Search Results (11,044)

DaxibotulinumtoxinA for injection (DAXI) is a botulinum neurotoxin (BoNT) drug product comprising the 150 kDa pure BoNT/A1 as the drug substance formulated with a proprietary stabilizing excipient, RTP004. We hypothesized that RTP004 facilitates localization of BoNT/A1 to the neuronal membrane, resulting in increased BoNT internalization and cleavage of the synaptosomal-associated protein of 25 kDa (SNAP-25) within synaptic terminals. We characterized the interaction between RTP004 and BoNT/A1 using in silico and in vitro techniques. In vitro analyses revealed that negative charges on the BoNT/A1 surface were located on the light chain (LC, the catalytic domain) and the C-terminus of the heavy chain (H_C, the receptor-binding domain), potentially providing sites for interaction with the positively charged RTP004 peptide. RTP004 bound to BoNT/A1, but not to human serum albumin (HSA), in both static and dynamic conditions. RTP004, not HSA, enhanced binding of BoNT to artificial membranes and RTP004 dissociated from BoNT under conditions that mimicked physiological conditions of the synaptic vesicle. RTP004 also increased binding of BoNT to the synaptosomal cell membrane and enhanced cleavage of SNAP-25 in a dose-dependent manner. These findings demonstrate that RTP004, not the excipient HSA common in other BoNT/A1 drug products, enhances binding of BoNT to the cell surface, facilitates internalization of BoNT into the cell, and increases SNAP-25 cleavage. Full article

This study investigates the systemic effects of amoxicillin and tetracycline on healthy Mus musculus (Swiss albino) mice, focusing on food intake, body weight, and hematological parameters. Over a 14-day oral treatment period, both antibiotics significantly reduced weight gain and food efficiency, with sex-specific variations: tetracycline had stronger metabolic effects in males, while amoxicillin was more impactful in females. To explore underlying mechanisms, molecular docking and MM-GBSA analyses were performed on PPAR-γ and TLR2–TIRAP complexes. Both antibiotics showed negligible binding to PPAR-γ, suggesting their metabolic effects are not receptor-mediated. In contrast, tetracycline exhibited strong and stable binding to TLR2 (ΔG_bind = −27.87 kcal/mol), supported by extensive hydrogen bonding, implying potential immunomodulatory action. These findings suggest that antibiotic-induced metabolic and immune alterations are more likely driven by microbiota disruption and innate immune signaling, rather than direct metabolic receptor engagement. Full article

Endothelin-1 (ET-1) is a potent vasoconstrictor that exerts its numerous biological actions through two receptors, ET_A and ET_B. However, the implication and role of each receptor in ROS generation remain ambiguous. Previously, our group reported that blocking the basal activity of ET_A and ET_B receptors with their respective peptidic antagonists increased basal intracellular calcium (Ca²⁺⁾ levels, an effect inhibited by chelating extracellular Ca²⁺. Since a crosstalk between Ca²⁺ and reactive oxygen species (ROS) exists, the purpose of the present work was to investigate whether this increase in basal resting Ca²⁺ level induced by the blockade of ET_A and ET_B receptors is associated with an increase in resting ROS level. Our results showed that the basal activity of ET_A and ET_B receptors contributes negatively to the resting level of cytosolic and nuclear ROS, and that each receptor appears to act as the other’s physiological antagonist. Furthermore, our results showed that ET-1 receptor blockade increases ROS via a receptor insensitive to ET_A and ET_B receptor antagonists. This type of receptor could be the one reported by our group, ET_C, or simply a heterodimeric ET_A/ET_B receptor. Moreover, blocking the heterodimerized ET_A/ET_B binding site is sufficient to unblock the physiological antagonism that each receptor exerts on the other. Furthermore, our results showed that blocking both ETA and ETB receptors, thereby preventing heterodimerization, prevented the increase in resting ROS, supporting the existence of a heterodimerized ET-1 receptor. Since human vascular smooth muscle cells (VSMCs) express only ETB receptors at the nuclear membrane, it is possible to suggest that nuclear ETB receptors are homodimers that regulate the resting nuclear ROS level. In conclusion, our results showed that the regulation of resting ROS levels by ET-1 and its receptors can be mediated by homodimerized and/or heterodimerized receptor activation; hence, the importance of developing drugs targeting this receptor type. Full article

Background/Objective: Coffee is the most highly consumed beverage worldwide, and coffee drinkers exhibit decreased mortality and protection from aging-related diseases. This study investigates the role of orphan nuclear receptor 4A1 (NR4A1) in mediating the effects of brewed coffee and the major polyphenolic and polyhydroxy compounds in brewed coffee and also in determining their binding to NR4A1. Methods: The interactions of brewed coffee and several of the major individual compounds in brewed coffee with the ligand-binding domain of NR4A1 were determined using a fluorescent binding assay. For specific compounds, binding was also carried out by surface plasmon resonance, and molecular docking studies were also performed. NR4A1-responsive Rh30 cancer cells were used as models to determine NR4A1-dependent transactivation, cell growth inhibition and inhibition of specific gene products, and in some studies, knockdown of NR4A1 by RNA interference was also determined. Inhibition of lipopolysaccharide-induced IkBα by key polyphenolics was also investigated in RAW264.7 macrophages. Results: Brewed coffee and several polyphenolics, including caffeic acid, ferulic acid, chlorogenic acid, p-coumaric acid, several cinnamic acid derivatives, kahweol, and cafestrol, bound NR4A1 in binding assays, and most Kd values were <10 µM. Brewed coffee and the major polyphenolics inhibited growth of NR4A1-responsive Rh30 cells, and this was attenuated in NR4A1-deficient Rh30 cells. These same compounds also exhibited NR4A1-dependent effects on transactivation and gene product responses in Rh30 and RAW264.7 macrophages and exhibited inverse NR4A1 agonist activity. In contrast, the NR4A1-dependent activity of caffeine and quinic acid was highly variable, suggesting that they are selective NR4A1 ligands. Conclusions: The results of this study demonstrate that brewed coffee and its major polyphenolics and polyhydroxy constituents are NR4A1 ligands and that NR4A1 may play an important role in the health-protective effects of coffee. These results, coupled with recent studies, indicate that NR4A1 and its ligands may play an important role in diet and health. Full article

Periodontitis is a chronic infectious disease characterized by the destruction of the tooth-supporting tissues, including the gingiva, periodontal ligament, and alveolar bone, which may ultimately lead to tooth loss. However, blood-based biomarkers reflecting systemic inflammation in periodontitis remain poorly defined. We analyzed plasma proteomic data from the UK Biobank using Olink Explore proteomics to identify systemic protein signatures distinguishing chronic periodontitis patients (n = 90) from healthy controls (n = 2234). Among 2151 proteins passing quality control, 29 proteins showed significant differential expression (FDR < 1.0 × 10⁻⁵). Growth differentiation factor 15 (GDF15) exhibited the strongest upregulation (mean NPX: −0.183 to 0.157, effect size = 0.337, FDR = 2.82 × 10⁻¹²), followed by N-terminal pro-B-type natriuretic peptide (NT-proBNP) (effect size = 0.594), Interleukin-6 (IL-6) (effect size = 0.450), and Insulin-like growth factor binding protein-(4IGFBP4) (effect size = 0.269). Multiple TNF receptor superfamily members (TNFRSF1A/1B, TNFRSF10A/10B) and proteins involved in extracellular matrix remodeling (COL6A3, ADAM12) and vascular stress (ADM) were significantly elevated. In contrast, EGFR and DNER showed decreased expression. Protein–protein interaction network analysis revealed IL-6 as a central hub protein forming a tightly interconnected cluster with TNF receptor family members. These findings indicate systemic plasma protein profiles associated with chronic periodontitis within this population-based cohort. The identified proteins may provide a basis for future evaluation of blood-based biomarkers for chronic periodontitis, pending further validation. Full article

Background: Latent tuberculosis infection (LTBI) is the principal reservoir for active tuberculosis, with >85% of cases attributable to reactivation. Bacillus Calmette-Guérin fails to block this transition, leaving a critical gap in prevention. Methods: An immunoinformatics/reverse-vaccinology pipeline was applied to seven dormancy-related antigens retrieved from Mycobrowser. T-cell epitopes were predicted with NetMHCI/IIpan-4.1 and B-cell epitopes with ABCpred; antigenicity, allergenicity, and toxicity were evaluated with VaxiJen, AllerTOP, and ToxinPred. Secondary/tertiary structures were modeled with PSIPRED and AlphaFold-3; docking to Toll-like receptors (TLR) 2/4 and 100 ns molecular dynamics simulations assessed complex stability. Immune responses were simulated with C-ImmSim, and the mRNA sequence was human-codon-optimized using ExpOptimizer. Results: The resulting construct, RP14914P, encodes 14 cytotoxic T lymphocyte, 9 helper T lymphocyte, and 14 B-cell epitopes within an 866-aa, 90.4 kDa polypeptide. Antigenicity score = 0.7797, immunogenicity score = 8.58629. and no toxicity or allergenicity was predicted. Physicochemical analysis: instability index = 28.65, and solubility = 0.513. Estimated population coverage is 82.35% and 99.67% for Human Leukocyte Antigen (HLA)-I and HLA-II globally. Docking energies: −1477.8 kcal/mol (TLR2) and −1480.1 kcal/mol (TLR4). Molecular dynamics trajectories confirm stable binding. Immune simulation predicts potent activation of Natural Killer cells, macrophages, and dendritic cells, Th1 polarization, high interferon-γ/interleukin-2 secretion, and durable memory. Conclusions: In silico analyses predict that RP14914P exhibits favorable immunogenicity, safety, and broad population coverage, suggesting its potential as a promising mRNA vaccine candidate to prevent LTBI reactivation. However, these computational predictions require thorough experimental validation to confirm the vaccine’s immunogenicity and protective efficacy. Full article

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), results in substantial economic losses for the global pig farming industry. A critical step in the infection process is the binding of PRRSV to the CD163 receptor on the surface of porcine alveolar macrophages. This study successfully generated CD163−/− Landrace pigs using CRISPR/Cas9 gene editing technology. Following an experimental challenge with two distinct Type II PRRSV strains, the edited pigs exhibited complete resistance to infection. Virological and pathological examinations confirmed the absence of viral replication and the presence of characteristic pulmonary lesions and other organ damage in CD163−/− pigs. In contrast, wild-type control pigs exhibited high viral loads and severe pulmonary lesions, as well as damage to other organs. Our findings provide direct evidence that CD163 is an essential receptor for PRRSV infection in vivo. The CD163−/− pig model offers an effective genetic strategy for breeding pigs with an inherent resistance to PRRSV. Full article

Reproductive management in goats remains challenging due to seasonal breeding and the use of hormones that raise concerns about immunogenicity, cost, sustainability, and animal welfare. In this study, we evaluated date palm pollen (Phoenix dactylifera L.) (DPP) as a natural source of estrogenic compounds capable of modulating reproductive function. DPP was extracted using methanol, ethanol, acetone, and hexane, and the extracts were analyzed by ultra-performance liquid chromatography. Quercetin and coumestrol were detected in the methanolic and ethanolic extracts at comparable levels (quercetin 0.043–0.044 mg/g; coumestrol 0.987–1.015 mg/g of extract) (p > 0.05). The acetone extract contained significantly lower concentrations (quercetin 0.017 mg/g; coumestrol 0.033 mg/g of extract), while the hexane extract showed no detectable amounts. Molecular docking using the crystallographic structure of estrogen receptor alpha (PDB:6PIT) showed that both compounds interact with key residues of the receptor’s ligand-binding domain. Coumestrol exhibited the highest affinity (−9.3 kcal/mol), surpassing 17-β estradiol (−8.9 kcal/mol), forming several hydrogen bonds and hydrophobic contacts. Quercetin showed a lower affinity (−7.2 kcal/mol) but maintained stabilizing interactions compatible with partial agonist activity. Overall, methanol and ethanol were the most effective solvents for extracting phytoestrogens from DPP, and the findings support their potential as natural alternatives to hormones for estrus induction in goats. Full article

Goat reproductive performance is a key determinant of the productivity and economic value of goat farming, especially in meat and milk production. In a previous study, to investigate the genetic basis of prolificacy, we divided goats into groups according to their consistent reproductive performance (producing either single kids or twins) over five consecutive kidding cycles, and performed whole-genome resequencing and RNA-seq analysis on their ovarian tissues. Through integrated analysis, we identified three candidate genes—IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1), CDC25A (cell division cycle 25A), and RXFP2 (relaxin family peptide receptor 2)—as potential key regulators of reproductive capacity. Using goat ovarian granulosa cells, we systematically assessed the impact of each gene through gain- and loss-of-function experiments. Overexpression of IGF2BP1 promoted cell proliferation and suppressed apoptosis, underscoring its role in maintaining cellular viability. Conversely, its knockdown significantly impeded growth and induced cell death. Similarly, CDC25A enhanced granulosa cell proliferation, whereas its knockdown led to marked growth impairment and increased apoptosis. Proliferation was also enhanced by RXFP2 overexpression but impaired upon its knockdown, suggesting that RXFP2 is functionally important for follicular development. Collectively, these findings establish IGF2BP1, CDC25A, and RXFP2 as fundamental regulators of granulosa cell dynamics and ovarian follicular development, providing crucial functional insights and promising targets for genetic selection to enhance reproductive efficiency in goats. Full article

Retromer is an evolutionarily conserved protein complex first identified in budding yeast. It was originally described for its essential role in endosome-to-Golgi retrieval of lysosomal hydrolase receptors. Retromer is now known to mediate trafficking of many endosomal cargoes. The mammalian retromer is constituted by a core heterotrimer encoded by the vacuolar protein sorting (VPS) gene products VPS26, VPS35, and VPS29. To mediate cargo recognition and endosomal sorting into various pathways, this trimer can cooperate with phosphoinositide-binding sorting nexin family members. Defective retromer functioning has been associated with alterations in cellular homeostasis, leading to disease. To gain insights into how it may mediate these broad processes, a proteomic strategy in polarized Madin-Darby canine kidney cells was devised to identify retromer-interacting proteins. Subsequent validation of one of the candidates, i.e., cytokeratin 19, led to the unexpected finding that retromer localizes to the pericentriolar region in dividing cells and subsequently translocates to the midbody during cytokinesis. Retromer was found interacting with CK19, and its antisense depletion led to delocalization from CK19. Subcellular fractionation and live cell monitoring of depleted cells provided evidence of a role by retromer in post-metaphase progression and in epithelial cell migration, thereby connecting retromer with key processes of cellular dynamics. Full article

The immune system maintains homeostasis through coordinated innate and adaptive responses, and its imbalance increases disease susceptibility. The immunomodulatory effects of 6-methoxykaempferol (6MK), a methoxylated flavonoid found in sweet cherries, were studied in a mouse model of cyclophosphamide (CPA)-induced immunosuppression. The expression of key signaling proteins in the NF-κB and MAPK pathways was studied to explore the underlying molecular mechanisms. The Toll-like receptor-4/myeloid differentiation factor-2 receptor complex (TLR4/MD2), which can stimulate the immune response by activating these pathways, was used to study possible interactions with 6MK using docking analysis. 6MK administration significantly restored immune organ integrity (spleen up to 15.1% and thymus up to 16.8%), enhanced NK cell function (up to 43.8%), promoted T (up to 24.5%) and B cell proliferation (up to 26.4%), increased pro- and anti-inflammatory cytokine (IL-1β, IL-6, TNF-α, IL-4, IL-10, and TGF-β) levels, and elevated NO (up to 25.6%) and immunoglobulin (IgG, IgA, and IgM) concentrations. Additionally, 6MK upregulated antioxidant enzymes (CAT, HO-1, and SOD) and reactivated suppressed NF-κB and MAPK pathways. The docking-supported hypothesis, based on putative interactions and the estimated free energy of binding, suggests that 6MK possesses agonistic potential for the TLR4/MD2. Changes in the gut microbiome due to 6MK treatment, such as an increase in alpha diversity, abundance of Dorea longicatena, and the upregulation of formaldehyde-consuming pathways, may also contribute to immune enhancement. These findings show that 6MK may alleviate immunosuppression, suggesting its potential for future studies targeting immune-related diseases and conditions. Full article

Hippocalcin (HPCA), a neuron-enriched calcium-binding protein, plays a critical role in brain function, but its role in neural precursor cells remains unclear. N-methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors essential for neurodevelopment and synaptic plasticity, and their function has been implicated in neurological conditions. In this study, we investigated the role of HPCA in regulating NMDA receptor expression and function in mouse hippocampal neural precursor cells (mHNPCs). HPCA knockdown significantly reduced the expression of NMDA receptor-related genes, including Grin2C, Shank1, Serpine2, and selectively attenuated NMDA-induced calcium signaling. Transcriptomic analysis identified ELAV-like RNA-binding protein 3 (Elavl3), a neuron-enriched factor associated with neuronal activity, as a downstream candidate affected by HPCA knockdown. Consistently, Elavl3 suppression phenocopied HPCA deficiency, resulting in impaired NMDA receptor activity and reduced neuronal differentiation. Furthermore, hippocampal HPCA knockdown in vivo led to alterations in locomotor activity, contextual memory, and affective behaviors. Taken together, these findings demonstrate that HPCA supports NMDA receptor function and neuronal development, in part through Elavl3-associated pathways, and highlight HPCA as an important regulator of hippocampal function. Full article

Marine lectins function as pattern recognition receptors in innate immunity through carbohydrate-binding mechanisms. However, mechanistic evidence detailing intracellular signaling cascades (e.g., MAPK/NF-κB/JAK-STAT activation linked to defined cytokine outputs) remains taxonomically uneven. Bivalve mollusks—particularly the Mytilectin family—represent the most extensively characterized group, whereas lectins from other marine phyla (echinoderms, cnidarians, fish, algae) have been studied primarily for structural and glycan-binding properties alongside phenotypic antimicrobial outcomes. Signaling-level resolution in native immune-cell contexts, while present in some cases, remains comparatively limited. This review synthesizes mechanistic insights dominated by bivalve-derived lectins, while integrating cross-taxa comparisons at evidence-supported levels. Specific bivalve lectins induce macrophage activation and pro-inflammatory cytokine production through reactive oxygen species-dependent activation of key signaling pathways including MAPK, NF-κB, and JAK-STAT cascades. These lectins exhibit context-dependent properties, promoting inflammatory responses in resting cells while inducing endotoxin tolerance in pre-activated macrophages through epigenetic reprogramming. Functional outcomes include broad-spectrum antiviral activity through viral envelope glycoprotein binding, anti-inflammatory effects in pain models, and cancer-associated immune responses through tumor glycan recognition and macrophage polarization. Critical gaps include uncharacterized effects on adaptive immunity, limited understanding of dendritic cell and natural killer cell interactions, and incomplete evaluation of cancer immunotherapy potential. Future research should prioritize mechanistic characterization of marine lectin-based immunotherapeutics. Full article

Background: Brucella outer membrane proteins (Omps) are an important part of its cell wall and major virulence-related factors. Omp16 and Omp19 proteins are the advantageous antigens of Brucella and have been widely used in research on vaccines against brucellosis. As an emerging vaccine, the mRNA vaccine has unique advantages in the fight against intracellular parasitic bacteria. Methods: In this study, mRNA encoding the omp16 and omp19 genes of Brucella. melitensis (B. melitensis) was synthesized using in vitro transcription. The target mRNA was transfected into HEK 293T cells to evaluate protein expression levels and assess its immunogenicity. Finally, bioinformatic approaches were employed to analyze potential antigenic epitopes. Results: In this study, the successfully constructed recombinant plasmids pIVTRup-omp16 and pIVTRup-omp19 were utilized to synthesize omp16-mRNA and omp19-mRNA, each approximately 600 nt in length. Western blot analysis detected the expression of proteins with molecular weights of 16 kDa and 19 kDa in HEK 293T cells at 24 h post-transfection with mRNA. Purified rOmp16 and rOmp19 had good immunogenicity, which could specifically bind to serum antibodies of brucellosis patients. rOmp16 had stronger immunogenicity than rOmp19. Epitope prediction showed that Omp16 contained seven epitopes and Omp19 contained six epitopes. In addition, Omp16 and Omp19 could form stable complexes with target receptors. Simulated immunization with Omp16 and Omp19 proteins significantly activated both CD4⁺ and CD8⁺ T cells. Conclusions: The immunogenic proteins were successfully expressed in cells based on the mRNA fragments synthesized from omp16 and omp19 genes of B. melitensis, which was a preliminary exploration for the preparation of B. melitensis mRNA vaccine. Full article

Diabetic kidney disease (DKD) is a major complication of diabetes mellitus that contributes substantially to chronic kidney failure and increased cardiovascular risk. Beyond progressive deterioration of renal function, DKD is associated with disturbances in endocrine and vascular regulation. Among these, alterations in vitamin D homeostasis and blood pressure (BP) control represent clinically relevant, yet incompletely integrated aspects of DKD pathophysiology. This narrative review synthesizes current evidence on the multidirectional relationships between DKD, vitamin D deficiency, and arterial hypertension (AH). Attention is given to renal mechanisms responsible for reduced vitamin D availability in DKD, including proteinuria-related loss of vitamin D-binding proteins, impaired proximal tubular reabsorption, decreased renal activation of vitamin D, and hormonal regulators such as fibroblast growth factor-23. It further discusses how insufficient vitamin D signaling may influence renal and vascular pathways involved in BP regulation. Mechanistic links between vitamin D deficiency and AH in DKD are discussed, with emphasis on maladaptive activation of the renin–angiotensin–aldosterone system (RAAS), persistent inflammation, oxidative stress, endothelial dysfunction, and insulin resistance. These interdependent processes promote both renal injury progression and sustained elevations in BP, forming a self-reinforcing pathogenic loop. Finally, available data on vitamin D-based therapeutic strategies in DKD are reviewed, including native vitamin D supplementation, active vitamin D metabolites, and vitamin D receptor agonists. Although experimental and observational studies suggest potential nephroprotective and vasculoprotective effects, evidence from randomized clinical trials remains heterogeneous. Further well-designed prospective studies are required to clarify the clinical utility of vitamin D interventions in patients with DKD and coexisting AH. Full article