Plant-mediated nanoparticles are being investigated for metabolic disorders, but therapeutic gains from full-dose co-administration should be distinguished from true pharmacological synergy. This study compared the antidiabetic effects of Cymbopogon citratus leaf ethanolic extract (LEE), biosynthesized silver nanoparticles (AgNPs), and their combination in alloxan-induced diabetic mice. AgNPs were synthesized using LEE as a reducing and capping agent and characterized by UV-Vis spectroscopy, dynamic light scattering, zeta potential analysis, transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. The AgNPs showed a surface plasmon resonance peak at 432 nm, mean hydrodynamic diameter of 38.2 ± 4.6 nm, zeta potential of -28.4 ± 3.1 mV, and predominantly spherical morphology. Diabetic mice received metformin, LEE, AgNPs, or LEE + AgNPs for 28 days, followed by evaluation of glycaemic, oxidative, lipid, hepatorenal, haematological, pancreatic histopathological, and integrated biomarker recovery responses. The combination group recorded fasting blood glucose of 128.0 ± 3.0 mg/dL and HbA1c of 6.5 ± 0.1%, compared with 141.0 ± 5.7 mg/dL and 7.3 ± 0.1%, respectively, in the AgNP group. Although combined administration produced the highest integrated biomarker recovery index (80.61 ± 0.61%), additional improvements over AgNP monotherapy were limited or nonsignificant across several endpoints. Pancreatic histopathology also showed improved islet area, cellular density, and lesion scores following treatment. Co-administration produced endpoint-specific additional benefits but did not demonstrate pharmacological synergy. Further route-matched dose-response studies, mechanistic validation, nanoparticle biodistribution, and long-term biosafety assessment are required.
Crosslinking of the high affinity IgE receptor (FcεRI) by multivalent antigen increases intracellular Ca2+ concentration ([Ca2+]i) through two sequential steps: (i) Ca2+ release from the endoplasmic reticulum (ER) and (ii) store-operated Ca2+ entry (SOCE) mediated by stromal interaction molecule 1 (STIM1)-Orai1 interaction. This process is essential for basophil and mast cell activation. We previously showed that FcεRI disaggregation by monomeric hapten rapidly and persistently inhibited [Ca2+]i increase although upstream signal disruption was transient in rat basophilic leukemia cells. However, the underlying inhibitory mechanisms remain unclear. In this study, we found that monomeric hapten dissociates STIM1-Orai1 interaction by promoting Orai1 re-phosphorylation. Although Ca2+ release from ER, the first step in [Ca2+]i increase, was blocked by monomeric hapten addition, phosphorylation of phospholipase Cγ1, a critical step for Ca2+ release from the ER, was inhibited only when the monomeric hapten was added immediately after multivalent antigen stimulation, but not when added several minutes later. However, STIM1-Orai1 interaction was dissociated by the monomeric hapten, regardless of the timing of its addition. Multivalent antigen induced Orai1 dephosphorylation, which is necessary for SOCE. In contrast, monomeric hapten addition promoted its re-phosphorylation. These results suggest that cessation of [Ca2+]i increase following FcεRI disaggregation primarily arises from immediate suppression of SOCE via STIM1-Orai1 dissociation.
PTEN plays diverse tumor-suppressive roles, including inhibition of PI3K-AKT signaling and maintenance of genomic integrity in the nucleus. Elucidating the molecular mechanisms regulating its subcellular localization is therefore essential for understanding PTEN functions. PTEN350, a fragment comprising the N-terminal phosphatase and C2 domains of PTEN, preferentially localizes to the nucleus, although the residues responsible for this localization remain unclear. Previously, we demonstrated that Thr348 contributes to the prominent nuclear accumulation of the PTEN350 fragment and PTENA4 carrying alanine substitutions in the Ser380/Thr382/Thr383/Ser385 (STTS) motif. Since our previous findings suggested that Phe347 also contributes to PTEN nuclear localization, we investigated its role in the present study. Phe347 substitutions (F347A, F347Y, and F347L) were introduced into PTEN and its mutant or truncated derivatives, including PTEN350, PTEN, PTENA4, PTEN350,K13R, and PTENK13R,A4, either alone or in combination with T348D. The F347A substitution significantly attenuated the nuclear accumulation of PTEN350 and PTENA4, whereas F347L partially preserved nuclear accumulation and F347Y substitution exhibited an intermediate phenotype. Similar effects of the F347 substitutions were also observed in the T348D mutants, although the differences among the three substitutions were less pronounced. A similar pattern was observed for the plasma membrane localization of PTEN350,K13R and PTENK13R,A4, with F347A causing the greatest reduction, F347L retaining partial membrane localization, and F347Y exhibiting an intermediate phenotype. The effects of these substitutions were consistently observed in both HEK293T cells and HeLa cells. Collectively, these findings indicate that Phe347 is an important determinant of PTEN subcellular localization.
Nuclear pore complexes (NPCs) contribute to genome organization and cell identity, yet how post-mitotic NPC assembly is coordinated with chromatin architecture remains unclear. Here, we show that the nucleoporin ELYS preferentially associates with chromatin regions displaying distinct intrinsic DNA sequence features that are not explained by the repressive histone marks examined here. ELYS-bound regions are enriched for AT-rich sequences, whereas ELYS binding at super-enhancer-associated loci shift toward GC-rich sequence composition, revealing distinct sequence environments. These findings indicate that ELYS localization is associated with distinct intrinsic DNA sequence features and suggest a mechanism by which nuclear pore-associated architecture restores transcriptional programs after mitosis.
High-mobility group box 1 (HMGB1) is a chromatin-associated protein and a prototypical damage-associated molecular pattern whose dual intracellular and extracellular functions are increasingly implicated in cancer progression. Because viral proteins can harness HMGB1 to facilitate their own replication and remodel the microenvironment of transformed cells, human oncogenic viruses provide an instructive model for examining this duality. In this conceptual review, we organized the available evidence around two functional nodes. At the first node, intracellular HMGB1 supports viral replication, acting on viral chromatin in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus, and on structured viral RNA in hepatitis C virus. At the second node, viral infection or specific viral oncoproteins induce HMGB1 secretion, which promotes infected-cell survival and remodels the tumor microenvironment, as reported for KSHV, hepatitis B virus, and human T-cell leukemia virus type 1. Human papillomavirus engage a receptor-level variant of this node through the HMGB1-TLR4 axis. Only KSHV currently supports both nodes in matched experimental systems. Therefore, we present a sequential two-node arrangement as a hypothesis, instead of an established property of oncogenic viruses. We further considered how viruses reverse the tumor-suppressive, genome-stabilizing functions of nuclear HMGB1, with conserved and divergent strategies apparent across viral families; why the absence of HMGB1 data for Merkel cell polyomavirus is a tractable and informative gap; and which HMGB1- and RAGE-directed agents are realistically positioned for evaluation in virus-associated cancers.
Perfluorooctane sulfonate (PFOS), a persistent member of the per- and polyfluoroalkyl substances (PFAS), has been associated with adverse neurodevelopmental outcomes. However, the direct effects of PFOS on the developing human hippocampus remain incompletely understood due to the limited availability of physiologically relevant human models. In the present study, we utilized human hippocampal organoids (hHOs) derived from human pluripotent stem cells to investigate the developmental effects of PFOS exposure. The organoids exhibited molecular characteristics consistent with hippocampal identity, including increased expression of the medial pallium-associated markers compared with human cortical organoids. Exposure to PFOS resulted in a dose-dependent reduction in MAP2 immunoreactivity together with increased cleaved caspase-3-positive cells, suggesting neuronal toxicity. Gene expression analyses further demonstrated increased expression of progenitor-associated genes accompanied by reduced expression of neuronal markers DCX and MAP2. Bulk RNA sequencing identified broad transcriptional alterations following PFOS exposure, including enrichment of proliferation-associated biological processes and downregulation of neuronal differentiation-related pathways. In addition, multiple genes involved in mitochondrial oxidative phosphorylation and the tricarboxylic acid cycle were reduced in PFOS-treated organoids. Collectively, these findings suggest that PFOS induces neuronal toxicity in human hippocampal organoids and is accompanied by alterations in developmental transcriptional programs and mitochondrial metabolism-related gene expression.
Mitochondrial calcium homeostasis is critical for bioenergetics, cell signaling, and cell survival and death, but its regulatory mechanism remains largely unknown. Here, a mitochondria-targeted genetically encoded calcium indicator has revealed that physiological concentrations of ascorbic acid (vitamin C) suppress mitochondrial calcium uptake in both intact living cells and permeabilized cells and enhance intracellular calcium signaling compared with ascorbate-deprived conditions. Mechanistic analyses indicate that this effect is mediated by a reduction in mitochondrial membrane potential, the primary driving force for mitochondrial calcium uptake. These findings uncover an unrecognized role of ascorbic acid in mitochondrial calcium homeostasis. Given the roles of mitochondrial calcium in neurodegeneration and cancer cell bioenergetics, our findings provide new insights into disease pathophysiology and potential therapeutic strategies.
This study attempts to repurpose ivermectin (IVM) for AD by inhibiting the PTP1B enzyme. IVM is a widely known, commercially approved antiparasitic drug. The PTP1B enzyme dephosphorylates multiple kinase substrates and is responsible for metabolic regulation by the PI3K/AKT downstream signaling cascade. A similar signaling pathway is present in the brain. We assessed binding affinity in BIOVIA Discovery Studio Visualizer version 2021 and inhibitory activity using a commercially available ELISA kit. The streptozotocin (3 mg/kg, intracerebroventricularly)- induced AD mouse model was used to study the biological effects of IVM using NOR and Y-maze behavioral assays. Multiple disease physiologies, such as oxidative stress, mitochondrial complex (I-IV) dysfunction, brain insulin resistance, neuroinflammation, Aβ aggregation, apoptosis, and autophagy signaling cascade, were studied by assessing various biomarkers to generate a proof-of-concept. The docking study demonstrated potential interactions of IVM with the catalytic pocket of PTP1B enzyme, yielding a docking score of -8.0. To validate the docking protocol, the re-docked ligand was superimposed on the crystallographic ligand, resulting in an RMSD of 0.000 Å, therefore confirming the reliability of the computational approach. The calculated IC50 value of IVM by ELISA inhibitory assay was found 4.58 μM. The in vivo study of IVM (10 mg/kg) in the mouse model showed improvement in cognitive deficits on behavioral assays and was comparable to donepezil and DPM1001. The preliminary screening investigations through in vitro enzyme inhibition and molecular-level studies showed promising improvements in multiple physiological parameters that progressed to AD. The IHC showed a reduction in the Aβ plaque load and activated microglia cell count. These preliminary data demonstrate that IVM exhibits considerable PTP1B inhibitory potential and require further exploration in a robust, statistically powered preclinical study, followed by clinical trials, to repurpose it in AD and related conditions.
Thioredoxins (Trxs) are ubiquitous oxidoreductases that maintain cellular redox homeostasis through thiol-disulfide exchange reactions. Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain absent from the canonical thioredoxin EcTrx1, but the physiological significance of this domain has remained unclear. Here we show that EcTrx2 undergoes reversible, redox-dependent structural switching accompanied by a functional conversion under oxidative stress. Oxidative conditions promoted the formation of high-molecular-weight (HMW) oligomeric complexes, whereas reducing conditions favored low-molecular-weight (LMW) species. Increased surface hydrophobicity of oxidized EcTrx2 correlated with a marked enhancement of holdase chaperone activity and a concomitant reduction in disulfide reductase activity. Size-exclusion chromatography coupled with transmission electron microscopy further revealed that the HMW oligomers were the predominant chaperone-active species, whereas the LMW form primarily retained reductase activity. Deletion of the N-terminal zinc-binding domain abolished the redox-dependent structural transition and impaired chaperone activation, demonstrating that this domain is required for stress-responsive functional switching. These findings identify EcTrx2 as a redox-regulated molecular chaperone and provide a mechanistic basis for reversible structural and functional switching in a bacterial thioredoxin during oxidative stress.
INTRODUCTION:The increasing prevalence of antibiotic-resistant bacteria, particularly Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), necessitates the development of more effective antibacterial strategies. Gold nanoparticles (AuNPs) have emerged as promising nanocarriers to improve antibiotic delivery and antibacterial efficacy. METHODS:Amox-AuNPs were synthesized using the Turkevich method and characterized by UV-Vis spectroscopy, FTIR, particle size analysis, zeta potential, FE-SEM, TEM, and stability evaluation. Antibacterial activity was assessed using microdilution and disk diffusion assays. Cytocompatibility and safety were evaluated using the MTT assay on MCF-10A cells and the Fish Embryo Toxicity (FET) test. The selectivity index (SI) was determined from the estimated IC50 and MIC90 values. RESULTS:Characterization confirmed successful conjugation of amoxicillin onto AuNPs with high entrapment efficiency (76.89%) and good colloidal stability over 20 days. Amox-AuNPs exhibited significantly enhanced antibacterial activity against S. aureus and MRSA, with an estimated MIC90 of 2.38 ppm against MRSA. Cytotoxicity testing demonstrated cell viability above 70%, while the estimated SI of 14.78 and FET results indicated favorable biocompatibility and safety. CONCLUSION:Amox-AuNPs effectively enhanced the antibacterial activity of amoxicillin while maintaining good physicochemical stability, favorable biocompatibility, and a wide therapeutic window, highlighting their potential as a nanocarrier for combating antibiotic-resistant bacteria.
OBJECTIVE:To systematically identify RING finger protein (RNF) biomarkers with diagnostic value in sepsis and to explore their potential as therapeutic targets. METHODS:Sepsis peripheral blood transcriptome datasets from Gene Expression Omnibus (GEO) were integrated. Sepsis-associated RNF genes were identified via differential expression analysis and weighted gene co-expression network analysis (WGCNA). Feature genes were selected using LASSO, SVM-RFE, and SHAP. Summary-data-based Mendelian randomization (SMR) assessed causality, and diagnostic performance was validated by receiver operating characteristic (ROC) curves in three independent cohorts. Single-cell RNA sequencing resolved cellular localization and perturbations. DrugReflector deep learning predicted repurposable drugs, molecular docking evaluated binding affinity, and PheWAS assessed target safety. RESULTS:A total of 13 sepsis-related RNF genes were identified, and three feature genes (ZFP36L2, RNF125, RNF175) were further selected. SMR showed that only elevated RNF175 expression significantly increased sepsis risk (OR = 1.267, 95% CI 1.002-1.603, P = 0.048). The diagnostic AUCs of RNF175 were 0.931, 0.776, and 0.835 in the training and two validation cohorts, respectively. Single-cell atlas revealed predominant RNF175 expression in T cells with significant upregulation in sepsis (P < 0.001). Deep learning predicted tadalafil as the top candidate, with a molecular docking binding energy of -9.072 kcal/mol for RNF175. PheWAS found no significant phenotype associations, indicating low off-target risk. CONCLUSIONS:RNF175 is an early sepsis biomarker with both diagnostic value and causal relevance, potentially acting through T-cell modulation. Tadalafil may be a pathway-targeted repurposable drug. This study provides new perspectives for precision medicine in sepsis.
The misfolding and subsequent aggregation of transthyretin (TTR) into amyloid fibrils, which deposit in tissues such as the heart and peripheral nerves, underlies a series of progressive and fatal diseases. Consequently, molecules that inhibit protein misfolding represent a promising therapeutic strategy. Structural analysis of the TTR-quercetin complex indicates that quercetin stabilizes the TTR tetramer. In this study, Thioflavin T fluorescence assays, transmission electron microscopy, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy demonstrated that quercetin significantly delays the aggregation rate of TTR. This leads to a decrease in β-sheet content and fibril formation. Furthermore, the inhibition of fibrillogenesis by quercetin significantly attenuated the cytotoxicity induced by TTR fibrils. These findings suggest that quercetin holds promise as a natural product for treating transthyretin amyloidosis. However, further in vivo studies and clinical trials are required to validate these findings and explore its translational potential.
