Search Results (3,672)

To explore an alternative detection approach for the pesticide imidacloprid (IMI), this study repurposed the quinoline-benzimidazole fluorescent probe DQBM-B—previously developed for Co²⁺ recognition—and investigated its detection performance and interaction mechanism toward IMI in the aggregated state. The optimal working conditions of the probe were determined by optimizing key detection parameters, and the sensing performance and matrix compatibility were evaluated through selectivity tests and proof-of-concept spiked cucumber extract analysis. The DQBM-B aggregates interact with IMI synergistically through intermolecular hydrogen bonding and π–π stacking, which enrich IMI at the aggregate surface to create a local enrichment layer. The observed fluorescence quenching arises from the synergistic contribution of static quenching (due to ground-state complex formation) and the inner filter effect (IFE). Under the optimal conditions, the system exhibited a detection limit of 0.75 μmol L⁻¹ for IMI with favorable anti-interference ability. The matrix effect evaluation in cucumber extract demonstrated good recovery and precision, demonstrating the feasibility of the aggregation-regulated IFE strategy in complex food matrices. This study expands the application scope of the DQBM-B probe from metal-ion sensing to pesticide detection and provides a metal-free, aggregation-regulated strategy for the fluorescence detection of neonicotinoid pesticides. Full article

Background/Objectives: Human teeth naturally fluoresce due primarily to organic components within dentine, contributing to their vitality and appearance under ultraviolet (UV) light. Contemporary resin composites increasingly incorporate fluorescent agents to reproduce this property and improve aesthetic integration with natural dentition. This narrative review examines the fluorescence characteristics of natural teeth and direct resin composites, evaluates methods used to assess dental fluorescence, and identifies factors influencing fluorescence stability and biomimetic performance. Methods: Peer-reviewed studies published between 2004 and 2025 investigating fluorescence in human teeth and direct resin-based composites under UV, visible or infrared illumination were reviewed. Studies focused on indirect restorative materials, non-human specimens or caries detection alone were excluded. Results: Natural teeth generally exhibit fluorescence emission peaks between 410 and 500 nm, with dentine demonstrating greater fluorescence intensity than enamel. Composite resins commonly incorporate rare-earth oxide fluorophores and often display similar emission wavelengths (approximately 450–485 nm), although fluorescence intensity varies considerably among brands and shades. Material fluorescence is influenced by composition, aging and environmental exposure. Spectrofluorometry provides highly accurate quantitative assessment, whereas photographic and quantitative light-induced fluorescence (QLF) methods offer greater clinical applicability. Despite advances in fluorescence mimicry, variability among contemporary materials generally continues to permit differentiation from natural tooth. Conclusions: Although substantial progress has been made in reproducing natural tooth fluorescence, significant variability and temporal instability remain among contemporary composite materials. Fluorescence-based methods therefore remain useful for restoration identification. However, continued improvements in biomimetic performance may reduce their future effectiveness. Greater standardisation of fluorescence assessment and investigation of alternative detection approaches are needed to support clinical and forensic applications. Full article

Streptococcus salivarius-derived extracellular vesicles (SsEVs) have emerged as important mediators of host–microbe communication, but their role in oral squamous cell carcinoma (OSCC) remains unclear. In this study, SsEVs were isolated and characterized by transmission electron microscopy and nanoparticle tracking analysis, and their uptake by CAL-27 cells was confirmed by fluorescence imaging. Functional assays demonstrated that SsEVs inhibited the proliferation, migration, and invasion of CAL-27 cells in a concentration-dependent manner. RNA sequencing revealed substantial transcriptional reprogramming following SsEV treatment, with enrichment analyses indicating the suppression of pathways associated with cell adhesion, extracellular matrix remodeling, lipid metabolism, and particularly Notch signaling. Gene set enrichment analysis (GSEA) and gene set nariant analysis (GSVA) consistently identified Notch signaling as significantly downregulated. Further validation showed that SsEVs markedly decreased the expression of JAG1, NOTCH1, and HEYL at both the mRNA and protein levels. In a CAL-27 xenograft model, SsEV treatment significantly inhibited tumor growth and reduced JAGGED1, NOTCH1, and HEYL expression in tumor tissues, as confirmed by immunohistochemistry. Collectively, these findings demonstrate that SsEVs suppress OSCC progression both in vitro and in vivo by inhibiting the JAG1–NOTCH1–HEYL signaling axis, suggesting that microbiota-derived extracellular vesicles may represent a promising therapeutic approach for OSCC. Full article

Anti-CD226 antibody-mediated stimulation of NK-92 cells (sNK-92) represents a potential immunotherapeutic approach; however, its cytotoxic and apoptosis-associated effects in hormone-independent solid tumors remain insufficiently characterized. This study investigated the activity of sNK-92 cells against PC3 castration-resistant prostate cancer and SH-SY5Y neuroblastoma cell lines, using PNT1A normal prostate epithelial and BJ normal dermal fibroblast cells as non-malignant controls. Cytotoxicity was assessed by CCK-8 assay at target-to-effector (T:E) ratios of 1:1, 1:5, and 1:10, and markers historically associated with the intrinsic (BAX, caspase-9), extrinsic (caspase-8), and executioner (caspase-3) apoptotic pathways were evaluated quantitatively by ImageJ-based corrected total cell fluorescence (CTCF) analysis. sNK-92 cells produced significant ratio-dependent cytotoxicity against PC3 and SH-SY5Y cells, reaching 23.76% and 26.29%, respectively, at the 1:10 T:E ratio, with sNK-92 producing significantly greater cytotoxicity than unstimulated NK-92 at this ratio in both cell lines and additionally at the 1:5 ratio in SH-SY5Y cells; no significant reduction in CCK-8 viability was detected in PNT1A or BJ cells at any ratio. Quantitative immunofluorescence analysis demonstrated substantially increased relative fluorescence intensity of all four apoptosis-associated markers in sNK-92-treated PC3 and SH-SY5Y cells compared with their corresponding controls and, in most comparisons, with NK-92-treated cells, whereas changes observed in the PNT1A and BJ non-malignant models examined were markedly smaller. These findings provide preliminary quantitative evidence that anti-CD226-stimulated NK-92 cells exert a preferential cytotoxic effect on the tumor cell models examined, relative to the non-malignant models tested, and induce changes in apoptosis-associated markers consistent with engagement of apoptotic signaling. Further orthogonal validation is warranted. Full article

Carbon nanoparticles (CNPs) have attracted considerable attention for biomedical applications owing to their excellent biocompatibility, tunable optical properties, and low toxicity. In this study, we report a simple, rapid, and cost-effective one-pot bottom-up synthesis of fluorescent carbon nanoparticles using propanoic acid as a novel liquid carbon precursor. Unlike conventional approaches that often require high temperatures, lengthy reaction times, or complex procedures, the proposed method enables efficient nanoparticle synthesis under mild thermal conditions. The use of propanoic acid enabled carbonization under relatively mild conditions, yielding nanoparticles with excellent aqueous dispersibility. Following the synthesis of CNPs using propanoic acid as the carbon precursor, the nanoparticles were treated with ethylenediamine (EDA) as a nitrogen source and surface-passivating agent, yielding nitrogen-doped carbon nanoparticles (NCNPs). The resulting NCNPs exhibited approximately fourfold higher fluorescence intensity than the undoped CNPs. The NCNPs exhibited strong visible photoluminescence with a dominant emission band in the green spectral region (490–540 nm), which is advantageous for cellular imaging due to reduced background interference and improved imaging selectivity. Furthermore, the NCNPs demonstrated high aqueous solubility, cellular permeability, and excellent photostability under the in vitro imaging conditions investigated. The bioimaging capability of the NCNPs was evaluated using human lung fibroblasts and lung cancer cells. Confocal fluorescence microscopy revealed efficient cellular uptake and successful nuclear labeling without observable photobleaching and morphological alterations. These findings demonstrate the feasibility of using propanoic acid-derived NCNPs as fluorescent probes for cellular imaging and suggest their potential for future applications in bioimaging, biosensing, and related biomedical research. Further studies are warranted to evaluate their long-term biological safety and performance. Full article

Polyphenol oxidase (PPO) is a key enzyme responsible for enzymatic browning in fresh-cut fruits and vegetables, severely compromising their quality and shelf life. This study aimed to investigate the combined inhibitory mechanism of oxyresveratrol (OXY) and epigallocatechin gallate (EGCG) on PPO through multi-spectroscopic analyses, molecular docking, TEM, and XRD, with the goal of developing a natural and effective anti-browning strategy for fresh-cut fruits. The results showed that the optimal combined effect was achieved at an OXY:EGCG ratio of 1:2, where the inhibition rate was significantly enhanced by 43.72% and 14.36% compared to using OXY or EGCG single treatment, respectively. The combined treatment exhibited enhanced chelation capacity of copper ion and DPPH radical scavenging activity, and enhanced hydrogen-bonding interactions while lowering binding energy, exhibiting characteristics of mixed inhibition kinetics. Structural characterization showed that the combined treatment drastically reduced the enzyme’s fluorescence intensity to 39.81% of that of the native enzyme, induced rearrangements in α-helix and random coil structures, triggered obvious protein aggregation, and weakened the intensity of crystal diffraction peaks. Importantly, the combined treatment effectively delayed browning in fresh-cut pear slices, demonstrating its practical application potential. These findings provide a promising natural combined approach for controlling enzymatic browning and extending the shelf life of fresh-cut produce. Full article

Phycobiliproteins are water-soluble photosynthetic pigments extracted mainly from microalgae and cyanobacteria with many potential biotechnological applications, such as healthy food colorants, nutraceuticals, fluorescent tags, or non-toxic therapeutic agents. We have recently devised a green innovative two-step ultrasound-based process (named UltraBlu) to obtain blue phycocyanin (a phycobiliprotein) with a high-purity grade from the cyanobacterium Limnospira platensis. To assess the possibility of a wider use of the method, this study evaluates the UltraBlu process on an organism of a different taxonomic domain, the red microalga Porphyridium cruentum, for extracts rich in B-phycoerythrin (B-PE), the main phycobiliprotein produced by this organism. The UltraBlu process is characterized by the extraction of phycobiliproteins decoupled from biomass cell lysis, although the process is entirely carried out in an aqueous medium. Conversely, cell lysis is integrated with the purification step and is carried out by ultrasonication in ammonium sulfate solution before the pigment extraction/recovery step. B-PE, significantly purer than that obtained via conventional one-step direct ultrasound-assisted extraction, was recovered within a few hours from fresh biomass, only isolating the B-PE extract from the leftover biomass by centrifugation. Yields generally exceeded 9%, approaching the highest pigment contents reported for P. cruentum in the literature. Full article

Background: Pathogenic variants in SLC6A1, which encodes the γ-aminobutyric acid (GABA) transporter GAT-1, cause developmental and epileptic encephalopathies (DEEs) through reduced GABA uptake, impaired transporter trafficking, and functional haploinsufficiency. 4-phenylbutyrate (PBA) is a clinically available small molecule with chemical-chaperone and histone-deacetylase-inhibitor activities that can rescue misfolded GABAergic proteins, but variant-level rescue data are needed to guide precision treatment. Methods: We report a novel de novo missense mutation p.Ala305Val in GAT-1 encoding SLC6A1, in a patient with myoclonic-atonic epilepsy and a developmental and epileptic encephalopathy phenotype. Ala305Val was compared with the residue-matched comparator p.Ala305Thr (Ala305Thr). Variant effects were evaluated by (i) protein-structure prediction across nine stability-prediction algorithms using the cryo-EM-derived human GAT-1 template (PDB 7Y7W); (ii) 3H-GABA uptake assays in HEK293T cells and in human iPSC-derived astrocytes and cortical neurons; (iii) live-cell confocal microscopy of ER colocalization; (iv) pharmacologic rescue with PBA, TUDCA and salubrinal (v) and GAT-1 cDNA gene-augmentation, alone and in combination with PBA. Results: AI-based stability predictors uniformly indicated destabilization of GAT-1 p.Ala305Val and GAT-1 p.Ala305Thr. GAT-1 p.Ala305Val reduced 3H GABA uptake across HEK293Ts, astrocytes, and neurons. The mutant transporter accumulated within the endoplasmic reticulum (ER), with ER colocalization rising from approximately 30% in wildtype to ~80% in GAT-1 p.Ala305Val; PBA reduced ER retention to approximately ~40% and restored total GAT-1 fluorescence toward wildtype levels. Pharmacochaperones (PBA, TUDCA) restored GABA uptake for the mutant transporters. Wildtype GAT-1 gene augmentation improved GABA uptake in the heterozygous condition but combined PBA plus wildtype allele augmentation produced rescue greater than either intervention alone in the available dose-response ranges. Conclusions: GAT-1 p.Ala305Val is a trafficking-impaired, loss-of-function variant whose dysfunction is amenable to two convergent therapeutic axes: pharmacologic correction of folding and trafficking, and augmentation of functional transporter expression. These findings support a two-pronged precision-medicine framework for SLC6A1-related DEEs in which PBA increased the transporter function augmented by genetic approaches. Full article

Breast cancer cells exhibit a reversed pH gradient and metabolic plasticity that promote proliferation, invasion, and resistance to therapy. Natural products such as chlorogenic acid (CGA) and cinnamaldehyde (CA) have shown emerging anticancer potential. However, their effects on intracellular pH and metabolic transport systems remain undefined. Therefore, the aim of this study was to characterize these parameters in breast cancer and non-tumorigenic breast cells. This study evaluated the physiochemical properties of CGA and CA using LC–MS, under pH conditions (pH 1.2, 7.4, and 9.0) mimicking the gastrointestinal track (GIT). Additionally, LC–MS-based human liver microsome (HLM) assays with NADPH were used to evaluate susceptibility to CYP-mediated metabolism to evaluate first-pass metabolic stability. Intracellular uptake kinetics were quantified at multiple time points using LC–MS. Following CGA:CA treatment, intracellular pH (pH_i) was measured in cancerous MDA-MB-231 and non-tumorigenic MCF-10A breast cell lines using SNARF-1 targeted ratio-metric fluorescence approach. Expression of OATP1B1, GLUT1, and MCT1 were analyzed by Western and immunofluorescence respectively, to assess potential cellular uptake of CGA:CA through OATP1B1 and their effects on glucose uptake and lactate and proton transport. Physiochemical results demonstrated that the compounds ranged from fully stable (pH 1.2 and 7.4) to completely unstable (pH 9.0). HLM incubation indicated no CYP-mediated hepatic metabolism. Treatment results showed that there was rapid intracellular uptake of CGA and CA in cancer cells and that CGA:CA lowered pH_i in both MDA-MB-231 and MCF-7 cells, while pH_i remained mostly unchanged in MCF-10A cells. Protein analysis revealed that CGA:CA treatment downregulated GLUT1 and MCT1 expression in cancer cells, suggesting impaired glycolytic activity and lactate shuttling. OATP1B1 expression was significantly suppressed in cancer cells, suggesting feedback inhibition of the solute carrier protein. Collectively, these findings indicate that CGA and CA exhibit favorable biochemical stability and disrupt intracellular pH regulation and metabolic transporter expression in breast cancer cells. Importantly, normal cells are not significantly affected. Thus, CGA:CA demonstrates therapeutic potential for breast cancer through pH_i and metabolic modulation. Full article

Macrophages are highly plastic innate immune cells that integrate diverse microenvironmental cues to adopt pro-inflammatory (M1) or anti-inflammatory (M2) functional states, which critically influence the pathogenesis of infectious, autoimmune, inflammatory, and malignant diseases. This review provides an overview of current concepts of macrophage ontogeny, functional heterogeneity, and disease-associated phenotypes, with a specific focus on experimental approaches used as M1/M2 test systems for macrophage phenotyping. The scope of the review encompasses commonly used experimental models, induction protocols for M1- and M2-like polarization, and key readouts, including gene-expression signatures and metabolic parameters. Particular emphasis is placed on reporter-based platforms (luciferase, BRET, fluorescent nanoparticle probes), label-free biophysical methods such as electrical impedance monitoring and metabolic profiling, and their application to dynamic, real-time assessment of macrophage phenotype in the context of tumor microenvironments and chemotherapeutic exposure. The potential of integrating reporter systems with single-cell omics, spatial transcriptomics, and patient-derived ex vivo platforms is considered, with a view to transforming M1/M2 test systems into clinically oriented assays capable of tracking macrophage programs during therapy and supporting the development of macrophage-targeted diagnostics and treatments. Full article

Objectives: Initial caries lesions (ICLs) are the earliest manifestation of dental caries, characterized by subsurface porosity that affects enamel’s optical properties while maintaining its surface integrity. Resin infiltration is a minimally invasive approach to managing these lesions by penetrating and sealing pores with low-viscosity resin, potentially modifying their visual appearance. In order to provide a quantifiable, objective assessment of this optical behavior, this study aimed to evaluate the modulation of color coordinates and VITA Classical shade transitions within the initial lesions immediately following infiltration with Icon Smooth Surface (DMG, Hamburg, Germany). Materials and Methods: Forty-nine extracted human premolars with naturally occurring proximal ICLs (subsurface demineralization) were selected. Lesions were assessed for fluorescence level using 655 nm laser fluorescence (DIAGNOdent pen). Reflectance spectrophotometry (SpectroShade Micro) was employed to record color coordinates in the CIELab and CIELCh spaces, alongside VITA Classical shades, before and after resin infiltration. Results: The infiltration procedure resulted in a statistically significant increase in b* (yellowness) and subsequent C* (chroma) parameters (p < 0.05). VITA Classical shade guide analysis showed an insignificant shift in color distribution according to value (p > 0.05). Conclusions: The present exploratory study provides quantitative insights into the immediate color-modifying effects of a resin infiltration procedure on natural initial proximal caries lesions, complementing the existing literature with objective CIELab/CIELCh and VITA Classical measurements. The p-values should be interpreted as descriptive indicators of potential trends rather than definitive statistical proof. Further research with larger cohorts and long-term follow-up is needed to validate the durability and clinical relevance of these esthetic outcomes. Full article

Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis–Menten constants (K_M) and reaction rates (V_max) were calculated using nonlinear regression. For both enzymes, K_M values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. K_M values for fully methylated duplexes were in the range of (4.4–7.0)·10² nM for GlaI and 2.4–55 nM for BlsI. K_M values were significantly lower for the hemimethylated duplexes: (1.9–8.0)·10² nM for GlaI and (0.7–15.0)·10² nM for BlsI; and even lower for unmethylated duplexes: (27–46)·10² nM for GlaI and (0.28–23)·10² nM for BlsI. Maximum reaction rates varied within relatively narrow limits: V_max values were in range (4.5–19.5)·10⁻⁴ nM/s for GlaI and (1.4–18)·10⁻⁴ nM/s for BlsI, respectively. V_max values were depended weakly on the degree of methylation compared to the K_M. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels. Full article

Background: Microtubules play a crucial role in neuronal structure and function, with their stability and dynamics regulated by posttranslational modifications (PTMs) such as polyglutamylation. In Alzheimer’s disease (AD), the microtubule-associated protein TAU becomes mislocalized into the somatodendritic compartment (‘TAU missorting’), dissociates from microtubules, aggregates into neurofibrillary tangles, and contributes to microtubule destabilization and neuronal death. Objectives and Methods: Here, we investigated the role of tubulin tyrosine ligase-like proteins (TTLLs) in TAU missorting and microtubule dysregulation using human-induced pluripotent stem cell (hiPSC)-derived cortical neurons treated with oligomeric amyloid-beta (oAβ) to replicate AD-like conditions. TTLL1, TTLL4, and TTLL6 were selectively knocked down (KD) to assess their impact on TAU missorting and microtubule stability. Fluorescence resonance energy transfer (FRET) microscopy was used to examine proximities between TAU and TTLL proteins. Results: We observed TAU missorting, increased tubulin polyglutamylation, decreased tubulin acetylation associated with microtubule destabilization, and synaptic declustering in oAβ-treated neurons. TTLL1 KD significantly reduced TAU missorting, tubulin polyglutamylation, and synaptic disintegration, while TTLL4 KD showed moderate effects, and TTLL6 KD restored microtubule acetylation. Importantly, TTLL KD did not impair neuritic networks, dendritic complexity, or neuronal activity. FRET microscopy in HEK293T cells revealed a close molecular proximity between TAU and TTLL1 consistent with a potential direct or complex-mediated association, but not with other TTLLs, suggesting a direct role of TTLL1 in TAU-mediated toxicity. Conclusions: Our findings identify TTLL1 as a promising therapeutic target for limiting TAU-associated cytoskeletal pathology in AD. These results support further development of pharmacological or genetic strategies targeting TTLL1 as a disease-modifying approach for AD and related tauopathies. Full article

Food spoilage along the supply chain represents a major global challenge, contributing to economic losses, environmental impacts, and food safety concerns. Graphene-based materials have emerged as promising platforms for real-time food freshness monitoring owing to their high surface area, electrical conductivity, chemical sensitivity, and compatibility with flexible sensing architectures. This review critically examines graphene-based sensing strategies for food freshness and spoilage monitoring, including chemiresistive, dielectric, field-effect, optical/fluorescence, photoelectrochemical, mass-sensitive, and colorimetric approaches. Representative sensing platforms are compared in terms of analytical performance, including detection range, limit of detection, selectivity, response and recovery times, calibration, reproducibility, and stability. Particular attention is given to machine-learning-assisted sensing, multifunctional platforms for temperature, humidity, and gas monitoring, and the challenges associated with real-world implementation, including environmental interference, sensor fouling, signal drift, long-term stability, and cross-matrix validation. Finally, commercialization readiness, integration into intelligent packaging, and safety and regulatory considerations related to graphene-based food-contact applications are discussed. Overall, the review identifies the main technological gaps and future research priorities toward robust, scalable, and practical graphene-based systems for real-time food freshness monitoring. Full article

Fluorescence-based immunoassays are widely used for sensitive and specific biomarker detection; however, further improvements in sensitivity remain desirable for detecting low-abundance analytes without increasing assay complexity. In this work, we present a proof-of-concept demonstration of guided electrokinetic assembly of functionalized latex beads as a post-assay signal enhancement strategy using alternating-current electro-osmosis (ACEO). Carboxyl-modified 1 μm polystyrene beads conjugated with Alexa Fluor 647-labeled anti-IgG were localized within lithographically defined windows on carbon interdigitated electrode arrays, producing localized fluorescence enhancement through physical bead localization without enzymatic amplification or additional labeling chemistries. Compatibility of the approach with fluorescence-based immunoassays was demonstrated through adaptation of a TNF-α ELISA workflow. Electro-osmotic localization of functionalized bead conjugates was achieved within 120 s, producing an approximately 12-fold increase in corrected total fluorescence relative to the corrected signal of the pre-electro-osmosis condition while demonstrating negligible enrichment of unbound fluorescent protein. Application of the platform to a TNF-α sandwich immunoassay yielded an approximately 5.5-fold enhancement in fluorescence signal, and robust bead localization was maintained across anti-IgG concentrations ranging from 1 to 4 μg/mL. These findings demonstrate that guided electrokinetic bead localization provides an effective signal enhancement strategy for fluorescence-based immunoassays and represents a promising approach for improving the detection of low-abundance analytes. Full article