP1/8-sec versus PyMSP1/8-mem mRNA vaccines. Results: Immunogenicity of the PyMSP1/8-sec mRNA vaccine was superior to the recombinant formulation, inducing higher antibody titers against both vaccine components. Following challenge with P. yoelii 17XL pRBCs, all PyMSP1/8-sec-immunized animals survived, with 50% of these showing no detectible pRBCs in circulation (<0.01%). In addition, mean peak parasitemia in PyMSP1/8-sec mRNA-immunized mice was significantly lower than that in the rPyMSP1/8 vaccine group. Both PyMSP1/8-sec and PyMSP1/8-mem were protective against P. yoelii 17XL challenge, with PyMSP1/8-mem immunization providing a significantly higher level of protection than PyMSP1/8-sec immunization considering the number of animals with no detectable pRBCs in circulation and the mean peak parasitemia in animals with detectable parasitemia. Conclusions: mRNA vaccines were highly immunogenic and potently protective against blood-stage malaria, outperforming a similar recombinant-based vaccine. The membrane-bound antigen was more effective at inducing protective antibody responses, highlighting the need to consider antigen localization for mRNA vaccine design. Full article
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