Search Results (6,211)

Rice protein has limited gelation properties, restricting its food applications. This study added four polyphenols—catechin (C), epicatechin (EC), tannic acid (TA), and proanthocyanidins (PC)—to rice protein to investigate their effects on gel rheology, in vitro digestibility, and microstructure. Multi-spectroscopy and molecular docking were used to explore interaction mechanisms. During the temperature sweep (95 °C), PC- and TA-composite gels (GRP-PC, GRP-TA) showed storage moduli slightly higher than the pure rice protein gel (GRP), while GRP-C and GRP-EC (C- and EC-composite gels) were similar to GRP. In frequency sweep (25 °C), GRP had the highest modulus, followed by GRP-PC > GRP-TA > GRP-EC > GRP-C. Polyphenols reduced total digestibility (from 77.4% to 67.6–75.2%). All polyphenol-complexed gels showed markedly improved ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activities. C and EC induced loosely crosslinked microstructures, whereas TA and PC formed sheet-like aggregates. Fluorescence quenching was predominantly static, with quenching rates TA > PC > EC > C. Binding constants followed the same order. Thermodynamic parameters (ΔH > 0, ΔS > 0, ΔG < 0) indicated hydrophobic interactions as the driving force. Molecular docking revealed that PC formed the most hydrogen bonds (8) with rice glutelin, followed by TA (4), C (5), and EC (3). These findings provide data support for designing rice protein-based functional foods. Full article

Currently, the most commonly used methods for detecting Mycoplasma are the culture method and the indicator cell culture method. However, both approaches exhibit low sensitivity and are incapable of detecting low-concentration contamination. In addition, the detection period may extend up to 28 days, which is unsuitable for rapid screening and may delay timely contamination control measures. To address these limitations, a Mycoplasma detection method based on nucleic acid amplification technology (NAT) was developed following a comparative analysis of gene sequences from various Mycoplasma species. The method was validated with respect to its detection performance and its applicability to biological product samples. DNA was extracted from Mycoplasma-contaminated samples using a magnetic bead-based nucleic acid extraction method. Universal primers were designed based on the highly conserved 16S rRNA gene sequence of Mycoplasma, and amplification was performed using multiplex quantitative PCR (qPCR) with fluorescent probes. The limit of detection (LOD) was established based on statistics of 24 replicates. Method specificity and robustness were evaluated according to the guidelines set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2), while sample applicability was assessed in accordance with the European Pharmacopoeia (EP) <2.6.7>. The NAT-based Mycoplasma detection method enabled rapid, qualitative identification of Mycoplasma contamination. The validated LOD was 10 CFU/mL, and the method met predefined requirements for sensitivity, specificity, and robustness. To assess applicability, real biological product samples, including monoclonal antibodies, antibody fusion proteins, bispecific antibodies, and trispecific antibodies, were spiked with 10 CFU/mL of standard Mycoplasma strains. All spiked samples tested positive. These findings confirm that the NAT-based Mycoplasma detection method is suitable for process control and product release testing in the production of biological products. Full article

Pregabalin (PGB) exerts its therapeutic effects by binding to the α₂δ auxiliary subunits of voltage-gated calcium channels and modulates synaptic transmission in the brain. However, its influence on cerebellar parallel fiber–Purkinje cell (PF–PC) synaptic transmission remains unclear. In the present study, we investigated the effects of PGB on PF–PC synaptic transmission using whole-cell patch-clamp recording, glutamate fluorescence imaging, immunohistochemistry, co-immunoprecipitation, Western blotting, and pharmacological approaches. Micro-application of PGB to the cerebellar molecular layer induced a concentration-dependent inhibition of PF–PC excitatory postsynaptic currents (EPSCs), accompanied by an increased paired-pulse ratio. The inhibitory effect of PGB on PF–PC EPSCs was abolished by extracellular blockade of N-methyl-D-aspartate receptors (NMDAR) or their GluN2A subtype, as well as by disruption of α₂δ-1–NMDAR complexes, but not by intracellular NMDAR inhibition. Glutamate sensor imaging further showed that PGB markedly reduced the fluorescence intensity of glutamate release evoked by PF stimulation. In the presence of tetrodotoxin (TTX) and a gamma-aminobutyric acid type A (GABAA) receptor antagonist, PGB reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude. The PGB-induced reduction in mEPSC frequency was fully abolished by extracellular blockade of GluN2A-containing NMDARs or disruption of α₂δ-1–NMDAR complexes. Similarly, the inhibitory effects of PGB on PF–PC EPSCs and mEPSCs were eliminated by extracellular PKA inhibition, but not by intracellular protein kinase A (PKA) inhibition. Western blot analysis showed that PGB significantly increased PKA phosphorylation in the molecular layer of the cerebellar cortex. Immunoreactivity for GluN2A and α₂δ-1 subunits was colocalized within the molecular layer and abundantly distributed around the dendrites and somata of PCs. Co-immunoprecipitation further verified that α₂δ-1 was co-precipitated with GluN1 in cerebellar molecular layer tissue samples. The results indicate that PGB depresses glutamate release from parallel-fiber terminals in the mouse cerebellar cortex through the presynaptic α₂δ-1-coupled GluN2A-containing NMDAR/PKA signaling pathway, thereby attenuating PF–PC synaptic transmission. Full article

Peanut (Arachis hypogaea L.) production faces persistent threats from various infectious diseases. Planting healthy varieties with robust botanical defense networks is critical for minimizing future costs. Non-expressor of pathogenesis-related (NPR) regulators are involved in immune activation and act as key targets for deeper stress adaptation, and are thus promising targets for genetic enhancement. In this study, we characterized the peanut NPR4B protein and demonstrated its local subcellular binding to the nucleus. Ectopic overexpression of AhNPR4B in Arabidopsis thaliana significantly enhanced resistance to the necrotrophic pathogen Botrytis cinerea and enhanced cold tolerance, as supported by quantitative and statistical analyses (p < 0.05). As regards underlying molecular events, Y2H (Yeast 2-Hybrid) analysis revealed a binding in vitro physical relation of AhPR2-like to AhNPR4B. This binding was demonstrated in vivo through BiFC (Bimolecular Fluorescence Complementation). These results suggest that the AhNPR4B-AhPR2-like complex may act as a key regulatory module associated with biotic and abiotic stress signaling, potentially contributing to broad-spectrum stress resistance. These findings provide foundational insights into the functional roles of AhNPR4B and its interaction with AhPR2-like in regulating stress resistance and support its potential as a candidate target for future genetic improvements to enhance stress resilience in peanuts. Full article

Background/Objectives: The present study aims to test three different types of PMMA (Fotodent Guide—3D printed (M1), Aidite Temp—milled (M2), Duracryl—self-polymerized (M3) on HFIB-G and on OECM-1. Methods: The two cell types (HFIB-G and OECM-1) were kept in contact with the materials, Fotodent Guide, Aidite Temp, and Duracryl (n = 6), for 24 and 48 h, and subsequently subjected to the following tests: MTT, LDH, NO (according to ISO 10993-5:2009), and immunofluorescent detection of proteins associated with autophagy and apoptosis (mitochondria and caspases 3/7; detection of autophagosomes). Statistical interpretation was made using t-test and ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001). Results: The MTT assay revealed a reduction in cell viability for all tested materials on gingival fibroblasts compared to control cells, with the most pronounced decrease observed for the 3D-printed material (M1 viability 66.77% for 24 and 52.45% 48 h—p < 0.001), while the self-polymerizing resin (M3 viability 85.92% for 24 h and 85.63% for 48 h) showed the highest level of cellular tolerance (p < 0.001 at 24 h and p < 0.01 at 48 h). Regarding OECM-1 cells, all materials reduced cell viability, particularly M3 after 48 h of incubation (viability 61.79%—p < 0.001). LDH levels generally indicated low membrane damage for all materials. Statistically significant increases in NO levels were recorded for both cell types, suggesting a mild proinflammatory response, especially for M2 OECM-1 48 h—p < 0.05 and M3 (HFIB-G 48 h—p < 0.05, OECM-1 48 h p < 0.05). For both 24 and 48 h, fluorescence analysis demonstrated a significant increase in mitochondrial activity in gingival fibroblasts (p < 0.001), whereas tumor cells exhibited a significantly decreased mitochondrial activity (p < 0.001), particularly for the 3D-printed material M1 (p < 0.001). Caspase-3/7 expression increased in gingival fibroblasts incubated with materials for 24 and 48 h (p < 0.001), while tumor cells showed reduced caspase activity both after 24 and 48 h (p < 0.001). Autophagosome formation decreased initially in fibroblasts at 24 h (p < 0.001) but increased significantly after 48 h (p < 0.001), while tumor cells generally showed enhanced autophagic activity under most experimental conditions (p < 0.001). Conclusions: Our results suggest that all three PMMA-based materials exhibit acceptable biocompatibility profiles, of more than 70%, according to ISO 10993-5:2009, although cellular responses vary depending on the manufacturing technique and the cellular model used. In our study conditions, self-polymerized resin (M3) was the most compatible with gingival fibroblasts, while the 3D-printed and CAD/CAM milled materials (M1 and M2) had a more pronounced impact on cells’ viability and metabolic activity. Full article

Interactions between resveratrol and biological or carrier systems play a key role in determining its bioavailability, stability, and delivery performance. These interactions involve proteins, lipids, cyclodextrins, nucleic acids, polysaccharides, and other formulation matrices, and are governed by noncovalent forces such as hydrogen bonding, hydrophobic interactions, π–π stacking, and desolvation effects. This review examines how complementary spectroscopic, calorimetric, structural, and computational techniques are used to characterize resveratrol interactions. Fluorescence, UV–visible spectroscopy, circular dichroism, FTIR, NMR, ITC, DSC, X-ray diffraction, molecular docking, and molecular dynamics simulations are discussed according to their contribution to binding analysis, conformational assessment, thermodynamic interpretation, structural organization, and complex stability. By integrating these approaches, this review provides a technique-oriented framework for understanding resveratrol binding and guiding the development of more stable resveratrol-based carrier systems and bioactive formulations. Full article

The present study aimed to compare the composition structure, interfacial, and antioxidant activities of flaxseed protein isolates (FPIs) in different flaxseed varieties. The results showed that apparently intact protein bodies (PBs) were manifested as densely staining cytoplasmic inclusions with distinct boundaries and varying diameter ranges among different flaxseed varieties. Through alkali extraction with isoelectric precipitation, FPIs exhibited a relatively small and irregular lamellar strip structure with varying sizes and shapes packed with spherical particles in studied flaxseed varieties. The different composition structures of FPIs among studied flaxseed varieties were also obtained, involving the protein subunits’ intrinsic fluorescence properties, secondary structures, and amino acid profiles. These structural differences also led to differential purities, aqueous solubility, dispersion properties, and surface charges. Moreover, the varying emulsifying and foaming properties of FPIs from different flaxseed varieties were also observed due to the formation of coarse lipid droplets (5~40 μm) and foams (20~100 μm) with the specific structure of the oil/gas–water interface and bulk aqueous phase. The retention of phenolic compounds into FPIs still displayed evident variety specificity from 323 to 478 mg/100 g and 210 to 347 mg/100 g, which definitely led to escalated antioxidant activities. Thus, FPIs from Longya 13# and Neiya 9# flaxseed varieties were screened for favorable emulsifying and foaming properties due to the balanced molecular rigidity/unfolding and interfacial adsorption/stabilization behavior. Full article

SUMOylation is a rapid and dynamic process that orchestrates the switch between complex assembly and disassembly and between protein stabilization and turnover, making it particularly suitable for regulating stress responses. While proteomic methodologies exist for analyzing SUMOylated proteins under stress conditions, methods/tools for visualizing polySUMOylation dynamics have not been established. Here, we develop a polySUMOylation tracking tool by fluorescently labeling the polySIM domains derived from RNF4, which can reliably track polySUMO location and relate polySUMOylation levels to puncta number and intensity under various stress conditions, such as serum starvation, oxidative stress, and genotoxic stress. Furthermore, we extend its utility for tracking polySUMOylation across multiple cellular contexts in both control and stressed states. Collectively, this tracking tool enables deeper investigation of polySUMOylation dynamics and advances our understanding of how polySUMOylation regulates cellular processes in stress responses and disease pathogenesis. Full article

Introduction: Glioblastoma (GBM) is the most aggressive primary tumor of the central nervous system and is highly resistant to temozolomide (TMZ). Rutin is a potent antioxidant with immunomodulatory and anti-glioma effects in vitro, although its mechanisms of action remain incompletely understood. This study investigated the effects of rutin on morphology, viability, redox balance, and pro-tumoral signaling in GBM 2D cultures and 3D spheroids, as well as its association with TMZ sensitivity. Methods: GL15 and U343 human GBM cell lines and primary astrocytes were treated with rutin (5–30 μM) and/or TMZ (125–4000 μM). Cell metabolic activity and viability were assessed by MTT, PI/DiOC18(3) or PI/Hoechst. Cell migration was assessed from spheroid-derived cells, and extracellular matrix (ECM) components (fibronectin and laminin) were evaluated by immunofluorescence. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA fluorescence. IL-6, STAT3, NOS2, and IDO1 gene expression were determined by RT-qPCR, and protein expression of MMP2, fibronectin, STAT3, PI3K, and AKT by Western blotting. Nitric oxide (NO) and L-kynurenine levels were quantified in the supernatant by colorimetric assays. Results: Rutin reduced cell viability and enhanced TMZ cytotoxicity in both 2D and 3D cultures, while exerting selective effects by increasing metabolic activity and attenuating TMZ-induced effects in non-tumoral primary astrocytes. In 3D spheroids, rutin affected structural organization and reduced spheroid-derived cell migration, accompanied by changes in ECM components, including MMP2, fibronectin, and laminin. Rutin decreased intracellular ROS levels and suppressed the TMZ-induced increase in ROS and NOS signaling. These effects were accompanied by modulation of IL-6/STAT3 signaling, along with reduced STAT3, PI3K, and AKT protein levels. Rutin also modulated immunometabolic parameters, including extracellular L-kynurenine and nitric oxide levels, and enhanced TMZ responsiveness following pre-sensitization. Conclusions: Rutin enhances TMZ responsiveness by modulating interconnected pro-tumoral mechanisms, including redox balance, pro-survival signaling, ECM remodeling and migratory behavior, and immunometabolic pathways linked to chemoresistance, supporting its potential as an adjuvant therapeutic strategy. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating processes such as assembly, genome packaging, and virion maturation. Despite their biological importance, these interactions remain challenging to study and are relatively underexploited as therapeutic targets. Split reporter systems, based on protein-fragment complementation, provide quantitative platforms to measure PPIs by reconstituting reporter activity when interacting protein partners are brought into proximity. These systems can be applied in vitro and in live cells which enables detection of dynamic and multimeric interactions in physiologically relevant contexts. Major classes of split reporter systems include β-lactamase, alkaline phosphatase, luciferase-based platforms, green fluorescent protein, and horseradish peroxidase. Assay performance depends on factors such as fusion protein stability, expression levels, and reporter kinetics, which influence sensitivity, dynamic range, and reliability. These approaches have been applied to study viral protein interactions across diverse systems, including HIV-1 matrix and nucleocapsid proteins, flaviviral capsid proteins, hepatitis B virus core protein, and chikungunya virus capsid. Split reporter assays also enable high-throughput screening for small-molecule inhibitors that disrupt viral PPIs and multimerization. This provides a functional readout linked to viral replication. Despite the challenges that exist in assay optimization and protein stability, the sensitivity and versatility of these systems provide a framework to interrogate viral protein interactions and support the development of antiviral therapeutics.: Full article

The increasing fragmentation of plastic debris into nanosized particles represents a threat to freshwater ecosystems, yet the biological effects of nanoplastics (NPs) on freshwater invertebrates remain poorly understood. This study investigated tissue distribution, cellular effects and immune responses following acute exposure to polyethylene terephthalate nanoplastics (PET-NPs) in the freshwater gastropod Pomacea canaliculata, a species of high ecological relevance and physiological resilience. Adult snails were injected with PET-NPs at 5 or 10 mg/L and sampled after 24 and 72 h. PET-NPs accumulation in the anterior and posterior kidneys was assessed by fluorescence imaging and tissue morphology was evaluated. Stress- and inflammation-related genes (Pc-Heat Shock Protein (HSP)70, Pc-HSP90 and Pc-Allograft inflammatory factor 1) expression was quantified by RT-qPCR. PET-NPs uptake and phagocytic activity were analyzed in circulating hemocytes in vivo and ex vivo. PET-NPs were accumulated in renal tissues, persisting up to 72 h without histopathological alterations. Gene expression analyses revealed non-linear and dose/time-dependent responses. Hemocytes of different morphologies internalized PET-NPs in a dose-dependent manner and showed intercellular particle transfer. Overall, acute PET-NP exposure determines rapid immune handling and tissue sequestration with limited short-term physiological impact, underscoring the potential involvement of immune processes in NPs fate and highlighting the need for chronic exposure studies. Full article

Choy Sum (Brassica rapa subsp. chinensis var. parachinensis), also known as flowering Chinese cabbage, is an important stalk vegetable in Asia. However, the unique regulatory mechanism governing its “easy-bolting yet susceptible to premature bolting” trait remains poorly understood. The phosphatidyl ethanolamine-binding protein (PEBP) family serves as a central regulator of bolting, flowering, and growth development in plants. But this gene family has not been systematically identified and studied in Choy Sum yet. Therefore, this study systematically identified and analyzed the members of the PEBP gene family in Choy Sum using bioinformatics, transcriptomics, real-time fluorescence quantification, subcellular localization, and transgenic techniques. A total of 12 BcPEBP genes were identified and categorized into three subfamilies: FT-like, TFL1-like, and MFT-like. Phylogenomic analyses revealed family expansion through whole-genome duplication with strong purifying selection. Most members have highly conserved core motifs and gene structures. Protein sequence alignment showed that BcFT-2 and BcTFL-2 underwent non-synonymous mutations at key residues. The analysis of cis-acting elements suggests that the BcPEBP gene may be influenced by complex hormone and light regulatory networks. Expression profiling demonstrated leaf-specific upregulation of BcFT-1/2 during development and shoot apices-predominant expression of BcTFL1 genes, and the expression between homologous genes of BcTFL1-1/3 is more refined. Subcellular localization confirmed dual nuclear and plasma membrane targeting of BcFT-1/2 proteins. Overexpression of BcFT-1/2 in transgenic Arabidopsis promotes flowering. These findings establish BcPEBP genes as key bolting regulators and provide molecular targets for breeding-improved varieties. Full article

Despite regulations restricting asbestos use in many developed countries, asbestos-containing materials (ACMs) persist in aging buildings and can release airborne fibers during demolition and renovation. Therefore, continuous monitoring of airborne asbestos fibers is essential for environmental safety and exposure assessment. Fluorescence microscopy (FM) with fluorescently labeled asbestos-binding proteins offers greater sensitivity and selectivity in detection compared with conventional phase contrast microscopy (PCM). However, its practical application is limited by manual sample preparation and microscopic observations. This study introduces the conceptual design and initial development of an automated FM-based sensing system for monitoring airborne asbestos fibers. The system was constructed by modifying a commercial PM_2.5 continuous air sampling platform and integrating automated fluorescent staining, FM imaging, and AI-assisted image analysis for fiber recognition and counting. The system automatically reports airborne asbestos concentrations with corresponding fluorescence images and advances the membrane filter to enable continuous measurements. Performance evaluation using pulverized ACMs showed an overall agreement within 14.1% with PCM–scanning electron microscopy measurements at the group level. Although variability was observed at low fiber concentrations owing to stochastic sampling effects, the results validate the feasibility of automated FM-based sensing for continuous environmental monitoring of airborne asbestos fibers. Full article

Background/Objectives: Advanced renal cell carcinoma (aRCC) remains incurable, with no established optimal sequence of targeted therapies due to interpatient heterogeneity and acquired resistance. We developed a luciferase-enabled patient-derived orthotopic xenograft (PDOX) avatar platform to evaluate sequential targeted therapies in individualized aRCC models that recapitulate tumor architecture, proliferation, angiogenesis, metastasis, and PD-L1 expression. Methods: Tumor specimens from two renal cell carcinoma (RCC) patients were expanded subcutaneously in NOD/SCID mice, transduced with luciferase/red fluorescent protein (Luc/RFP), and orthotopically implanted into mouse kidneys (KiCa-Pt58: sarcomatoid RCC, pT3aN1M1, Fuhrman grade 4; KiCa-Pt118: clear cell RCC with sarcomatoid component, pT3aNxM0, Fuhrman grade 4, respectively). Tumor growth and metastasis were monitored weekly by bioluminescence imaging (BLI). Mice were randomized into vehicle control or four sequential treatment groups (Everolimus→Sunitinib [E→S], Sunitinib→Everolimus [S→E], Pazopanib→Sunitinib [P→S], Pazopanib→Everolimus [P→E]). Drugs were administered orally three times weekly until resistance (>200% BLI increase), with one switch. At necropsy, tumor burden, ex vivo BLI metastasis, weights, H&E histology, and immunohistochemistry (Ki67, CD44, CD31, PD-L1) were assessed. Results: Two independent experiments were performed. In dosing optimization, PDOX tumors recapitulated parental histology and proliferative indices, mirroring patient trajectories. KiCa-Pt58 (metastatic sarcomatoid RCC; deceased 1-month post-nephrectomy) showed aggressive features: rapid engraftment at low doses, early growth (week 2), and lung metastases in 78% of mice (sacrifice day 34), reflecting a fulminant course. KiCa-Pt118 (non-metastatic; patient recurrence-free >8 years post nephrectomy) exhibited indolent behavior: delayed engraftment requiring higher doses plus lymph node stromal (HK) support, slower growth (week 4), no metastases, and later sacrifice (day 78), consistent with remission. In sequential therapy evaluation, for KiCa-Pt58, P→E yielded greatest reductions in tumor weight (p < 0.01), lung metastases (p < 0.01), Ki67+ proliferation, CD31+ angiogenesis, and PD-L1 expression versus control; E→S and S→E were also effective. For KiCa-Pt118, S→E and P→E reduced tumor burden (p < 0.01) and Ki67⁺ proliferation; S→E lowered CD31 and PD-L1. Conclusions: This RCC PDOX platform faithfully preserves patient-specific biology—including metastatic propensity, engraftment efficiency, growth kinetics, and stromal dependency—while enabling real-time evaluation of sequential targeted therapies. Given the limited number of models tested, these findings provide proof-of-concept for individualized treatment exploration in advanced RCC and support future investigation of rational combinations with immune checkpoint blockade in humanized or immunocompetent systems. Full article

Improving the water-holding capacity (WHC) during the processing of rabbit meat can effectively enhance the texture of the final product, but it remains a practical challenge. This study aims to develop an ultrasound-assisted curdlan curing strategy to reduce the water loss of rabbit meat during the processing. Herein, the water retention performance, myofibrillar protein (MP) structure, and processing adaptability of rabbit meat as affected by the ultrasound-assisted curdlan curing treatment were investigated. Compared with the control group, ultrasound-assisted curdlan treatment increased WHC by 14.0% and reduced cooking loss by 15.4%. Moreover, this combined treatment showed significantly higher WHC and lower cooking loss than curdlan or ultrasound treatment alone (p < 0.05). Moreover, the ultrasound-assisted curdlan curing resulted in higher ultraviolet absorption and fluorescence intensity of myofibrillar proteins (MPs) in rabbit meat, but the intensity of the main protein band observed in SDS-PAGE was lower. Furthermore, the rabbit meat treated with the ultrasound-assisted curdlan curing maintains the highest water content (75.2% for steaming, 74.7% for boiling, 74.4% for microwaving, 70.1% for roasting, and 71.8% for air-frying) under various thermal processing methods. Therefore, the ultrasound-assisted curdlan curing offers a feasible route to improve water retention in rabbit meat, providing an applicable basis for reducing water loss in meat production. Full article