Search Results (109)

Particles exhibiting fluorescence after Nile Red staining have been increasingly investigated in human biological samples in the context of microplastic exposure, but their clinical relevance remains unclear. In this study, Nile Red-positive microparticles were quantified in whole blood from chronic users of different nicotine-delivery systems and non-user controls. The study included 73 nicotine users, including conventional cigarette smokers, e-cigarette users, and heated tobacco product users, as well as an age- and sex-matched control group. Only participants without diagnosed chronic somatic disease were enrolled. Microparticles were quantified by flow cytometry after Nile Red staining. Associations with age, nicotine dependence assessed with the FTND and FTQ, motivation to quit smoking assessed with the Schneider Motivation Test, and selected hematological and biochemical parameters were also analyzed. In the overall analysis, the mean number of microparticles was higher in nicotine users than in controls, but the difference was not statistically significant (p = 0.38). In subgroup analyses, higher particle counts were observed in conventional cigarette smokers and heated tobacco product users compared with controls. No significant difference was found for e-cigarette users. Higher particle counts were also observed in older participants and in participants with greater nicotine dependence. Participants with higher particle counts more often showed stronger nicotine dependence and lower motivation to quit smoking. These results suggest that circulating Nile Red-positive microparticle counts may differ according to the type of nicotine product used and selected behavioral characteristics. However, the subgroup findings should be interpreted with caution due to the exploratory design and limited statistical power. Full article

A modified Pitot-tube jet (PTJ) separation pump combines centrifugal phase separation with pressure buildup and enables compact oil–water treatment, where a water-rich stream can be discharged at elevated pressure. This work advances an existing laboratory PTJ configuration toward a turbomachinery-oriented rotor concept for systematic design studies and subsequent field-oriented prototypes. Starting from a centrifuge-like reference configuration without blades that prioritizes separation stability, an impeller with trimmed blades is introduced to increase pressure head while limiting blade interaction with the oil–water interface by operating primarily in the outer, water-rich annulus. Comparative experiments with and without the impeller show a pronounced increase in pressure head, up to about a factor of three at the maximum speed investigated. The results also indicate a purity penalty caused by blade-induced mixing and secondary flows. This exposes the central design trade-off of the PTJ machine. Higher specific work input increases pressure head but can reduce discharge quality. Hydraulic optimization, therefore, needs to be coupled to ppm-level purity constraints. Density-based monitoring lacks resolution in the relevant trace range, and chemical-based analyses are too slow for systematic investigations. An imaging-based fluorescence method using Nile Red as a selective tracer is, therefore, implemented as a rapid analysis tool. High-resolution imaging with automated region of interest evaluation provides a robust calibration from 5–500 ppm for safe, non-fluorescent model oils such as sunflower oil. This enables efficient operating-window mapping and comparative screening of rotor concepts under reproducible conditions. Full article

With the global increase in microplastic pollution, even environments considered pristine have shown signs of being affected by these contaminants. In this context, it becomes essential to conduct studies that identify and quantify the presence of microplastics in remote regions such as Antarctica. This continent is particularly relevant due to its low anthropogenic influence and its essential role in regulating planetary ecosystems and biodiversity. In this study, 49 Antarctic samples were analyzed using pretreatment techniques with NaCl and ZnCl₂ saline solutions, followed by fluorescence microscopy using Nile Red dye to estimate the microplastic abundance index. Both solutions showed good performance in the separation and identification of particles. Approximately 37% of the samples showed contamination by potential microplastics (PMPs), with a higher concentration of particles retained on paper filters and fibers observed in the supernatants. The results indicate that the presence of MPs in Antarctica is irregular and not ubiquitous, differing from other studies that suggest a wider distribution. It is speculated that the observed contamination results from oceanic transport from other regions of the planet and from sources associated with human activities on the Antarctic continent (e.g., tourism and research). Full article

Trace metals are persistent stressors in coastal ecosystems, yet most marine algal toxicity assessments still rely on freshwater model species and growth-based endpoints that provide limited mechanistic resolution. Here, we quantified the sensitivity of two ecologically contrasting marine diatoms—the benthic Cylindrotheca closterium and the planktonic Thalassiosira weissflogii—to ten environmentally relevant metals using a dual-endpoint approach that integrates chlorophyll fluorescence (photosystem function) and Nile Red-based lipid-body fluorescence (metabolic reallocation). Fluorescence-based EC₁₀ values revealed distinct species- and metal-specific patterns, with C. closterium consistently responding at lower concentrations and Hg producing the strongest inhibition in both species (EC₁₀ ≈ 0.04–0.06 mg L⁻¹). Lipid-body accumulation detected earlier metabolic disturbance for several metals, particularly Hg, As, Cr(VI), and Cd, and frequently occurred at concentrations where fluorescence remained minimally affected. These sequential thresholds indicate that pigment impairment and metabolic reallocation represent mechanistically distinct stages of the cellular stress response that differ among metals and between diatom guilds. Comparison with published toxicity data shows that the dual-endpoint sensitivities observed here fall within, or slightly above, the upper range of reported microalgal responses, underscoring the pronounced susceptibility of benthic diatoms to redox-active and thiol-reactive metals. The strong agreement between fluorescence-based EC values and traditional growth-derived benchmarks for key metals further supports fluorescence as an operationally efficient endpoint suitable for integration into emerging ISO marine algal bioassays. Overall, this study demonstrates that pairing a rapid functional marker with a mechanistically informative metabolic biomarker enables metal-specific toxicity fingerprinting and provides an ecologically grounded basis for incorporating benthic diatoms into coastal metal risk assessment frameworks. Full article

Microplastics (MPs) have been detected in aquatic environments worldwide; however, freshwater systems remain underexplored. MPs’ presence has not been reported yet in the Río Bravo/Grande at the Mexico–United States border. In 2022 and 2023, water (30 L) and sediment (~500 g) samples were collected at the river along the El Paso (U.S.)–Juárez (Mexico) municipalities. Water and sediment were digested using H₂O₂ or NaOH to isolate (CaCl₂), dye (Nile Red), quantify, and classify MPs by fluorescence microscopy. The number of microparticles detected in water and sediment ranged from 0.4 to 17 particles/L and 2.8 × 10³–1.0 × 10⁴ particles/kg, respectively. Similar concentrations of microparticles were estimated in water among sampled sites (ANOVA) in 2022 (p = 0.432, α = 0.05) and 2023 (p = 0.255, α = 0.05), but there were differences in sediments (2022, p < 0.01; 2023, p = 0.032. α = 0.05). Fibres, fragments, and films, with sizes ranging from 12.62 to 4282.25 µm, were found in the samples. Fibres were the most abundant shape in water (71.94%) and sediments (91.07%). MP concentrations varied in water and sediments in both years. The data generated by this study provide new insights into feasible methods for MP detection and its presence in the Río Bravo/Grande. Full article

The aim of the circular economy for plastics is to replace some of them with bio-based polymers in the future. In this work, second cheese whey (SCW) was used as a low-cost substrate for the production of the natural polyester poly(3-hydroxybutyrate)-hydroxybutyrate (PHB) by three Pseudomonas sp. phDV1 strains, namely, the wild type, a depolymerase PhaZ and PhaR knockout mutants. SCW has high polluting loads, characterized by high levels of lactose, phosphorus, nitrogen and salinity, as well as high turbidity due to the presence of whey solids. Initially, SCW was evaluated as the sole carbon source for the growth of the bacterial strains and the production of PHB. Fermentation conditions were screened to maximize polymer synthesis. Small-scale experiments showed that the strains could grow and produce PHB in SCW with and without enzymatic treatment. The formation and intracellular localization of PHB were determined with fluorescence microscopy, using Nile Red staining. Analytical HPLC was also used to quantify the PHB content in the cells and to optimize production. This study demonstrates the efficacy of Pseudomonas sp. phDV1 in SCW treatment and biomass valorization, providing a sustainable solution for dairy wastewater management while producing valuable resources. Full article

Obesity is among the top contributing factors for non-communicable chronic disease development and has attained menacing global proportions, affecting approximately one of eight adults. Phytochemicals that support energy metabolism and prevent obesity development have been the subject of intense research endeavors over the past several decades. Cycloastragenol is a natural triterpenoid compound and aglycon of astragaloside IV, known for activating telomerase and mitigating cellular aging. Here, we aim to characterize the effect of cycloastragenol on lipid metabolism in a glucose-induced obesity model in Caenorhabditis elegans. We assessed the changes in the body length, width, and area in C. elegans maintained under elevated glucose through automated WormLab system. Lipid accumulation in the presence of either cycloastragenol (100 μM) or orlistat (12 μM), used as a positive anti-obesity control drug, was quantified through Nile Red fluorescent staining. Furthermore, we evaluated the changes in key energy metabolism molecular players in GFP-reporter transgenic strains. Our results revealed that cycloastragenol treatment decreased mean body area and reduced lipid accumulation in the C. elegans glucose-induced model. The mechanistic data indicated that cycloastragenol suppresses the nuclear hormone receptor family member NHR-49 and the delta(9)-fatty-acid desaturase 7 (FAT-7) enzyme, and activates the 5′-AMP-activated protein kinase catalytic subunit alpha-2 (AAK-2) and the protein skinhead 1 (SKN-1) signaling. Collectively, our findings highlight that cycloastragenol reprograms lipid metabolism by down-regulating the insulin-like receptor (daf-2)/phosphatidylinositol 3-kinase (age-1)/NHR-49 signaling while simultaneously enhancing the activity of the AAK-2/NAD-dependent protein deacetylase (SIR-2.1) pathway. The anti-obesogenic potential of cycloastragenol rationalizes further validation in the context of metabolic diseases and obesity management. Full article

Background/Objectives: Transdermal formulations are widely utilized in the pharmaceutical and cosmetic fields because they enable non-invasive administration and sustained local drug delivery. Conventional ex vivo skin permeation experiments using Franz diffusion cells have limitations in capturing the spatial and temporal dynamics of skin penetration. This study aimed to develop a confocal laser scanning microscopy (CLSM)-based approach to visualize and semi-quantitatively assess the penetration behavior of fluorescent dyes with differing lipophilicities. Methods: Four fluorescent dyes with different Log P values—Rhodamine B (Rho-B), Rhodamine 123 (Rho-123), Fluorescein Sodium (Flu-Na), and Nile Red (NR)—were formulated into lotion-based vehicles and applied to excised human abdominal skin. CLSM imaging was performed from 10 min to 240 min post-application. Fluorescence intensities were extracted from depth-resolved regions (R1–R4, 30-μm intervals) to examine penetration kinetics and distribution. Results: CLSM imaging demonstrated that Rho-B penetrated through stratum corneum and entered deep into the skin via the hair follicles. Rho-123 and Flu-Na exhibited intercellular and follicular penetration; however, Flu-Na showed only a slight increase in intensity over time; NR showed negligible penetration into the deeper layers. The results of our analysis indicated that moderately lipophilic substances such as Rho-B and Rho-123 diffused deeply into the skin via both transdermal and follicular routes, whereas highly hydrophobic or lipophilic substances remained in the superficial layers. Conclusions: The CLSM-based approach enabled spatially and temporally resolved, semi-quantitative evaluation of transdermal penetration in a single, non-destructive experiment. Although restricted to fluorescent probes, this approach provides a practical early-stage screening tool for comparing route-specific and time-dependent penetration behaviors of compounds with different lipophilicities. Full article

The pervasive environmental contamination by micro- and nanoplastics (MNPs) presents a formidable analytical challenge, necessitating the development of rapid and sensitive detection methods. While conventional techniques often suffer from limitations in sensitivity and throughput, fluorescent probe-based technology has emerged as a powerful alternative. This review charts the evolution of these probes, from initial stains relying on hydrophobic adsorption to advanced molecular designs engineered for specific chemical recognition. We critically examine key operational mechanisms, including the solvatochromic response of Nile Red, polarity-discriminatory probes enabling a “microplastic rainbow,” and targeted systems achieving turn-on fluorescence via restriction of intramolecular rotation. Furthermore, we highlight cutting-edge signal enhancement strategies, such as plasmon- and metal-enhanced fluorescence, which amplify detection to the femtogram level. Special emphasis is placed on the distinct challenges posed by nanoplastics, including their propensity for aggregation in aqueous matrices that exacerbates false positives and their superior ability to breach biological barriers, and how AIE luminogens and PEF/MEF strategies mitigate these issues through enhanced signal-to-noise ratios and subcellular resolution, differing from their application to microplastics. Critically, we address the imperative for low-toxicity probe designs, emphasizing biocompatibility and biodegradability criteria to facilitate safe, long-term in vivo tracking and widespread ecological surveillance. The integration of these sophisticated probes with smart, “activate-on-target” systems is paving the way for next-generation MNP analysis, offering critical insights for environmental monitoring and toxicological assessment. Full article

Plastics play a pivotal role in various industries owing to their versatility in engineering their physical, mechanical, and chemical properties while exploiting their remarkable durability, light-weight nature, and cost-effectiveness. Yet, their widespread use has led to the pollution of Earth’s water systems. Over time, plastic waste degrades into microplastics, particles smaller than 5 mm. Recent studies have highlighted the growing concerns associated with microplastics, especially in bottled beverages, including bottled water, with associated hazards still in the very early stages of being fully understood. Furthermore, the global understanding of the extent of microplastic contamination in the environment and along the food chain remains limited. This study aimed to detect, quantify, and characterise microplastics in bottled drinking water produced and sold in Malta. Samples from five brands were filtered, stained with Nile red, and quantified using fluorescence microscopy. The average microplastic concentration was found to be 35,877 ± 23,542 particles per litre, with 84% of samples exhibiting contamination, which was noted to be statistically significant. The average particle diameter was measured to be 2.3696 ± 0.0035 µm. Raman spectroscopy was used to chemically characterise 10 larger particles per brand (i.e., 50 samples), identifying the presence of cellulose, polyurethane, polymethyl methacrylate, polyethylene, and smaller quantities of other polymers. Morphological analysis classified 36 of the larger particles as fragments and 14 as fibres. Excluding laboratory-introduced contamination, the primary source of microplastic contamination in the analysed bottled water was traced to the bottle caps. Full article

Background: Polyacrylamide gel electrophoresis (PAGE) has long been used for lipoprotein analysis, enabling the separation and profiling of lipoprotein fractions such as LDL and HDL. However, conventional disc PAGE systems are limited by low throughput and inability to directly compare multiple samples under identical conditions. Alternative methods, including high-performance liquid chromatography and agarose gel electrophoresis, require specialized equipment and expertise, limiting their clinical utility. Methods: We present a colorimetric and fluorescence-based histidine-imidazole PAGE (HI-PAGE) system that provides rapid, cost-effective, and reproducible separation and profiling of lipoproteins in human serum. By combining electrophoretic separation with lipid-specific fluorescent staining using Nile Red, the fluorescence-based HI-PAGE (fHI-PAGE) not only visualizes distinct migration patterns of lipoprotein fractions, but also enables the quantification of LDL-cholesterol (LDL-C). Clear resolution of LDL and other lipoprotein fractions was achieved within 1 h without band distortion, allowing for direct comparison of multiple samples on a single gel. Results: We validated fHI-PAGE using serum from healthy individuals and patients, demonstrating that its fluorescence-based detection was more sensitive than conventional Sudan Black B staining while providing LDL-C estimates concordant with values calculated by the Friedewald formula. Moreover, fHI-PAGE proved advantageous in cases of hypertriglyceridemia, where Friedewald calculations are unreliable. Conclusions: These findings establish fHI-PAGE as a practical and clinically applicable platform for simultaneous lipoprotein profiling and LDL-C quantification. Full article

The production of water-in-water emulsion droplets, the coalescence of which is prevented by adding oil-in-water micrometric droplets, is reported. Hexadecane (O) and cetyl trimethyl ammonium bromide (CTAB) were added to a W/W emulsion made of dextran (Dex)-enriched droplets in a Polyethyleglycol (PEG)-enriched continuous phase, and the mixture was further sonicated. Using Nile red to label the oil droplets enabled the observation of their presence at the surface of Dex droplets (5 µm), allowing for stabilizing them, preventing coalescence of the W/W emulsion, and mimicking W/O/W double emulsions. The addition of sulfate derivative of Dextran (DexSulf) allowed for stable droplets of a slightly larger diameter. By contrast, the addition of carboxymethyl Dextran (CMDex) destabilized the initial aqueous double-like emulsion, yielding sequestration of the oil droplets within the Dex-rich phase. Interestingly, addition of DexSulf to that unstable emulsion re-yielded stable droplets. Similar findings (destabilization) were obtained when adding sodium dodecyl sulfate (SDS) to the initial double-like emulsion, which reformed stable droplets when adding positively charged Dextran (DEAEDex) derivatives. The use of fluorescently (FITC) labeled derivatives of Dextran (Dex, CMDex, DEAEDex, and DexSulf) allowed us to follow their position within, out of, or at the interface of droplets in the above-mentioned mixtures. These findings are expected to be of interest in the field of materials chemistry. Full article

Hypertension and metabolic dysfunction-associated fatty liver disease (MAFLD) are both common chronic diseases globally. Nearly half of patients with hypertension are complicated by MAFLD. The mechanisms of the bidirectional promotion between the two remain unclear. The (pro) renin receptor ((P)RR) is one of the classic members of the renin–angiotensin system (RAS) and serves as the receptor for prorenin. Although the role of (P)RR in the induction and progression of hypertension has been extensively studied, its role and underlying mechanisms in MAFLD remain underreported. In this study, we aim to investigate the role of (P)RR in the pathogenesis of hypertension combined with MAFLD. In this study, SHRs were used for the model for hypertension combined with MAFLD. Liver lipid content analysis, liver H&E staining, the detection of (P)RR, ERK and downstream proteins related to fatty acid synthesis and transport, and RNA sequencing and data analysis were performed. In the in vitro experiments, we activated (P)RR using renin and established the lipid deposition model of HepG2 cells induced by renin for the first time. (P)RR was specifically blocked using handle region peptide (HRP), and Nile red fluorescence staining, (P)RR/ERK/PPARγ protein expression analysis, and immunofluorescence were performed to further verify the role of (P)RR in the pathogenesis of hypertension combined with MAFLD. Our results demonstrate that (P)RR plays a role in the development and progression of hypertension combined with MAFLD. The hepatic TG and FFA levels in the SHRs were increased, and the protein expression of the (P)RR/ERK/PPARγ pathway and downstream proteins related to fatty acid synthesis and transport were upregulated. HRP reversed the activation of these proteins and reduced intracellular lipid accumulation. In conclusion, our study first reveals that (P)RR is a potential therapeutic target for hypertension combined with MAFLD. And we found the (P)RR/ERK/PPARγ axis for the first time, which plays an important role in the progression of spontaneous hypertension combined with MAFLD. Full article

Stormwater runoff is a significant source of microplastics to surface water. This study addresses challenges in the sampling, treatment, and characterization of microplastics in existing stormwater control measures across various regions in the United States. Stormwater sediment samples were collected via traditional stormwater sampling approaches for particulate and inorganic contamination with portable automatic samplers, analyzed using visible and fluorescence microscopy with Nile red as a selective stain, and subsequently confirmed through Raman spectroscopy. The inclusion of laboratory and field blanks enabled the identification of contamination at key steps during sample handling. The results reveal that the filtration process is a significant source of laboratory contamination, while the sampling process itself could be a primary contributor to overall sample contamination. Additionally, it was found that using green fluorescence as the sole emission wavelength may underestimate MP quantities, as some particles emit fluorescence exclusively in the red spectrum. Raman analysis revealed interferences caused by pigments and additives in plastics, along with challenges evaluating particles in the low micron range (≤10 microns), which complicates a comprehensive analysis. The findings of this study emphasize the importance of implementing strong quality assurance and control measures when assessing the levels of microplastics in the environment, including sample collection, processing, and analysis. Full article

The increasing prevalence of microplastic (MP) pollution and the labor-intensive nature of existing identification methods necessitate improved large-scale detection approaches. Nile Red (NR) fluorescence, which varies with polarity, offers a potential classification method, but standardization of carrier solvents and fluorescence differentiation techniques remains lacking. This study evaluated eight NR-carrier solvents (n-hexane, chloroform, acetone, methanol, ethanol, acetone/hexane, acetone/ethanol, and acetone/water) across ten common MP polymers (HDPE, LDPE, PP, EPS, PS, PC, ABS, PVC, PET, and PA). Fluorescence intensity, Stokes shift, and solvent-induced polymer degradation were analyzed. The study also assessed HSV (Hue/Saturation/Value) color spaces for Stokes shift representation and MP differentiation. Fenton oxidation effectively quenched fluorescence in natural organic matter (e.g., eggshells, fingernails, wood, cotton) while preserving NR-stained MPs. Acetone/water [25% (v/v)] emerged as the optimal solvent, balancing fluorescence performance and minimal degradation. Full article