Search Results (6,125)

Treatment for large critical-sized bone defects and impaired fracture healing remain challenging. Clinically used protein-based osteoinductive factors, such as recombinant bone morphogenetic proteins (BMPs), can be effective; however, they are costly and limited by stability, dose-delivery issues, and safety concerns. Preclinical small molecules offer an alternative because they are chemically stable, scalable to manufacture, and readily integrated for systemic administration or localized release from scaffolds, hydrogels, cements, and implant coatings. With an emphasis on delivery formats and mechanistic themes, this review examines small molecules that have been shown to improve bone regeneration in preclinical models, contrasting those of biological origin with synthetic and repurposed compounds. Across studies, these selected compounds promote osteoblast commitment, differentiation, and matrix mineralization via BMP/Smad signaling and Wnt/beta-catenin (β-catenin) activation, often through glycogen synthase kinase-3 beta (GSK-3β) inhibition or relief of pathway antagonism or Hedgehog (Hh) pathway stimulation. Beyond osteoinduction, several candidates address issues that commonly limit repair, including angiogenesis, oxidative stress, inflammatory tone, osteoimmune regulation, and suppression of osteoclast-mediated resorption. Direct head-to-head comparisons are rare across both classes and reporting heterogeneity complicates interpretation. Key translational gaps include limited cytotoxicity and immunologic profiling, dose and release optimization, durability of benefit, and insufficient evaluation of rational combinations. More rigorous in vivo studies, including larger animal models and standardized outcome metrics, are needed to prioritize promising candidates and guide clinical development. Full article

Recombinogenic DNA damage can initiate chromosomal rearrangements that can alter gene expression or accelerate cancer progression in higher eukaryotes. Thus, there is a critical need to identify genes that suppress chromosomal rearrangements and environmental exposures that promote genetic instability. Cell cycle checkpoints modulate the cell cycle so that DNA repair occurs before the replication or segregation of damaged chromosomes. Saccharomyces cerevisiae (budding yeast) RAD9 was the first cell cycle checkpoint gene identified, which initiated intensive research studies into the mechanisms of checkpoint activation and the phenotypes of checkpoint mutants. The budding yeast Rad9 protein serves as both an adaptor and scaffold that facilitates downstream effector activation to orchestrate a DNA damage response at multiple stages of the cell cycle, which facilitates double-strand break (DSB) repair by sister chromatid recombination. However, the role of RAD9 in homologous recombination and in suppressing gross chromosomal rearrangements (GCRs) is not completely understood. In this review we discuss how RAD9 can promote genome instability resulting from aberrant DNA replication intermediates, while suppressing DSB-associated rearrangements. We also discuss possible mechanisms accounting for the synergistic increase in genomic instability in double mutants defective in both RAD9 and recombinational repair. We emphasize that while there is an overlap between checkpoint and recombinational repair pathways, RAD9 and checkpoint pathways can function independently to suppress chromosomal instability. These studies thus elucidate checkpoint mechanisms that control homologous recombination between repeated sequences. Full article

Fowl adenovirus serotype 4 (FAdV-4) has been identified as the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS), resulting in significant economic losses in major poultry-producing countries since 2015. Timely and accurate diagnosis of FAdV-4 infection is essential for the effective prevention and control of HHS. In this study, the two nonstructural genes of FAdV-4, 100K and 22K, were inserted into the expression vector pET-32a (+) respectively. The expressed recombinant proteins were used as coating antigens to develop two indirect ELISA methods, designated as 100K-ELISA and 22K-ELISA. Both ELISAs demonstrated high specificity, showing no cross-reactivity with serum samples positive for other avian diseases. Both ELISAs yielded positive results when applied to 50 serum samples from SPF chickens experimentally infected with FAdV-4 and negative results when applied to 50 serum samples from SPF chickens immunized with an inactivated FAdV-4 vaccine. Similarly, the field sample testing results demonstrated a significant ability to distinguish between vaccinated and infected samples. The 100K-ELISA and 22K-ELISA, which are based on nonstructural proteins, may be effective tools for differentiating between FAdV-4 infection and vaccination, offering a promising approach for differentiating infected from vaccinated animals (DIVA) strategies in poultry. Full article

The potential of water-soluble membrane proteins (wsMPs) has not been fully realized. In this article, we exploit the nearly identical functionality of wsMPs with their membrane-bound counterparts and show that we can create water-soluble membrane proteins that incorporate into the plasma membranes of cells and alter their fate. As a proof of concept, we demonstrate the functional properties of water-soluble engineered pore-forming proteins, K⁺ ionic channels (MthK), and constitutively active GPCRs—among them frizzled receptors—both in vitro and in vivo. We call this method in vivo deployment of recombinant viable MPs, iDRIVE. Furthermore, we demonstrate that our strategy mediates the unidirectional insertion of MPs into the plasma membrane, and through constitutively active receptors, we present evidence for similar signaling pathway activation between small molecules and our water-soluble proteins using model phenotypes and molecular signaling assays. We present three examples where wsMPs are functional in dictating cellular fate, both in vitro and in vivo. Lastly, we show the induction of similar differential methylation via the activation of the Wnt signaling pathway using the conventional small molecule agonist, CHIR99021, or our wsFrizzled receptors (iDRIVE-FZD) in human embryonic kidney (HEK 293) embryoid spheroids (ESs). Additionally, we show that Wnt activation via wsFrizzled receptors results in even more biologically relevant epigenetic changes than via the small molecule CHIR99021. Future work will employ iDRIVE to differentiate stem cells in the production of research and clinically relevant organoids. Full article

The emergence of variant strains of Avian orthoreovirus (ARV) has caused substantial economic losses in the poultry industry worldwide, but the molecular features of goose-origin strains and viral transmission among different avian species remain poorly understood. Here, we describe a goose-origin avian orthoreovirus strain, SD0407, associated with growth retardation and joint swelling. Complete genome analysis identified ten double-stranded RNA segments. Sequence comparison indicated that SD0407 is closely related to previously reported duck-origin reovirus strains. Phylogenetic and recombination analyses showed that most segments clustered with duck-origin strains, indicating close genetic relatedness among waterfowl-origin orthoreoviruses. Sequence and structural analysis of the σC attachment protein revealed ten unique amino acid substitutions, including D250 within the DE-loop region involved in receptor-binding. Molecular docking suggested that σC interacts with the conserved AnxA2-S100A10 heterotetrameric receptor complex, providing a possible structural basis for receptor compatibility across avian species. Although SD0407 replicated efficiently in goose embryo fibroblasts, it did not induce expression of type I, II or III interferons. Transcriptome profiling revealed weak activation of innate immune signaling and downregulation of metabolic and cytoskeletal genes, consistent with effective suppression of antiviral responses. These findings demonstrate that SD0407 combines structural variability with immune evasion to enhance host adaptability and underscore the importance of sustained ARV surveillance in waterfowl populations. Full article

Background: Mycoplasma pneumoniae (MP) is a major cause of respiratory tract infections in children and adolescents. Currently, there is no licensed vaccine, underscoring the urgent need for the development of safe and effective vaccines. Objective: The aim of this study is to develop a recombinant subunit vaccine candidate incorporating three antigens: the P1 protein, the P40/90 complex, and a detoxified mutant of community-acquired respiratory distress syndrome toxin. The protective efficacy of this vaccine candidate was also evaluated. Methods: Target genes were codon-optimized for expression in E. coli, and the recombinant proteins were successfully expressed and purified. The low-toxicity CARDS toxin mutant was screened based on TNF-α secretion levels in stimulated RAW264.7 cells. A three-component vaccine composed of P1, P40/90, and the mutant CARDS toxin was formulated and adjuvanted with either Al(OH)₃ alone or in combination with CpG. Mice were immunized, and immunogenicity was assessed by measuring antigen-specific IgG antibody titers. Protective efficacy was evaluated following challenge by analyzing lung histopathology, bacterial load, and inflammatory cytokine levels. Results: Seven high-purity recombinant proteins were successfully produced, including P1, the P40/90 complex, wild-type CARDS toxin, and four CARDS toxin mutants (E132A, E132Q, H36A, R10A). The E132A mutant was selected due to its significantly reduced toxicity while retaining immunogenicity. The three-component vaccine effectively elicited antibody responses against each of the included antigens. After three immunizations, IgG antibody titers in all groups reached approximately 10⁴. Immunized mice showed markedly reduced pulmonary pathology scores (control group: 2 or 2.67; immunized groups: 1.67, 1.33, and 0) and significantly decreased bacterial loads in lung tissue (control: 30.11 ± 10.40 cp/μL; immunized groups: 20.72 ± 4.37 cp/μL and 8.51 ± 8.32 cp/μL). Furthermore, the group receiving the alum + CpG adjuvant exhibited approximately a 10-fold higher antibody response compared with the alum-only group, indicating enhanced protective efficacy. Conclusions: The three-component candidate vaccine, MPtriV, adjuvanted with Al(OH)₃ + CpG, demonstrates promising immunogenicity, safety, and protective efficacy against Mycoplasma pneumoniae infection, providing a viable strategy and experimental foundation for the development of MP subunit vaccines. Full article

Factor XII (FXII) is a central mediator at the intersection of coagulation, fibrinolysis, inflammation, and immunity. It is activated upon contact with negatively charged surfaces, triggering the intrinsic coagulation pathway and driving thrombus formation and stabilization. Beyond clotting, FXII contributes to activation of the kallikrein–kinin system, generation of bradykinin, and modulation of inflammatory and immune responses. Congenital FXII deficiency does not increase bleeding risk, highlighting its unique role and making FXII inhibition an attractive strategy for anticoagulation and immune modulation with a potentially superior safety profile. Preclinical studies provide compelling evidence for this concept. In models of ischemic stroke and traumatic brain injury, FXII blockade significantly reduced infarct volume, improved neurological outcomes, and attenuated neuroinflammation without increasing hemorrhage. Similarly, in extracorporeal circulation and vascular stent implantation, FXII inhibition prevented thrombus formation and reduced fibrin deposition, achieving effects comparable to heparin but with markedly lower bleeding risk. Several classes of FXII inhibitors are currently in development, including antisense oligonucleotides, peptides, recombinant proteins, and monoclonal antibodies. Among them, Ixodes ricinus contact phase inhibitor (Ir-CPI) and recombinant human albumin-fused Infestin-4 (rHA-Infestin-4) have demonstrated strong antithrombotic efficacy in animal models. Most notably, garadacimab, a monoclonal anti-FXIIa antibody, has completed phase 3 trials and received regulatory approval for hereditary angioedema (HAE) prophylaxis, where it markedly reduces attack frequency with a favorable safety profile. This review summarizes current knowledge on FXII biology and evaluates its translational potential as a novel target for anticoagulant and anti-inflammatory therapies. Full article

Background/Objectives: Peroxiredoxins (Prxs) are key antioxidant enzymes involved in cellular redox homeostasis. Prx6 is a multifunctional member of the Prx family that has been reported in other organisms to possess glutathione peroxidase and phospholipase A₂ (PLA₂)-related activities. However, the structural and immunological roles of 1-Cys Prx6 in crustaceans remain poorly understood. This study aimed to identify and characterize a Prx6 gene from Penaeus vannamei (PvPrx6) and to evaluate its potential involvement in antioxidant defense. Methods: PvPrx6 cDNA was identified and analyzed using bioinformatics and AlphaFold2 modeling. Tissue distribution and transcriptional responses to lipopolysaccharide (LPS), poly(I:C), and peptidoglycan (PGN) were examined by RT-qPCR. Recombinant PvPrx6 (rPvPrx6) was expressed in Escherichia coli, and its antioxidant activity was evaluated in vitro using a metal-catalyzed oxidation (MCO) assay. Results: PvPrx6 encodes a 219-amino-acid protein containing conserved AhpC/TSA and 1-Cys Prx domains. Sequence comparison and 3D modeling revealed conserved peroxidase (Thr⁴¹, Cys⁴⁴, Arg¹²⁷) and residues (His²³, Lys²⁹, Asp¹³⁵) corresponding to the reported PLA₂-associated motif. Structural analysis suggested that Lys²⁹ occupies a position corresponding to the Ser³² residue of human Prx6, although this did not imply functional equivalence. PvPrx6 transcripts were highly expressed in the lymphoid organ and hepatopancreas and were significantly induced at 12 h following immune challenge. rPvPrx6 exhibited dose-dependent protection against hydroxyl radical-mediated DNA damage under the experimental conditions. Conclusions: Collectively, these findings suggest that PvPrx6 retains conserved structural characteristics of Prx6 proteins and may contribute to antioxidant defense in P. vannamei. However, further studies are required to validate its enzymatic activity and in vivo functional roles. Full article

Background: Rabies is a highly fatal zoonotic disease that causes approximately 59,000 human deaths worldwide each year. Current inactivated rabies vaccines require multiple doses and are associated with high costs. The full-length rabies virus glycoprotein (RVG), a membrane protein, exhibits substantial instability in its trimeric structure during recombinant expression. This instability makes it difficult to obtain high-purity, correctly folded antigens. Objectives: This study focuses on the preparation of a full-length recombinant RVG subunit vaccine candidate expressed in a glycoengineered Pichia pastoris system with mammalian-like glycosylation. Methods: The full-length RVG gene (including the transmembrane domain and cytoplasmic tail) from the Challenge Virus Standard-11 (CVS-11) strain was codon-optimized and inserted into the pPICZαA vector to construct the recombinant expression plasmid pPICZαA-RVG. The plasmid was transformed into glycoengineered Pichia pastoris X33-7 (low-mannose type) by electroporation for inducible expression. The target protein was purified by nickel affinity chromatography, anion-exchange chromatography, and Superdex-200 size-exclusion chromatography. The structural characteristics of the purified protein were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The purified antigen was formulated with the adjuvants AS03 or MF59. BALB/c mice (n = 5 per group) were immunized intramuscularly following a four-dose schedule (days 0, 7, 14, and 28). Antigen-specific IgG antibody titers were measured by ELISA, and neutralizing antibody titers were determined using the rapid fluorescent focus inhibition test (RFFIT). Results: Glycoengineered Pichia pastoris yeast strains expressing wild-type RVG (RVG-WT) or a mutant variant (RVG-M6: R84S, R199S, H270P, R279S, K300S, and R463S) were successfully constructed. The purified RVG antigen formed nanoparticles with an average particle size of approximately 75 nm. Immunized mice generated robust RVG-specific IgG responses, with titers reaching approximately 6.31 × 10⁵ for RVG-WT after the fourth immunization, compared to 3.16 × 10³ for RVG-M6 and 5.62 × 10³ for the RVG-WT-PEG control. Two weeks after the fourth immunization, RVG-WT formulated with AS03 or MF59 induced significant neutralizing antibody responses compared with the control group (p < 0.0001 and p < 0.01, respectively). The neutralizing antibody titers reached 1:79.43 in the AS03 group and 1:33.11 in the MF59 group, whereas the WT-PEG + AS03 control group showed a low titer of 1:3.72. In contrast, RVG-M6 formulated with MF59 failed to induce detectable neutralizing antibodies (1:3.02). Furthermore, RVG-WT + AS03 induced significantly higher neutralizing antibody responses than the WT-PEG + AS03 control group (p < 0.0001), and a significant difference was also observed between the RVG-WT + MF59 and RVG-M6 + MF59 groups (p < 0.01). Conclusions: The glycoengineered Pichia pastoris expression system successfully produced uniform full-length rabies virus glycoprotein nanoparticles with high purity. When formulated with the AS03 adjuvant, RVG-WT induced high-titer neutralizing antibodies in mice, suggesting a promising strategy for the development of recombinant subunit vaccines against rabies. However, this study is limited by the absence of challenge studies and validation in target animal species, which will be further investigated in future work. Full article

The moderate pathogenicity coupled with high host susceptibility of Eimeria maxima has precipitated substantial economic losses in the poultry industry. Addressing challenges such as emerging drug resistance underscores the imperative for innovative vaccine strategies. This study developed a novel DNA vaccine to solve this challenge by fusing E. maxima elongation factor-1α (EmEF1α) with chicken chemokine XCL1 (ChXCL1) in the pVAX1 vector. The recombinant plasmid, designated pVAX1-ChXCL1-EmEF1α, was successfully constructed and confirmed to express the ChXCL1-EmEF1α fusion protein in vitro. Immunization of chickens with this DNA vaccine elicited a robust and balanced immune response, characterized by significantly increased proportions of CD4⁺ (11.76%) and CD8⁺ (5.58%) T lymphocytes, elevated levels of Th1-associated cytokines (IFN-γ and IL-12), and strong antigen-specific IgG and IgA antibody responses. Following experimental challenge with E. maxima, vaccinated birds exhibited substantial protection: a 66.4% reduction in oocyst shedding, a 71.7% improvement in relative weight gain, marked attenuation of intestinal lesions, and an anticoccidial index (ACI) of 170. These findings demonstrate that the ChXCL1-EmEF1α DNA vaccine effectively enhances both cellular and humoral immunity. Collectively, this study validates ChXCL1 as a potent molecular adjuvant and establishes the “antigen–adjuvant” fusion DNA platform as a promising strategy for developing next-generation vaccines against avian coccidiosis. Full article

The coordination between transcription and mRNA processing is essential for eukaryotic gene regulation, yet the structural basis of this coupling remains poorly understood in Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. In this study, we characterized the interaction between the transcriptional coactivator EhPC4 and the polyadenylation factor EhCFIm25 through an integrated in vitro and in silico approach. Far-Western assays confirmed direct physical interaction between both recombinant proteins. To elucidate the molecular mechanism, we performed 500 ns Molecular Dynamics simulations of full-length EhPC4, identifying high flexibility in its N-terminal region. Protein–protein docking analysis revealed a stable EhPC4-EhCFIm25 complex (Cluster C4) with favorable binding energies (∆G = −11.4 kcal/mol). Notably, heatmap analysis of the interaction interface identified a conserved “hotspot” at the C-terminal end of EhCFIm25 (residues 249–255) that mediates the binding with PC4 without occluding DNA-binding domain (K127 in EhPC4) or RNA-recognition motifs in EhCFIm25. Our findings suggest that EhCFIm25 serves as a molecular scaffold that physically couples transcription and polyadenylation, providing a structural framework for the efficient regulation of virulence-related genes in this parasite. Full article

Background: Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne viral disease with a high fatality rate, and no licensed vaccines are currently available. The nucleoprotein (NP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) plays a critical role in viral replication and immune recognition, making it a promising target for vaccine development. This study aimed to design and evaluate a multiepitope recombinant DNA vaccine targeting the NP of CCHFV. Methods: Cytotoxic T lymphocyte (CTL) epitopes from the NP were predicted via immunoinformatics approaches and systematically assessed for antigenicity, allergenicity, toxicity, hydrophobicity, and global population coverage. The selected epitopes were incorporated into four DNA vaccine constructs driven by a cytomegalovirus promoter, adjuvanted with human β-defensin 3 (hBD3), and fused to the reporter protein mRuby3. The constructs were evaluated in vitro using a fluorescent reporter system designed to provide a readout of TCR signaling upon the co-culture of T lymphocytes with differentiated monocytic cells expressing antigens. In vivo immunogenicity and protective efficacy were assessed in BALB/c (exploratory pilot) and IFNAR^−/− mice, a highly susceptible model for viral infection. Cytokine responses were measured to assess immunogenicity. Results: In vitro assays showed predominantly antigen-independent T-cell activation, suggesting that nonspecific stimulation inherent to the reporter co-culture system likely obscured the detection of antigen-specific TCR signaling. In vivo analyses in BALB/c mice revealed that the constructs elicited only modest systemic cytokine profiles while CCHFV-specific IgG and IFN-γ secretion remained undetectable, indicating that antigen-specific T-cell and antibody responses were limited. In the IFNAR^−/− challenge model, several peptide groups achieved significant 2–3 log reductions in tissue viral RNA and infectious titers (p < 0.05 vs. sham). However, the observed viral modulations were insufficient to reach the protective threshold and did not translate to a survival benefit (0%). Conclusion: Despite a rational in silico foundation, the multiepitope DNA vaccine constructs demonstrated limitations in inducing potent, antigen-specific immunity across both mouse models. The lack of antigen-specific responses indicates limitations in epitope selection, construct design, and delivery strategies, requiring optimization of next-generation epitope-based vaccines. These findings highlight the complexity of translating computational epitope predictions into functional vaccines, and provide benchmark data as a framework to guide future optimizations. Full article

Collagen, the most abundant extracellular matrix (ECM) protein, has emerged as a cornerstone biomaterial in drug delivery and regenerative medicine due to its intrinsic biocompatibility, biodegradability, and low immunogenicity. Engineering collagen into microspheres transforms its functionality beyond bulk scaffolds by increasing surface area, enabling minimally invasive delivery, and providing precise control over degradation, mechanical properties, and therapeutic release. This review provides a comprehensive analysis of collagen-based microspheres, with a particular focus on their dual role as biomimetic microenvironments and delivery systems. Recent advances in fabrication strategies, including emulsification, microfluidics, spray-drying, and electrospraying, are discussed in the context of scalability, size control, and payload encapsulation. Composite approaches that incorporate bioactive minerals, polysaccharides, or synthetic polymers are highlighted for their ability to enhance mechanical performance and biological function. We further examine characterization frameworks that link microscale structure and physicochemical properties to biological outcomes, with emphasis on how collagen microspheres replicate key structural, mechanical, and signaling features of native tissue microenvironments. Collagen microspheres have demonstrated broad utility as controlled delivery platforms, cell-instructive microcarriers, and injectable systems for tissue regeneration, including applications in bone, cartilage, skin, and nerve repair, as well as advanced wound care and localized cancer therapy. Finally, we critically assess current challenges related to scalable manufacturing, sterilization compatibility, and batch reproducibility, and outline emerging solutions such as recombinant collagen, advanced biofabrication, and stimuli-responsive systems. Collectively, collagen microspheres represent a powerful and adaptable platform poised to advance next-generation regenerative and therapeutic technologies. Full article

Horses develop spontaneous tumors, typically in old age. Although local tumor control can be achieved using conventional therapies, systemic therapies are required to treat recurrent and/or metastatic tumors. Immune checkpoint inhibitors, such as anti-PD-L1 antibodies, have been approved for the treatment of various tumor types in humans; however, little is known about the immunosuppressive roles of the PD-1/PD-L1 pathway in horses, and the therapeutic potential of these inhibitors remains to be elucidated. Previously, we reported that the rat monoclonal anti-PD-L1 antibody 6C11-3A11 cross-reacts with horse PD-L1 to block the PD-1/PD-L1 interaction. To further develop antibodies for therapeutic purposes, their immunogenicity must be reduced to maximize efficacy and safety. To this end, we designed an equinized (equine-ized) anti-PD-L1 antibody, Eq6C11, using the complementarity-determining regions of 6C11-3A11. Eq6C11 had antigen-binding properties comparable to those of 6C11-3A11 and inhibited equine PD-L1 binding to PD-1 in a recombinant protein-based assay. Treatment with Eq6C11 significantly increased IFN-γ and IL-2 production in equine peripheral blood mononuclear cell cultures, suggesting its stimulatory activity on T-cell activation. Although further studies are needed to clarify its immunogenicity and clinical activity, these results encourage further development of Eq6C11 as a candidate immune checkpoint inhibitor for cancer immunotherapy in horses. Full article

Mitsugumin 53 (MG53, also TRIM72) is a muscle-enriched tripartite motif protein with a well-established role in acute membrane repair and cytoprotection in striated muscle and other stressed tissues. MG53 is a core component of cellular repair machinery, rapidly sensing membrane disruption and coordinating membrane resealing, mitochondrial preservation, and anti-inflammatory modulation. In contrast to its high expression in skeletal muscle, endogenous MG53 expression in the adult human heart is minimal, raising the question of how MG53 exerts cardioprotective effects in the human heart. Recent studies help address this by identifying MG53 as a circulating regenerative myokine. MG53 is secreted from skeletal muscle into the bloodstream and can reach distal organs, including the heart. These findings support a muscle-to-heart endocrine model in which MG53 mediates tissue crosstalk and helps provide repair capacity to the myocardium when intrinsic cardiac MG53 is low. Here, we summarize recent advances in MG53 biology, emphasizing molecular mechanisms and inter-organ communication underlying cardioprotection. We further highlight translational strategies leveraging recombinant MG53- and MG53-based therapeutics and discuss challenges that must be addressed for future clinical applications. Collectively, these insights support MG53 as an endocrine repair factor linking skeletal muscle to cardiac repair and a potential regenerative cardiovascular target. Full article