Search Results (419)

Human serum albumin (HSA), an endogenous protein, was employed in the synthesis of nanoparticles. The treatment of an HSA solution with ethanol and glutaraldehyde resulted in the formation of human serum albumin nanoparticles (HSA NPs), which exhibited a weak fluorescence emission peak at 515 nm upon excitation at 360 nm. Importantly, these synthesized HSA NPs displayed a pronounced fluorescence polarization (FP) signal under identical excitation and emission conditions. Furthermore, incubation of the HSA NPs with specific DNA aptamers targeting lysozyme and thrombin led to a significant decrease in the FP values. This reduction in FP was effectively reversed upon the addition of lysozyme and thrombin. Based on these observations, a label-free fluorescence polarization-based detection platform for lysozyme and thrombin was developed utilizing HSA NPs and a DNA aptamer system. Full article

Cervical cancer (CCa) is the fourth leading cause of cancer-related deaths among women worldwide, with nearly 90% of cases in low- and middle-income countries, especially in Sub-Saharan Africa. This study explores the roles of circular ribonucleic acids (circRNAs), hsa_circ_0001038 and circRNA_400029, and the impact of the serine/arginine-rich splicing factor 3 (SRSF3) inhibitor, theophylline, in CCa cell lines. We utilized cell cycle fluorescence-activated cell sorting (FACS) and Annexin V/propidium iodide (PI) assays to evaluate theophylline’s effects on SiHa and C33A cell lines. Results showed S-phase arrest in SiHa and G2/M arrest in C33A, with significant cytotoxic effects indicated by apoptosis analysis. Using CircAtlas, we identified micro ribonucleic acids (miRNAs) binding to hsa_circ_0001038, particularly miR-205-5p, which has a tumour-suppressive role. miRTarBase identified miR-16-5p as a key interacting miRNA for circRNA_400029. We constructed a competing endogenous ribonucleic acid (ceRNA) network, revealing multiple miRNA targets. Pathway analysis via the Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted critical signalling pathways involved in CCa oncogenesis. In conclusion, theophylline demonstrates cytotoxicity in CCa cells, suggesting its potential for repurposing in CCa theranostics, though further optimization is necessary. Full article

Background/Objectives: Extracellular Vesicles (EVs) have shown great promise as diagnostic and therapeutic tools, as well as pharmacological nanocarriers. Various strategies are being explored to develop EVs for monitoring, imaging, loading with pharmacological agents, and surface decoration with tissue-specific ligands. EVs derived from Mesenchymal Stromal Cells (MSC-EVs) are of particular interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this aim, canine MSCs were engineered to express the Green Fluorescent Protein (GFP) fused to CD63, Syntenin-1, TSG101, and the palmitoylation signal of Lck, which were expected to promote GFP incorporation into EVs. Overexpression of tagged GFP in canine MSCs was confirmed by Western blotting and examined by confocal microscopy and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed relatively high loading efficiency and secretion index but exhibited a diffuse localization pattern in the transfected cells. The palmitoylation signal showed low loading efficiency and localization specificity. TSG101 displayed a morphological pattern consistent with specific localization in endosomal structures, but its low expression level prevented further evaluations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 levels were approximately 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 emerged as the most suitable tag for canine MSC-EV engineering. Indeed, even if the secretion index favours Syntenin-1, CD63’s higher abundance in the lysate suggests its substantial post-secretion uptake. Further studies aimed at elucidating CD63’s specific contribution and identifying the domains involved in vesicle trafficking could provide valuable insights into EV bioengineering. Full article

With increasing attention to health, plant protein products have gained significant market potential due to their growing consumer demand. This study researches the influence of ultrasonic treatment on the structure and function of pea–oat composite protein gel (POPG) to enhance its elasticity and thermal stability. The ultrasonic treatment parameters were regulated to power (200–600 W for 30 min) and ultrasonic time (20–40 min at 400 W) during the preparation of POPG, and the properties and structure, including gel strength, rheological analysis, water-holding capacity (WHC), thermal characteristics, fluorescence performance, and microstructure, were further evaluated. The results showed that the POPG samples exhibited optimal values in WHC, gel strength, surface hydrophobicity, free sulfhydryl amount, and endogenous fluorescence at 400 W ultrasonic for 30 min compared with the untreated POPG. Rheological analysis indicated that POPG displayed the highest storage modulus and improved viscoelasticity. Ultrasonication resulted in an augmentation in β-sheet content, hence creating a more compact network structure. DSC and TGA revealed improved thermal stability, while SEM and CLSM exhibited a homogeneous and firm gel structure of POPG. This research offers the theory that ultrasonic technology can improve the performance of plant-based composite gels. Full article

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells during which parts of the cytoplasm are sequestered and subsequently delivered into lysosomes for degradation by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating the regulation and mechanism of autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. In addition, starvation-induced autophagy has not been characterized in detail using these reporters. Here, we describe the development of two endogenous autophagy reporters, gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. We demonstrate that these single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we reveal that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event because the vast majority of the detected fluorescent signals are autolysosome-specific, (ii) starvation triggers autophagy only after a measurable lag phase rather than immediately, and (iii) the regulation of starvation-induced autophagy depends on the actual life stage, and prevents excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to further dissect the physiological and pathological roles of autophagy in vivo. Full article

Background/Objectives: Urinary angiotensin-converting enzyme (uACE) activity has long been regarded as a promising biomarker for kidney and cardiovascular diseases; however, its clinical applicability has been limited by the presence of endogenous urinary inhibitors and technically demanding assay protocols. We aimed to establish a fast and reproducible method for measuring uACE activity to identify the inhibitory compounds responsible for previous assay failures and to define practical preanalytical conditions suitable for routine laboratory implementation. Methods: A fluorescence-based kinetic assay was optimized for urine samples. Endogenous inhibitors were isolated by membrane filtration and chemically characterized, while the effect of sample dilution was evaluated as a simplified alternative for eliminating inhibitory interference. We assessed the stability of ACE activity under various storage conditions to support reliable measurement. Results: Urea (IC₅₀ = 1.18 M), uric acid (IC₅₀ = 3.61 × 10⁻³ M), and urobilinogen (IC₅₀ = 2.98 × 10⁻⁴ M) were identified as the principal reversible inhibitors, jointly accounting for up to 90% suppression of uACE activity. Their inhibitory effect was effectively eliminated by a 128-fold dilution. ACE activity remained stable for 24 h at 25 °C but was completely lost after freezing. A strong positive correlation between uACE activity and creatinine concentration (r = 0.76, p < 0.0001) justified normalization. ACE activity-to-creatinine ratio turned out to be significantly lower in ACE inhibitor-treated patients than in untreated controls (6.49 vs. 36.69 U/mol, p < 0.0001). Conclusions: Our findings demonstrate that accurate measurement of uACE activity is feasible using a rapid dilution-based protocol. The normalized ACE activity can serve as a practical biomarker for detecting pharmacological ACE inhibition and monitoring therapy adherence in cardiovascular care and may also provide insight into renal pathophysiology such as tubular injury or local RAAS-related processes. Full article

This study aimed to investigate the effects of different freezing temperatures and storage durations on the digestive and structural properties of chicken meat proteins. The texture, protein digestibility, particle size, and microstructure of the digested samples were used to characterize their digestive properties. Conformational changes in the digested samples were confirmed by infrared spectroscopy, circular dichroism, and endogenous tryptophan fluorescence spectra. The results revealed that the moisture and protein contents decreased with extended storage and increased temperature. Compared to fresh chicken breasts, protein digestibility decreased as the duration of frozen storage increased. Moreover, the samples stored at −40 °C had higher digestibility than those stored at −18 °C, and the protein structure of the samples stored at −18 °C exhibited more damage and was more likely to aggregate compared to the protein of samples stored at −40 °C. Therefore, a higher freezing temperature and an extended frozen storage duration result in greater structural damage to the protein and lower protein digestibility. Full article

This study reports the construction and validation of a CAFLUX (Chemically Activated Fluorescent Expression) HepG2 reporter cell line engineered to express a histone H2B–green fluorescent protein (H2B–GFP) fusion protein under the control of a dioxin-responsive cytochrome P450 1A1 (CYP1A1) promoter. A lentiviral construct containing a synthetic promoter with multiple dioxin-responsive elements (DREs) upstream of the H2B–EGFP coding sequence was cloned into the pFUGW vector, packaged in human embryonic kidney (HEK) 293FT cells, and used to transduce HepG2 hepatocellular carcinoma cells. Stable clones obtained by limiting dilution were screened for GFP expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The resulting CAFLUX HepG2 cells exhibited dose-dependent nuclear GFP fluorescence when exposed to aryl hydrocarbon receptor (AhR) agonists, with limits of detection of approximately 0.01 pM for TCDD and 0.1 pM for benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH). This reporter activity correlated with endogenous CYP1A1 mRNA expression as determined by quantitative polymerase chain reaction (qPCR), confirming that GFP signals reflected native transcriptional responses. In functional assays, curcumin suppressed GFP expression in a concentration-dependent manner and induced apoptotic morphology at higher doses, while extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) significantly reduced both GFP fluorescence and CYP1A1 mRNA levels, suggesting an inhibitory effect on AhR-driven transcription. The CAFLUX HepG2 reporter system therefore provides a sensitive and reproducible platform for real-time, nuclear-localized monitoring of AhR-mediated gene expression. Its responsiveness to both agonists and antagonists underscores its potential utility in toxicological evaluation, drug discovery, and the investigation of EV-mediated signaling in liver cancer models. Full article

Accurate and detailed tissue characterization is a central goal in medical diagnostics, often requiring the combination of multiple imaging modalities. This study presents a multimodal imaging system that integrates mid-infrared (MIR) scanning with fluorescence imaging to enhance the chemical specificity and spatial resolution in biological samples. A motorized mirror allows rapid switching between MIR and fluorescence modes, enabling efficient, co-registered data acquisition. The MIR modality captures label-free chemical maps based on molecular vibrations, while the fluorescence channel records endogenous autofluorescence for additional biochemical contrast. Applied to mouse brain tissue, the system enabled the clear differentiation of gray matter and white matter, supported by the clustering analysis of spectral features. The addition of autofluorescence imaging further improved anatomical segmentation and revealed fine structural details. In mouse skin, the approach allowed the precise mapping of the layered tissue architecture. These results demonstrate that combining MIR scanning and fluorescence imaging provides complementary, label-free insights into tissue morphology and chemistry. The findings support the utility of this approach as a powerful tool for biomedical research and diagnostic applications, offering a more comprehensive understanding of tissue composition without relying on staining or external markers. Full article

Autophagy is a central pathway involved in maintaining cellular homeostasis during development, metabolism, and regeneration. The selective autophagy receptor p62/SQSTM1 is a multifunctional protein that plays a critical role in regulating autophagic activity by interacting with LC3/Atg8 proteins and ubiquitinated substrates. Aedes albopictus has rapidly spread worldwide and poses a serious threat to human health by transmitting dengue and other arboviral diseases. In the present study, we identified a putative p62/SQSTM1 homolog protein (AaSQSTM1) in Ae. albopictus, which contains conserved structural regions, including the Atg8 family interaction motif (AIM) and ubiquitin-associated (UBA) domain. The expression levels of AaSQSTM1 and AaAtg8 varied from the egg stage to the adult stage in Ae. albopictus. Quantitative analyses revealed that blood-feeding upregulated the expression levels of AaSQSTM1 and AaAtg8 in the ovaries of female adults. AaSQSTM1 was applied to monitor autophagic activity alone or with AaAtg8 by assaying endogenous protein levels and overexpressing fluorescent reporters in C6/36 cells. Our study revealed that AaSQSTM1 regulates the autophagic activity with AaAtg8, providing a basis for studying the autophagy process in mosquitoes. Full article

Stropharia rugosoannulata is a cultivated edible mushroom characterized by its nutritional composition and efficient cellulolytic enzymatic systems. However, the lack of genetic tools has significantly impeded the investigation of its molecular mechanisms, severely constraining the study of functional genomic and precision breeding in S. rugosoannulata. It was demonstrated in this study that the Agrobacterium-tumefaciens-mediated genetic transformation (ATMT) system is applicable for the transformation of S. rugosoannulata protoplasts. Through this proposal, we successfully achieved the expression of exogenous genes (mCherry gene encoding red fluorescent protein, hph gene encoding hygromycin B phosphotransferase, and GUS gene encoding β-glucuronidase) and the endogenous mutant gene SDI encoding the iron-sulfur protein subunit of succinate dehydrogenase in S. rugosoannulata. Furthermore, this study employed endogenous promoters of GPD encoding glyceraldehyde-3-phosphate dehydrogenase and SDI to enhance transformation efficiency and drive target gene expression. This study establishes the feasibility of ATMT in S. rugosoannulata systems, while achieving stable expression of a panel of selectable marker genes and reporter genes critical for genetic research in S. rugosoannulata. Full article

Background/Objectives: Glucagon-like peptide-1 (GLP-1) is an endogenous hormone with receptors widely expressed across multiple organs. GLP-1 receptor agonists (GLP-1RAs), primarily used for diabetes management, have demonstrated anti-inflammatory and antioxidant properties beyond glucose regulation. This study explores the protective effect of semaglutide, a GLP-1RA, in reducing oxidative stress and promoting wound healing in human dermal fibroblasts. Additionally, it assesses whether semaglutide offers the direct protection of retinal endothelial cells under oxidative stress. Methods: Human dermal fibroblasts and retinal endothelial cells were treated with semaglutide at concentrations ranging from 0 to 45 pg/mL for 24 h under oxidative stress induced by hydrogen peroxide (H₂O₂). Cell viability and ATP levels were measured via MTT and ATP assays. Apoptosis was evaluated using propidium iodide staining. Intracellular reactive oxygen species (ROS) and mitochondrial superoxide were assessed through confocal microscopy with specific fluorescent probes. Wound healing was tested using scratch assays, with closure monitored over time and quantified with ImageJ (version 1.51). Gene expression levels of antioxidants, extracellular matrix components, inflammatory cytokines, and MMPs (MMP3, MMP9) were determined via real-time PCR. Results: Semaglutide significantly improved cell viability and ATP production under oxidative stress (p < 0.001), while reducing apoptosis and intracellular ROS levels. It notably accelerated fibroblast wound closure, achieving near-complete restoration. Gene analysis revealed increased expression of antioxidant and ECM-related genes, along with decreased pro-inflammatory cytokines and MMPs, indicating reduced inflammation and enhanced tissue remodeling. Conclusions: Semaglutide offers robust antioxidative and cytoprotective effects in dermal fibroblasts and retinal endothelial cells, promoting wound healing. These findings highlight its therapeutic potential for diabetic foot ulcers and diabetic retinopathy, supporting further in vivo investigation. Full article

Distention of the urinary bladder wall during filling stretches the urothelium and induces the release of chemical mediators, including adenosine 5′-triphosphate (ATP) and prostaglandins (PGs), that transmit signals between cells within the bladder wall. The urothelium also releases soluble nucleotidases (s-NTDs) that control the availability of ATP and its metabolites at receptor sites in umbrella cells and cells deeper in the bladder wall, as well as in the urine. This study investigated whether PGs regulate the intravesical breakdown of ATP by s-NTDs. Using a murine decentralized mucosa-only bladder model and an HPLC technology with fluorescence detection, we evaluated the decrease in ATP and increase in ADP, AMP, and adenosine (ADO) in intraluminal solutions (ILS) collected at the end of physiological bladder filling. PGD₂, PGE₂, and PGI₂, but not PGF_2α, inhibited the conversion of AMP (produced from ATP) to ADO, likely due to a suppressed intravesical release of s-AMPases. The effects of exogenous PGD₂, PGE₂, and PGI₂ were mediated by DP1/DP2, EP2, and IP prostanoid receptors, respectively. Activation of either DP1 or DP2 receptors by endogenous PGD₂ also led to AMP increase and ADO decrease in ILS-containing ATP substrate. Finally, PGs produced by either COX-1 or COX-2 inhibited the hydrolysis of AMP to ADO. Together, these observations suggest that (1) endogenous PGs (chiefly PGD₂, and to lesser degree PGE₂ and PGI₂) allow release of s-NTDs like s-ATPases and s-ADPases but impede the formation of ADO from intravesical ATP by inhibiting the release of s-NTDs/s-AMPases; (2) it is possible that high concentrations of PGD₂, PGE₂ and PGI₂, as anticipated in inflammation or bladder pain syndrome, delay the ADO production and prolong the action of excitatory purine mediators; and (3) either COX-1 and COX-2 are constitutively expressed in the mouse bladder mucosa or COX-2 is induced by distention of the urothelium during bladder filling. Full article

Using in vitro models of hypoxia, episodes of short-term hypoxia/reoxygenation, and ischemia-like conditions, we were able to establish different sensitivities of hippocampal neurons to their damaging factors. Using a combination of fluorescence microscopy and immunocytochemistry methods, it was possible to show that GABAergic neurons are the most vulnerable to the damaging effects of hypoxia and ischemia-like conditions, as they have a special metabolism, which is characterized by increased production of reactive oxygen species and nitric oxide. It was shown that long-term hypoxia causes the death of GABAergic neurons due to the induction of a global [Ca²⁺]_i increase, whereas pyramidal neurons are resistant to 30-min hypoxia. Episodes of short-term hypoxia/reoxygenation activate the phenomenon of hypoxic preconditioning in glutamatergic neurons, while this endogenous protective mechanism is absent in GABAergic neurons. Selective activation of PI3K and PKG, combined with NOS inhibition, potentiates the preconditioning effect of hypoxia/reoxygenation in glutamatergic neurons and partially activates this neuroprotective mechanism in GABAergic neurons, as indicated by diminished post-hypoxic NMDA-induced Ca²⁺ transients. These findings suggest that pharmacological intervention can protect GABAergic neuronal populations. The expression of parvalbumin, calbindin, or calretinin in the cytosol of GABAergic neurons contributes to the suppression of the global [Ca²⁺]_i increase during hypoxia/reoxygenation episodes, which correlates with their survival even in the absence of the hypoxic preconditioning phenomenon. Additionally, all three studied calcium-binding proteins showed potential high efficiency in maintaining the bioenergetics of GABAergic neurons during ischemia-like conditions, limiting ROS production by mitochondria and in the cytosol, and reducing nitric oxide formation. In this case, parvalbumin showed the greatest efficiency. Full article

Fenpropathrin (FPT) residues in tea pose a threat to consumers’ health. Lateral flow immunoassay (LFIA) offers a rapid and convenient approach for FPT detection. However, existing LFIA formats, particularly those employing fluorescent nanoparticles, are susceptible to interference from the tea matrix’s endogenous fluorescence, limiting their accuracy for FPT analysis. Here, two complementary LFIAs based on gold nanoparticle (AuNP) and carbon nanoparticle (CNP) tags were developed, both of which avoid matrix fluorescence effects due to their intrinsic coloration under ambient light. We systematically evaluated five cleanup materials and identified polyvinylpolypyrrolidone as the optimal cleanup material. Following PVPP-based purification, green tea extracts required only a four-fold dilution prior to LFIA analysis, effectively minimizing matrix interferences, such as tea polyphenols, and enhancing assay robustness and sensitivity. The visual limit of detection was 0.64 μg/g and a quantitative limit of detection (qLOD) was 0.11 μg/g for FPT in green tea using the AuNP-based LFIA. The CNP-based LFIA further improved sensitivity, with a visual limit of detection of 0.16 μg/g and a qLOD of 0.017 μg/g, a 6.4-fold enhancement in qLOD compared to the AuNP assay. Together, these two assays provide an efficient and accurate strategy for on-site screening of FPT residues in green tea. Full article