Nile Red Staining Research Articles

Standardization of FTIR-Based Methodologies for Microplastics Detection in Drinking Water: A Meta-Analysis Indeed and Practical Approach

The detection of microplastics (MPs) in drinking water presents significant environmental and public health challenges. This study comprises two stages: a meta-analysis aimed at standardizing Fourier Transform Infrared Spectroscopy (FTIR) methods for MP detection, followed by the practical implementation of these findings in the laboratory. The review of studies conducted from 2019 to 2023 identifies 0.45 μm cellulose nitrate filters and Nile red staining as the most effective techniques for fluorescent detection. Experimental results demonstrate the superior retention capabilities of cellulose nitrate filters and the uniformity of Nile red staining. This dual approach not only optimizes water treatment processes but also enhances the accuracy of MP detection. The findings contribute to improved water quality management and public health protection by establishing robust protocols for MP analysis.

Microplastics in different nasal irrigation options.

We aimed to assess the presence of microplastics in nasal irrigation methods commonly used in the treatment of sinusitis and rhinitis, and to evaluate human exposure. A total of 150 samples were included in the study, consisting of nasal wash bottles containing nasal irrigation solution, seawater spray, syringes for nasal irrigation with isotonic solution. The amount of microplastics per millilitre in the samples and patient exposure during single use were assessed separately for each method and product. All samples were filtered using a stainless steel vacuum filter on filter paper with a pore size of 1.2μm, washed at least three times with distilled water and incubated at 45°C for 24h to prevent mould growth. Identification and counting of microplastics was performed using a Leica Flexacam C1 camera connected to an M80 stereomicroscope. The presence of microplastics was confirmed by the hot needle method and Nile red staining. An average of 6.49 ± 13.08 microplastics/product was detected in all filtered samples. The lowest microplastic count was 0 microplastics/product in syringes and the highest was 92 microplastics/product in nasal wash bottles. Significant differences in the amount of microplastics individuals were exposed to during a single use were found between nasal wash bottles and seawater brands, while no significant differences were found between syringe brands. When nasal wash kits, seawater sprays and isotonic nasal rinses were evaluated separately, significant differences were found in the number of microplastics, the microplastics/ml ratio and the number of microplastics exposed during a single use. The highest microplastic exposure was found in nasal irrigation bottles. The exposure of individuals to microplastics increases with medical support treatments, regardless of intranasal or intravenous administration. Due to the inflammation, oxidative stress and proliferation caused by microplastics, new regulations and inspections of production conditions should be implemented worldwide to reduce exposure.

Pervasive Microplastic Ingestion by Commercial Fish Species from a Natural Lagoon Environment

Microplastics have emerged as a significant global environmental concern in the recent decade. The aim of this study was to elucidate microplastic contamination of commercial fish species in a natural lagoon environment. Microplastic contamination was examined in the gastrointestinal tracts and gills of 157 commercial fish from 18 species with varying feeding habits in a vital and sensitive lagoon ecosystem, which connects to the Indian ocean. Microplastics were extracted using digestion, followed by stereomicroscopic inspection using Nile Red stain, and identified via μ-FTIR analysis. Over half of studied fishes ingested microplastics (54.14%). Filaments (50%) and blue items (43%) were the most commonly ingested. Of all the fish species, Eubleekeria splendens had the highest average concentration of microplastics in GIT (1.41 ± 2.52 items/g w.w. tissues), although no statistically significant difference in amount of ingested microplastics (items/g w.w. tissues) was observed among species. The highest concentrations of inhaled microplastics were recorded in Sillago vincenti (1.38 ± 1.30 items/g w.w. tissues). The majority of the extracted microplastics (33%) belonged in the size class 500–1500 μm with rayon, polyethylene terephthalate, and polypropylene as the primary polymers. This study found no correlation between microplastic ingestion and fish species and feeding habits, but a positive correlation with fish size was observed. These findings reveal widespread microplastic contamination in edible fish, posing potential risks to commercially important species due to increasing pollution in lagoon ecosystems.

Nosema ceranae infection reduces the fat body lipid reserves in the honeybee Apis mellifera

Nosema ceranae is an intestinal parasite frequently found in Apis mellifera colonies. This parasite belongs to Microsporidia, a group of obligate intracellular parasites known to be strongly dependent on their host for energy and resources. Previous studies have shown that N. ceranae could alter several metabolic pathways, including those involved in the nutrient storage. To explore the impact of N. ceranae on the fat body reserves, newly emerged summer bees were experimentally infected, and we measured (1) the lipid percentage of the abdominal fat body at 2-, 7- and 14-days post-inoculation (p.i.) using diethyl ether lipid extraction, (2) the triglyceride and protein concentrations by spectrophotometric assay methods, and (3) the amount of intracellular lipid droplets in trophocytes at 14- and 21-days p.i. using Nile Red staining. Comparing the three methods used to evaluate lipid stores, our data revealed that Nile Red staining seemed to be the simplest, fastest and reliable method. Our results first revealed that the percentage of fat body lipids significantly decreased in infected bees at D14 p.i. The protein stores did not seem to be affected by the infection, while triglyceride concentration was reduced by 30% and lipid droplet amount by 50% at D14 p.i. Finally, a similar decrease in lipid droplet reserves in response to N. ceranae infection was observed in bees collected in fall.

Tire rubber-derived contaminant 6PPD had the potential to induce metabolism disorder in early developmental stage of zebrafish

The increasing release of tire-derived particles, particularly those containing N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), into the environment has raised concerns regarding their ecological impact. This study aims to elucidate the toxicological effects of 6PPD on the metabolism in early developmental stage of zebrafish. Larval zebrafish were exposed to 10 and 100 μg/L 6PPD, and some endpoints in biochemical parameters, gene expression, and metabolism were analyzed. The results showed that 6PPD exposure disrupted glucolipid metabolism in zebrafish larvae, evidenced by increased triglyceride (TG) levels and decreased glucose content. Nile red staining indicated significant lipid accumulation in the liver and intestines. Additionally, RT-qPCR analysis revealed the upregulation of genes involved in lipid synthesis and metabolism, such as ppar-γ and fas, and downregulation of glycolysis-related genes like pk and gk. Furthermore, the untargeted metabolomics technique was used to identify a total of 220 differentially expressed metabolites (DEMs) with changes in amino acid metabolism, lipid metabolism, and the TCA cycle. KEGG pathway enrichment analysis highlighted disruptions mainly in Taurine and hypotaurine metabolism, Arginine and proline metabolism, and Histidine metabolism, which played very important roles on energy metabolism in zebrafish. The results provided some critical insights into the ecological risks associated with 6PPD.

Dendrobium Officinale Kimura & Migo Attenuates High Cholesterol Diet-Induced Atherosclerosis

Objectives: This study aimed to investigate the potential of Dendrobium officinale Kimura & Migo (DOKM) in ameliorating high cholesterol-induced atherosclerosis (AS) in zebrafish, focusing on the development of safe and effective drugs with low toxicity for AS treatment. Methods: A zebrafish AS model was established by feeding zebrafish a high cholesterol diet (HCD). Treatment was administered using 1 mg/L and 10 mg/L DOKM water extract. Fluorescence microscopy was utilised to observe the effects of DOKM on HCD-induced cholesterol accumulation, neutrophil accumulation, and macrophage infiltration in the vasculature. The impact of DOKM on HCD-induced disorders of lipid metabolism was assessed using Nile red staining. Total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) levels in zebrafish were measured using kits. The expression of pcsk9, srebf2, and hmgcr genes in zebrafish was determined using RT-PCR. HE staining and Elvitegravir (EVG) staining were employed to assess the effect of DOKM on plaque formation in the blood vessels of AS zebrafish. Results: A total of 145 compounds were collected from Dendrobium officinale, along with 680 corresponding targets. Additionally, 1583 atherosclerosis-related targets were identified, with 202 representing interaction targets between Dendrobium officinale and atherosclerosis-related targets. Key compounds identified included Gigantol, Chrysotobibenzyl, Naringenin Chalcone, Hesperetin, and Tangeretin. The core targets of Dendrobium officinale for atherosclerosis treatment were STAT3, PTPN11, JAK2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor alpha (PDGFRA), proto-oncogene tyrosine-protein kinase Src (SRC), STAT1, TP53, ESR1, and AKT1. GO functional enrichment analysis revealed a total of 2090 GO biological functional entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed 200 AS signalling pathways associated with DOKM treatment for atherosclerosis. DOKM significantly attenuated cholesterol accumulation, neutrophil aggregation, and macrophage infiltration in the blood vessels of AS zebrafish, ultimately leading to reduced plaque formation. AS zebrafish exhibited lipid metabolism disorders, characterised by significant increases in TC, TG, and LDL-C levels, as well as mRNA expression of pcsk9, srebf2, and hmgcr genes. Treatment with DOKM significantly improved these lipid metabolism disorders. Conclusion: DOKM effectively attenuates HCD-induced AS, and this therapeutic effect on AS may be related to its role in improving lipid metabolism dysfunction.

Multifaceted analysis of microplastic pollution dynamics in the Yamuna river: Assessing anthropogenic impacts and ecological consequences

Microplastics (MPs) are pervasive contaminants that pose significant ecological and human health risks, emerging as one of the most widespread anthropogenic pollutants in natural environments. This study investigates the abundance, characteristics, and distribution of microplastics (MPs) in the Yamuna River, encompassing 29 sampling points across urban, rural, and industrial zones in and around Delhi, Mathura, Haryana, and Agra. Microplastics were identified and quantified using Nile red dye staining and Micro-Raman spectroscopy, with particle size distribution predominantly between 2 μm to 80 μm and the largest detected particle measuring 256.5 μm. The average MPs concentration was 14,717 ± 4444 L−1, with a significant abundance of hazardous polymers such as polyethylene terephthalate (PET), polypropylene (PP), and polystyrene (PS). The study found that MPs were predominantly fragments and films (65.6 %) and fibers (30.6 %), with transparent particles being the most prevalent. The Pollution Load Index (PLI) consistently indicated high-risk levels (PLI > 100) at all sampling sites, highlighting substantial MP contamination. These results underscore the urgent need for continuous monitoring and the development of robust management strategies to address microplastic pollution in the Yamuna River. This study provides valuable insights into MPs spatial distribution and persistence, contributing to an improved understanding of their environmental impacts and guiding future mitigation and regulatory efforts.

Impact of seasonal changes and environmental conditions on suspended and inhalable microplastics in urban air

Microplastics (MPs) are ubiquitous environmental pollutants extensively detected in atmospheric environments. Airborne MPs have raised concerns due to their transport and potential health risks of inhalation exposure. However, the factors influencing airborne MPs, particularly their concentrations and shapes suspended in urban air, remain unclear. We investigated MPs in total suspension particles with one-year measurements in Taipei City and identified their features using Nile Red staining combined with fluorescence microscopy and micro-Fourier transform infrared (μFTIR) spectroscopy. This study quantified the mean number concentration of total MPs as approximately 6.0 #/m³. We observed that MP abundance varied seasonally, with higher levels in the warm season than in the cold. A similar trend was noted for polymer types. Fragment-like MPs were the predominant shape, mainly found in polystyrene (PS), polyethylene (PE), and polypropylene (PP), while fibrous MPs, detected mostly as polyethylene terephthalate (PET) and polyamide (PA), were primarily observed at sizes greater than 300 μm. Both fiber and fragment-like MPs were positively associated with particle mass concentration, temperature, ultraviolet (UV) index, and wind speed, but negatively correlated with relative humidity and rainfall. Fibrous MPs were more affected by environmental factors than fragment-like MPs. Meteorological changes significantly influenced suspended MPs more than human activity within the city.

Anemarrhena asphodeloides Bunge total saponins lower lipid via modulating MAOA activity to enhance defense mechanisms in mice and C. elegans

Ethnopharmacological relevanceWithin Anemarrhena asphodeloides Bunge (AAB), the pivotal bioactive constituents are identified as Anemarrhena asphodeloides Bunge total saponins (ABS). In traditional pharmacology, ABS has exhibited notable anti-inflammatory, hypoglycemic, and cardioprotective properties. Despite these observed effects, the specific protective mechanisms of ABS against metabolic diseases and improving the endocrine system remain largely uncharted. Aim to studyThis work intends to shed light on the effects and intrinsic mechanisms of ABS on metabolic diseases. Materials and methodsThe characterization of ABS components was achieved through High-Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS). To evaluate ABS's anti-inflammatory efficacy, mouse macrophages underwent analysis using the Griess method. Induced differentiation of mouse fibroblasts was assessed through Oil Red O staining. In an obesity model with C57BL/6 N mice, ABS administration prompted measurements of glucose and insulin tolerance. Western blot analysis quantified lipolysis and anti-inflammatory protein expression. Nile red staining gauged body fat content in C. elegans post-ABS treatment. The mechanism of ABS action was elucidated through mRNA sequencing, further validated using RNA interference technology, and nematode mutants. ResultsABS showcased the ability to diminish Nitric Oxide (NO) production in inflammatory macrophages and shrink adipocyte lipid droplets. In mice experiments, ABS was effective in alleviating fat accumulation and affecting serum lipid metabolism in diabetic mice. It enhanced oral glucose tolerance and insulin tolerance while increasing lipolysis-associated protein expression. ABS notably reduced fat content in C. elegans. Mechanistically, ABS downregulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and monoamine oxidase A (MAOA) expression while enhancing UGT, ilys-2, and ilys-3. Lipolysis emerged as a pivotal pathway for ABS in the therapeutic intervention of metabolic diseases. ConclusionsOur investigation has revealed that ABS exert a role in combating metabolic diseases by enhancing the body's defense mechanisms. ABS activate the NLRP3-neurotransmitter-visceral adipose pathway in mice, thereby bolstering resistance and diminishing fat accumulation. In C. elegans, ABS downregulated the expression of MAOA, bolstered resistance, and augmented glucuronidase activity, consequently leading to a reduction in fat content.

Vanillin, ferulic acid and their 1:1 combination inhibit lipid accumulation in 3T3 L1 adipocytes and 3D spheroids

BackgroundObesity is poised to be a major healthcare crisis worldwide. Genetic predisposition, inadequate activity, changing lifestyle and dietary patterns are cited as major causes for obesity. Even as a number of anti-obesity medications hit the market, there is still an ongoing quest to explore natural compounds, which are perceived as safer alternatives, for their anti-obesity activity. This study explores the anti-obesity potential of dietary polyphenols vanillin, ferulic acid and their combination using 3T3 L1 adipocytes and their 3D spheroids. MethodsStudies were conducted on differentiated 3T3 L1 adipocytes and their 3D spheroids. Assays conducted on 3T3 L1 adipocytes include Oil red O, fluorescent Nile Red staining and triglyceride quantification to assess effect on lipid droplet accumulation. 2 NBDG was used to assess glucose uptake following drug treatment. 3D spheroid cultures were generated and triglyceride content was quantified. Effect of drug treatment on gene expression was analysed using qRT-PCR. Results of monolayer culture were compared with 3D spheroid models. ResultsVanillin, ferulic acid and their combination lower intracellular triglyceride content and lipid droplet accumulation, inhibiting glucose uptake and conversion to triglycerides in 3T3 L1 adipocytes and their 3D spheroids. Compounds and their combination downregulated mRNA expression of C/EBP α and PPAR ɣ, FAS, ACC1, GLUT4, LPL, aP2. Vanillin treatment upregulated leptin mRNA expression. ConclusionVanillin, ferulic acid and their combination lower lipid accumulation and glucose uptake in 3T3 L1 adipocytes and 3D spheroids.

CLDN6 inhibits breast cancer growth and metastasis through SREBP1-mediated RAS palmitoylation

BackgroundBreast cancer (BC) ranks as the third most fatal malignant tumor worldwide, with a strong reliance on fatty acid metabolism. CLDN6, a candidate BC suppressor gene, was previously identified as a regulator of fatty acid biosynthesis; however, the underlying mechanism remains elusive. In this research, we aim to clarify the specific mechanism through which CLDN6 modulates fatty acid anabolism and its impact on BC growth and metastasis.MethodsCell function assays, tumor xenograft mouse models, and lung metastasis mouse models were conducted to evaluate BC growth and metastasis. Human palmitic acid assay, triglyceride assay, Nile red staining, and oil red O staining were employed to investigate fatty acid anabolism. Reverse transcription polymerase chain reaction (RT–PCR), western blot, immunohistochemistry (IHC) assay, nuclear fractionation, immunofluorescence (IF), immunoprecipitation and acyl–biotin exchange (IP-ABE), chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP) were applied to elucidate the underlying molecular mechanism. Moreover, tissue microarrays of BC were analyzed to explore the clinical implications.ResultsWe identified that CLDN6 inhibited BC growth and metastasis by impeding RAS palmitoylation both in vitro and in vivo. We proposed a unique theory suggesting that CLDN6 suppressed RAS palmitoylation through SREBP1-modulated de novo palmitic acid synthesis. Mechanistically, CLDN6 interacted with MAGI2 to prevent KLF5 from entering the nucleus, thereby restraining SREBF1 transcription. The downregulation of SREBP1 reduced de novo palmitic acid synthesis, hindering RAS palmitoylation and subsequent endosomal sorting complex required for transport (ESCRT)-mediated plasma membrane localization required for RAS oncogenic activation. Besides, targeting inhibition of RAS palmitoylation synergized with CLDN6 to repress BC progression.ConclusionsOur findings provide compelling evidence that CLDN6 suppresses the palmitic acid-induced RAS palmitoylation through the MAGI2/KLF5/SREBP1 axis, thereby impeding BC malignant progression. These results propose a new insight that monitoring CLDN6 expression alongside targeting inhibition of palmitic acid-mediated palmitoylation could be a viable strategy for treating oncogenic RAS-driven BC.Graphical

The importance of stratum corneum ω-linoleoyloxyacylceramides in human skin barrier health: their biochemistry, processing enzymes and metabolites involved in corneocyte lipid envelope maturation.

Over the past 50 years there have been great strides made in the discovery of the composition and relevance of the total stratum corneum (SC) ceramide matrix. However, the focus of this review is on the free intercellular class of ω-linoleoyloxyacylceramides, corneocyte-bound ceramides and associated lipids known as the corneocyte lipid envelope (CLE) together with their processing enzymes involved in aiding ceramide attachment the corneocyte protein envelope (CPE). Two structural models and partially shared biosynthetic pathways have been proposed for the attachment of CPE-bound O-ceramides (ω-hydroxyceramides attached to glutamate residues of proteins in the (CPE) using the 12R-lipoxygenase (12R-LOX)/epidermal lipoxygenase-3 (eLOX3)/epoxide hydrolase-3 (EPHX3)/unknown esterase/ transglutaminase-1 (TG1) attachment pathway) and CPE-bound EO-ceramides (epoxy-enone ceramides attached to cysteine residues of proteins in the CPE using the 12R-LOX/eLOX3/short chain dehydrogenase/reductase family 9C member 7 (SDR9C7)/non-enzymatic attachment pathway), i.e. there is a bifurcation step beyond epidermal eLOX3. Their formation and structures will be discussed as well as their relevance in compromised skin barrier conditions together with our own work on SC maturation examined by proteomics, lipidomics, enzyme immunolocalization studies, mechanical fragility assays and Nile red staining of corneocyte envelopes (CE). Reduced levels of 12R-LOX, eLOX3, SDR9C7 and TG1 were observed in photodamaged skin of the cheeks that were associated with reduced SC maturation as evidenced by Nile red staining and increased CE fragility. In the severely photodamaged cheeks of Albino African SC we also observed increased levels of acylceramides. Concomitantly by reducing the activity of 12R-LOX by antibody inhibition and TG1 inhibition with a known chemical inhibitor, we demonstrated in a humidity-based exvivo SC maturation model that these enzymes contributed to increased CE hydrophobicity and mechanical integrity. We hypothesize that at least the CPE-bound O-ceramide pathway is operational in the SC. Nevertheless, our understanding of the full complexity of ω-linoleoyloxyacylceramides and the composition of the CLE is limited particularly on cosmetically relevant body sites such as the face.

Epoxiconazole disturbed metabolic balance and gut microbiota homeostasis in juvenile zebrafish

Epoxiconazole (EPX) is a broad-spectrum fungicide extensively used in agricultural pest control. Emerging evidence suggests that EPX can adversely affect different endpoints in non-target organisms. Here, the toxicity of EPX was assessed using earlier developmental stage of zebrafish as a model to investigate its effects on metabolism and intestinal microbiota after 21 days of exposure. Our findings indicated that EPX exposure resulted in physiological alterations in juvenile zebrafish, including increase in triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and glycose (Glu). Nile red staining demonstrated enhanced lipid accumulation in juvenile, accompanied by a marked upregulation in the expression of genes associated with TG synthesis. Moreover, EPX led to alterations in amino acids and carnitines levels in 21 dpf (days post fertilization) zebrafish. We also observed that EPX disrupted intestinal barrier function in juvenile zebrafish, manifested by decreasing mucus secretion and changing in genes related to tight junctions. Moreover, for a more comprehensive analysis of the intestinal microbiota in 21 dpf zebrafish, the intestine tissues were dissected under a microscope for 16S rRNA sequencing analysis. The results revealed that EPX altered the structure and abundance of intestinal microflora in zebrafish, including decreased alpha diversity indices and shifted in bacteria at phylum and genus levels. Notably, the correlation analysis demonstrated strong associations between alterations in various pathogenic bacterial genera and levels of amino acids and carnitines. Overall, these findings confirm that the fungicide EPX promotes metabolic disorders and alterations in the intestinal micro-environment in 21 dpf zebrafish, shedding light on the toxicologic effects of chemicals to aquatic organisms during the development stage.

Embryology of a Lady’s Slipper Orchid, Paphiopedilum spicerianum and Cytokinin Requirements for Seed Germination and Protocorm Development

In this study, we document the primary structural changes that occur during the seed development of Paphiopedilum spicerianum (Rchb.f.) Pfitzer, an endangered species with high horticultural value. Within a defined timeline, our results offer insights into the connection between these structural changes in seeds and their germination percentage. The optimum germination was recorded for immature seeds collected at 180 to 210 days after pollination (DAP), during which the embryos are in the late globular stage and the suspensor begins to degenerate. As seeds continued to mature by 240 DAP, there was a gradual decline in germination. Histochemical staining of mature seeds reveals that only the inner seedcoat and the surface of the embryo exhibit positive reactions to the Nile red stain, suggesting a relatively weak coat-imposed dormancy. This weaker dormancy may contribute to the higher germination observed in mature seeds of P. spicerianum compared with other challenging-to-germinate species. Of the cytokinins examined, 6-(γ,γ-dimethylallylamino)purine (2iP), kinetin (KN), and 6-benzylaminopurine (BA) exhibited a stimulating effect on germination, concurrently enhancing the formation of amorphous protocorms.

Juvenile hormone-induced microRNA miR-iab-8 regulates lipid homeostasis and metamorphosis in Drosophila melanogaster.

Metamorphosis plays an important role in the evolutionary success of insects. Accumulating evidence indicated that microRNAs (miRNAs) are involved in the regulation of processes associated with insect metamorphosis. However, the miRNAs coordinated with juvenile hormone (JH)-regulated metamorphosis remain poorly reported. In the present study, using high-throughput miRNA sequencing combined with Drosophila genetic approaches, we demonstrated that miR-iab-8, which primarily targets homeotic genes to modulate haltere-wing transformation and sterilitywas up-regulated by JH and involved in JH-mediated metamorphosis. Overexpression of miR-iab-8 in the fat body resulted in delayed development and failure of larval-pupal transition. Furthermore, metabolomic analysis results revealed that overexpression of miR-iab-8 caused severe energy metabolism defects especially the lipid metabolism, resulting in significantly reduced triacylglycerol (TG) content and glycerophospholipids but enhanced accumulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In line with this, Nile red staining demonstrated that during the third larval development, the TG content in the miR-iab-8 overexpression larvae was continuously decreased, which is opposite to the control. Additionally, the transcription levels of genes committed to TG synthesis and breakdown were found to be significantly increased and the expression of genes responsible for glycerophospholipids metabolism were also altered. Overall, we proposed that JH induced miR-iab-8 expression to perturb the lipid metabolism homeostasis especially the TG storage in the fat body, which in turn affected larval growth and metamorphosis.

Spatial and temporal trends in microplastic pollution in the Eastern Oyster, Crassotrea virginica, in Tampa Bay, Florida

Microplastic pollution is an increasingly alarming concern with widespread global distribution in aquatic environments. Spatial and temporal differences in microplastic abundance were evaluated in the Eastern Oyster, Crassostrea virginica, at six sites within Tampa Bay. Oyster tissue was digested using 30 % hydrogen peroxide (H2O2) and microplastics were quantified using Nile Red stain and fluorescent particle excitement. A total of 3025 microplastics were found throughout six study sites over two seasons (winter 2021 and summer 2022) with varying site types. Microfragments (n=2867) made up the majority of microplastics, as compared to microfibers (n=158). Significant differences were observed among the sites studied, site type, and their location in the bay. Outflow and marina areas had significantly higher (p<0.01) amounts of microplastics compared to preserve areas, and the east bay had significantly higher (p<0.05) amounts than the west bay. Findings suggest microplastic contamination is associated with higher urbanization, proximity to drainage basins, and recreation.

Release Characteristics of Small-Sized Microplastics in Bottled Drinks Using Flow Cytometry Sorting and Nile Red Staining

Small-sized microplastics (MPs) pose concerns about potential risks to both the environment and human health. However, research on MP pollution is hampered by limitations in the detection techniques. Also, few studies have provided insight into the release of small-sized MPs from disposable polyethylene terephthalate (PET)-bottled drinks for outdoor usage. Thus, PET bottles’ potential to release small MPs sized 1–100 μm outdoors was studied in relation to physical and chemical parameters (temperature, sunlight irradiation, and drink characteristics) using flow cytometry and Nile Red dye. The results showed that temperatures below 80 °C had little effect on the release of MPs from PET bottles. Sunlight irradiation and alkalinity were prone to promote the generation of MPs, mostly 1–5 μm in size. Moreover, the combined impact test implied that two pairings—acidity with temperature and alkalinity with sunlight—positively affected MP release, with maximum releases of 21,622 ± 2477 particles/L and 31,081 ± 7173 particles/L, respectively. Based on the rapid quantification of small-sized MPs using flow cytometry after Nile Red selection, the results hereby presented will assist researchers in reducing MP release and aid them in the evaluation of MPs’ contamination of aquatic environments.

The protective effects of Kefir extract (KE) on intestinal damage in larval zebrafish induced by Oxytetracycline: Insights into intestinal function, morphology, and molecular mechanisms

The antibiotic oxytetracycline (OTC) can be detected in contemporary natural aquatic environments and has been implicated in causing intestinal damage in humans exposed to OTC-contaminated food or water. The irreversible damage caused by high concentrations of OTC to the intestine suggests that treatment through dietary means could still be necessary. This study proved the effectiveness of kefir extract (KE) in reversing intestinal damage caused by oxytetracycline (OTC) exposure. Following a 24-hour KE treatment subsequent to OTC exposure from 3 to 8 days post-fertilization of zebrafish larvae, molecular-level and microbiomic assessments revealed significant improvements. These included reduced expression of proinflammatory factors (IL-8 and IL-1β), increased antioxidant levels, and reversed unhealthy distribution of intestinal microbiota. Furthermore, KE supplementation showed potential in enhancing intestinal motility in the experiment of Nile red staining and fluorescent microbead transit. However, histological analysis showed that this short-term treatment with KE only partially reversed the intestinal morphological changes induced by OTC, suggesting that a longer treatment period might be necessary for complete restoration.

Plastic takeaway food containers may cause human intestinal damage in routine life usage: Microplastics formation and cytotoxic effect

The microplastics and organic additives formed in routine use of plastic takeaway food containers may pose significant health risks. Thus, we collected plastic containers made of polystyrene, polypropylene, polyethylene terephthalate, polylactic acid and simulated two thermal usages, including hot water (I) and microwave treatments (M). Nile Red fluorescence staining was developed to improve accurate counting of microplastics with the aid of TEM and DLS analysis. The quantity of MPs released from thermal treatments was determined ranging from 285.7 thousand items/cm2 to 681.5 thousand items/cm2 in containers loaded with hot water with the following order: IPS>IPP>IPET>IPLA, while microwave treatment showed lower values ranging from 171.9 thousand items/cm2 to 301.6 thousand items/cm2. In vitro toxicity test using human intestinal epithelial Caco-2 cells indicated decrease of cell viability in raw leachate, resuspended MPs and supernatants, which might further lead to cell membrane rupture, ROS production, and decreased mitochondrial membrane potential. Moreover, the leachate inhibited the expression of key genes in the electron transport chain (ETC) process, disrupted energy metabolism. For the first time, we isolate the actually released microplastics and organic substances for in vitro toxicity testing, and demonstrate their potential impacts to human intestine. SynopsisPlastic take-out containers may release microplastics and organic substances during daily usage, both of which can cause individual and combined cytotoxic effects on human colon adenocarcinoma cells Caco-2.

Optimization of sample preparation, fluorescence- and Raman techniques for environmental microplastics

Microspectroscopic imaging techniques based on spontaneous Raman scattering, Stimulated Raman Scattering (SRS), or fluorescence (with a selective dye) can be used to detect environmental microplastics (MPs) and determine their chemical as well as physical properties. The present study first focuses on optimizing the sample preparation, including a new design for a density separation apparatus and optimization of the Nile Red staining procedure. Tests were carried out with both white and colored reference materials, as well as environmental MPs in a suspended matter sample from the Rhine river. The new ‘MESSY’ system has a mean recovery of 95 ± 5.5 % (three polymer materials, in duplicate). The optimized Nile Red staining allows coarse categorization of MPs into “polar” vs. “non-polar” materials based on their Fluorescence Index (emission wavelength), but fluorescent additives in the polymer can cause misclassification. For unambiguous identification of the polymer type, Raman spectroscopy can be used. Even colored polymers, with or without Nile Red staining, were readily identified by Raman spectroscopy using a red laser (785 nm), except for particles containing carbon black. A Deep-UV Raman microscope (ex = 248.6 nm) was constructed, which allowed identification of all colored plastics, even those pigmented with carbon black. Since unsupervised mapping with spontaneous Raman is very slow, point measurements are preferably used after preselection of particles of interest based on fluorescence imaging. SRS is several orders of magnitude faster than spontaneous Raman mapping but requires multiple scans at different z-heights and at multiple wavenumber settings to detect and identify all particles. The results are expected to contribute to the development of suitable methodologies for the detection and identification of environmental microplastics.