Nile Red Staining Research Articles

Size Distributions of Microplastics in the St Louis Estuary and Western Lake Superior.

Identifying the sources and fate of microplastics in natural systems has garnered a great deal of attention because of their implications for ecosystem health. This work characterizes the size fraction, morphology, color, and polymer composition of microplastics in western Lake Superior and its adjacent harbor sampled in August and September 2021. The results reveal that the overall microplastic counts are similar, with the harbor stations ranging from 0.62 to 3.32 microplastics per liter and the lake stations ranged from 0.83 to 1.4 microplastics per liter. However, meaningful differences between the sample locations can be seen in the size fraction trends and polymer composition. Namely, the harbor samples had relatively larger amounts of the largest size fraction and more diversity of polymer types, which can be attributed to the urbanized activity and shorter water residence time. Power law size distribution modeling reveals deviations that help in the understanding of potential sources and removal mechanisms, although it significantly underpredicts microplastic counts for smaller-sized particles (5-45 μm), as determined by comparison with concurrently collected microplastic samples enumerated by Nile Red staining and flow cytometry.

Anti-adipogenesis effect of indole-3-acrylic acid on human preadipocytes and HFD-induced zebrafish.

Obesity, defined as excessive or abnormal body fat accumulation, which could significantly increase the risk of cardiovascular disease, type 2 diabetes mellitus (T2DM) diseases and seriously affect people's quality of life. More than 2 billion people are overweight, and the incidence of obesity is increasing rapidly worldwide, it has become a widely concerned public health issue in the world. Diverse evidence show that active metabolites are involved in the pathophysiological processes of obesity. However, whether the downstream catabolite of tryptophan, 3-indole acrylic acid (IA), is involved in obesity remains unclear. We collected the samples of serum from peripheral blood of obesity and health controls, and liquid chromatography-mass spectrometry (LC-MS) was performed to identify the plasma levels of IA. Additionally, we verified the potential benefits of IA on human preadipocytes and HFD- induced zebrafish by cell viability assay, flow cytometry assay, Oil red O staining, total cholesterol (T-CHO), triglyceride (TG) and nonesterified free fatty acids (NEFA) measurements and Nile Red staining. RNA-Seq, functional analysis and western blot revealed the mechanisms underlying the function of IA. We found that the content of IA in peripheral blood serum of overweight people was significantly lower than that of normal people. In addition, supplementation with IA in zebrafish larvae induced by a high fat diet (HFD) dramatically reduced HFD induced lipid accumulation. IA had no effect on proliferation and apoptosis of preadipocytes, but significantly inhibited adipogenesis of preadipocytes by down-regulate CEBPα and PPARγ. RNA-Seq and functional analysis revealed that IA regulated the adipogenesis of preadipocytes through stimulate the phosphorylation of STAT1. Taken together, IA has been identified as a potent metabolite for the prevention or treatment of obesity.

Microplastics in personal care products and cosmetics in Sri Lanka

In the Sri Lankan context, the lack of baseline studies to mitigate microplastic emissions through personal care and cosmetic products poses a huge problem. Hence this study serves as the first scientific investigation to analyze and characterize microplastics in selected personal care and cosmetic items available in the Sri Lankan markets. Fifteen brands representing five categories (face wash, facial scrubs, baby creams, shaving creams, and skin creams) of personal care and cosmetic items served as the basis for this investigation. Based on a questionnaire survey, from each category, three highly utilized brands were chosen and triplicates from each brand were used for the analysis. All samples were treated with the Fenton reagent to extract microplastics. Then through Nile red staining suspected microplastic were screened and characterized through FT-IR spectroscopy. The Nile Red analysis revealed seven brands of the fifteen to be stained with Nile Red and demonstrate luminance properties under UV light. However, FT-IR analysis proved only six brands contained actual microplastics. Low-density polyethylene and ethylene-propylene copolymer were the dominant types of microplastic. Most microplastics were irregularly shaped and white in color with sizes ranging from 238.55 ± 50.74 to 450.69 ± 174.9 μm. An emission estimation revealed that products FS-01 and FW-03 contain 3.36 ± 0.20 g and 0.2 ± 0.05 g of isolatable microplastics per product. While the present study provides scientific evidence for the availability of microplastics in products in Sri Lankan markets, it also provides a great opportunity to develop relevant policies and regulations to control them.

Anti-Diabetic Activity of an Extract of Syzygium Jambolanum – A Review

Syzygium jambolanum is a promising natural treatment for diabetes. The potential benefits of S jambolanum for diabetes include lowering blood sugar levels, increasing insulin sensitivity, protecting pancreatic beta cells, and slowing the absorption of glucose into the bloodstream. The anti-diabetic activity of the crude extract of S jambolanum was evaluated in L6 myotubes and the lipid deposition in tissue was measured using Nile red Staining. Nile red staining confirmed that a considerable quantity of lipids had been deposited in the tissue treated with a crude extract of S jambolanum, comparable to the quantity of lipids deposited with a standard drug known as Rosiglitazone. This study analyzed the anti-diabetic activity of a crude extract of S jambolanum to understand its potential as a feedstock for extracting bioactive constituents to screen for bioactive molecules in the treatment of diabetes.

RBP7 functions as a tumor suppressor in HR + breast cancer by inhibiting the AKT/SREBP1 pathway and reducing fatty acid

BackgroundIncreasing evidence proves that RBP7 plays a significant role in breast cancer (BC). The present study was aimed to investigate the mechanism of RBP7.MethodsWestern Blotting and qRT-PCR were performed for evaluating the expression levels. CCK8, colony forming, xenograft mouse model, wound healing and transwell assays were conducted to examine cell ability of proliferation, invasion and migration. Nile red staining and Oil red O staining were used for testing the lipid.ResultsRBP7 was related to overall survival (OS) in patients with HR + BC. RBP7 protein was significantly decreased in HR + BC tissues and cells. RBP7 suppressed HR + BC cell proliferation in vitro and in vivo, and inhibited migration and invasion. RBP7 reduced fatty acid in HR + BC cells by inhibiting the AKT/SREBP1 pathway.ConclusionsRBP7 may function as a tumor suppressor in HR + BC by inhibiting the AKT/SREBP1 pathway and reducing fatty acid.

Ticagrelor, but Not Clopidogrel, Attenuates Hepatic Steatosis in a Model of Metabolic Dysfunction-Associated Steatotic Liver Disease.

Previous studies have suggested that platelets are associated with inflammation and steatosis and may play an important role in liver health. Therefore, we evaluated whether antiplatelet agents can improve metabolic disorder-related fatty liver disease (MASLD). The mice used in the study were fed a high-fat-diet (HFD) and were stratified through liver biopsy at 18 weeks. A total of 22 mice with NAFLD activity scores (NAS) ≥ 4 were randomly divided into three groups (HFD-only, clopidogrel (CLO; 35 mg/kg/day), ticagrelor (TIC; 40 mg/kg/day) group). And then, they were fed a feed mixed with the respective drug for 15 weeks. Blood and tissue samples were collected and used in the study. The TIC group showed a significantly lower degree of NAS and steatosis than the HFD group (p = 0.0047), but no effect on the CLO group was observed. Hepatic lipogenesis markers' (SREBP1c, FAS, SCD1, and DGAT2) expression and endoplasmic reticulum (ER) stress markers (CHOP, Xbp1, and GRP78) only reduced significantly in the TIC treatment group. Inflammation genes (MCP1 and TNF-α) also decreased significantly in the TIC group, but not in the CLO group. Nile red staining intensity and hepatic lipogenesis markers were reduced significantly in HepG2 cells following TIC treatment. Ticagrelor attenuated NAS and hepatic steatosis in a MASLD mice model by attenuating lipogenesis and inflammation, but not in the CLO group.

Citrus sunki Peel Extract, Containing Nobiletin and Tangeretin, Enhances Proliferation and Differentiation in 3T3-L1 Adipocytes

In this experiment, effects of Citrus sunki peel extract (CPE) on proliferation and differentiation of 3T3-L1 cells were analyzed. Citrus sunki peel was extracted with ethanol to obtain CPE. Results of measuring DPPH and hydrogen peroxide radical scavenging activity revealed that CPE had an antioxidant ability. The 3T3-L1 cells were cultured in the basal medium (C) or with 0.05% dimethyl-sulfoxide (CDMSO) or with 50, 100, 200, 300, and 400 μg/mL CPE (CPE50, CPE100, CPE200, CPE300, and CPE400, respectively). As a result of cell counting and MTS assay, CPE significantly enhanced proliferation capacity of 3T3-L1 cells. To analyze the effect of CPE on the differentiation capacity of 3T3-L1 cells, relative gene and protein expression levels of Cebpb, Cebpa, and FASN related to adipogenesis were measured by RT-qPCR and Western blot. CPE increased adipogenesis-related gene and protein expression in 3T3-L1 cells, with CPE300 being especially effective. In Oil Red O staining, adipogenesis was significantly greater in CPE200, CPE300, and CPE400 than in C. This was confirmed image wise through Nile Red staining. Through HPLC analysis, it was confirmed that nobiletin and tangeretin were most abundant in CPE. To analyze effects of nobiletin and tangeretin on 3T3-L1 cells, the cells were cultured with various concentrations of nobiletin, tangeretin, and both. As a result of cell counting and MTS assay, nobiletin and tangeretin significantly decreased the proliferation capacity of 3T3-L1 cells. In RT-qPCR, western blot, Oil Red O staining, and Nile Red staining to analyze differentiation capacity, nobiletin and tangeretin significantly enhanced the expression of genes and proteins related to adipogenic differentiation compared to the control group. In conclusion, CPE, which had high antioxidant capacity, enhanced proliferation and differentiation in 3T3-L1 adipocytes. This improvement in differentiation is due to the influence of nobiletin and tangeretin contained in CPE.

Yunvjian decoction mitigates hyperglycemia in rats induced by a high-fat diet and streptozotocin via reducing oxidative stress in pancreatic beta cells

Ethnopharmacological relevanceYunvjian (YNJ), a traditional Chinese herbal formula first reported in Jing Yue Quan Shu, is commonly used in the clinical treatment of type 2 diabetes mellitus (T2DM). However, the mechanism by which YNJ affects T2DM remains unclear. Aim of the studyThis study aimed to assess the therapeutic effects of YNJ on T2DM and explore the potential mechanism involved. Materials and methodsHigh-performance liquid chromatography (HPLC) was used to identify the chemical compounds of YNJ. The anti-T2DM effects of YNJ were observed in a high-fat diet/streptozotocin induced rat model. The type 2 diabetic rats were prepared as follows: rats were fed a high-fat diet for four weeks and then intraperitoneally injected with a low dose (30 mg/kg) of streptozotocin. YNJ and the positive control metformin were used in these experiments. Biochemical assays were implemented to determine the fasting blood glucose, glucose tolerance, insulin sensitivity, serum lipid levels, and oxidative stress index of the pancreas. Hematoxylin-eosin (H&E) staining was used to assess histopathological alterations in the pancreas. The mechanism by which YNJ affects T2DM was evaluated in INS-1 cells treated with glucose and high sodium palmitate. YNJ-supplemented serum was used in these experiments. Methyl thiazolyl tetrazolium assays, enzyme-linked immunosorbent assays, Nile red staining, flow cytometric analysis, and Western blotting were used to assess apoptosis, insulin secretion, lipid accumulation, reactive oxygen species production, and protein levels. ResultsFive major compounds were identified in YNJ. In high-fat diet/streptozotocin-induced diabetic rats, YNJ-M notably decreased fasting blood glucose and lipid levels; ameliorated glucose tolerance, insulin sensitivity, and islet morphology; reduced Malondialdehyde levels; and restored superoxide dismutase activity in the pancreatic islets. Furthermore, the effect of YNJ-M was significantly greater than that of YNJ-L, and YNJ-H had little effect on diabetic rats. In vitro experiments revealed that YNJ-supplemented serum (10%, 15%, and 20%) dramatically suppressed apoptosis, mitigated intracellular lipid accumulation and reduced intracellular oxidative stress levels in a dose-dependent manner. Additionally, YNJ-supplemented serum increased the protein expression of Nuclear factor erythroid 2-related factor 2, Heme oxygenase-1, and superoxide dismutase 1 and inhibited the protein expression of Kelch-like ECH-associated protein 1. ConclusionYNJ ameliorates high-fat diet/streptozotocin induced experimental T2DM. The underlying mechanism involves reducing oxidative stress in pancreatic beta cells. The findings of this study provide scientific justification for the application of the traditional medicine YNJ in treating T2DM.

Pennogenin 3-O-β-Chacotrioside Attenuates Hypertrophied Lipid Accumulation by Enhancing Mitochondrial Oxidative Capacity.

Excessive lipid accumulation in adipocytes is a primary contributor to the development of metabolic disorders, including obesity. The consumption of bioactive compounds derived from natural sources has been recognized as being safe and effective in preventing and alleviating obesity. Therefore, we aimed to explore the antilipidemic effects of pennogenin 3-O-β-chacotrioside (P3C), a steroid glycoside, on hypertrophied 3T3-L1 adipocytes. Oil Red O and Nile red staining demonstrated a P3C-induced reduction in lipid droplet accumulation. Additionally, the increased expression of adipogenic and lipogenic factors, including PPARγ and C/EBPα, during the differentiation process was significantly decreased by P3C treatment at both the protein and mRNA levels. Furthermore, P3C treatment upregulated the expression of fatty acid oxidation-related genes such as PGC1α and CPT1a. Moreover, mitochondrial respiration and ATP generation increased following P3C treatment, as determined using the Seahorse XF analyzer. P3C treatment also increased the protein expression of mitochondrial oxidative phosphorylation in hypertrophied adipocytes. Our findings suggest that P3C could serve as a natural lipid-lowering agent, reducing lipogenesis and enhancing mitochondrial oxidative capacity. Therefore, P3C may be a promising candidate as a therapeutic agent for obesity-related diseases.

Lipid metabolism-related long noncoding RNA RP11-817I4.1 promotes fatty acid synthesis and tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of tumors. The influence of lipid metabolism disruption on the development of HCC has been demonstrated in published studies. To establish an HCC prognostic model for lipid metabolism-related long non-coding RNAs (LMR-lncRNAs) and conduct in-depth research on the specific role of novel LMR-lncRNAs in HCC. Correlation and differential expression analyses of The Cancer Genome Atlas data were used to identify differentially expressed LMR-lncRNAs. Quantitative real-time polymerase chain reaction analysis was used to evaluate the expression of LMR-lncRNAs. Nile red staining was employed to observe intracellular lipid levels. The interaction between RP11-817I4.1, miR-3120-3p, and ATP citrate lyase (ACLY) was validated through the performance of dual-luciferase reporter gene and RIP assays. Three LMR-lncRNAs (negative regulator of antiviral response, RNA transmembrane and coiled-coil domain family 1 antisense RNA 1, and RP11-817I4.1) were identified as predictive markers for HCC patients and were utilized in the construction of risk models. Additionally, proliferation, migration, and invasion were reduced by RP11-817I4.1 knockdown. An increase in lipid levels in HCC cells was significantly induced by RP11-817I4.1 through the miR-3120-3p/ACLY axis. LMR-lncRNAs have the capacity to predict the clinical characteristics and prognoses of HCC patients, and the discovery of a novel LMR-lncRNAs, RP11-817I4.1, revealed its role in promoting lipid accumulation, thereby accelerating the onset and progression of HCC.

Exosomes from adipose-derived stem cells activate sebocytes through the PI3K/AKT/SREBP-1 pathway to accelerate wound healing.

Sebocyte regeneration after injury is considered a key element of functional skin repair. Exosomes from adipose-derived stem cells (ADSCs-EXO) accelerate wound healing by promoting the proliferation of fibroblasts. However, the effects of ADSCs-EXO on sebocytes are largely unknown. In this study, the effects of ADSCs-EXO on sebocyte proliferation and migration were evaluated. The levels of phosphorylated AKT (p-AKT), AKT, sterol regulatory-element binding protein (SREBP), and perilipin-1 (PLIN-1) were detected with immunofluorescence, quantitative PCR, and western blot analysis. RNA-Seq was used to analyze the differential gene expression between the ADSCs-EXO group and the control group under anaerobic conditions. Lipogenesis was assessed with Nile red staining. In animal studies, full-thickness skin wounds in BALB/c mice were treated with gelatin methacrylate (GelMA) hydrogel-loaded sebocytes alone or in combination with ADSCs-EXO. Histopathological assessments of the wound tissues were performed Masson Trichrome staining, Immunohistochemical staining and so on. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway blocker LY294002 inhibited the effects of ADSCs-EXO on p-AKT and sebocytes proliferation. ADSCs-EXO also regulated the expression of SREBP-1 and PLIN-1 through the PI3K/AKT pathway in an oxygen level-dependent manner. In BALB/c mice, ADSCs-EXO accelerated sebocyte-assisted wound healing and regeneration. These in vitro and in vivo results supported that ADSCs-EXO can promote the regeneration of fully functional skin after injury through the PI3K/AKT-dependent activation of sebocytes.

High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most complex endocrine disorders in women of reproductive age. Abnormal proliferation of granulosa cells (GCs) is an important cause of PCOS. This study aimed to explore the role of fatty acid-binding protein 5 (FABP5) in granulosa cell (GC) proliferation in polycystic ovary syndrome (PCOS) patients. The FABP5 gene, which is related to lipid metabolism, was identified through data analysis of the gene expression profiles of GSE138518 from the Gene Expression Omnibus (GEO) database. The expression levels of FABP5 were measured by quantitative real-time PCR (qRT‒PCR) and western blotting. Cell proliferation was evaluated with a cell counting kit-8 (CCK-8) assay. Western blotting was used to assess the expression of the proliferation marker PCNA, and immunofluorescence microscopy was used to detect Ki67 expression. Moreover, lipid droplet formation was detected with Nile red staining, and qRT‒PCR was used to analyze fatty acid storage-related gene expression. We found that FABP5 was upregulated in ovarian GCs obtained from PCOS patients and PCOS mice. FABP5 knockdown suppressed lipid droplet formation and proliferation in a human granulosa-like tumor cell line (KGN), whereas FABP5 overexpression significantly enhanced lipid droplet formation and KGN cell proliferation. Moreover, we determined that FABP5 knockdown inhibited PI3K-AKT signaling by suppressing AKT phosphorylation and that FABP5 overexpression activated PI3K-AKT signaling by facilitating AKT phosphorylation. Finally, we used the PI3K-AKT signaling pathway inhibitor LY294002 and found that the facilitation of KGN cell proliferation and lipid droplet formation induced by FABP5 overexpression was inhibited. In contrast, the PI3K-AKT signaling pathway agonist SC79 significantly rescued the suppression of KGN cell proliferation and lipid droplet formation caused by FABP5 knockdown. FABP5 promotes active fatty acid synthesis and excessive proliferation of GCs by activating PI3K-AKT signaling, suggesting that abnormally high expression of FABP5 in GCs may be a novel biomarker or a research target for PCOS treatment.

Microplastics Trophic Transfer in Seafood Varieties Caught from the Coastal Waters off Negombo

Microplastics are a pervasive global pollutant that poses risks to marine organisms including commercially important species and poses threats even to humans. However, the trophic transfer of microplastics is yet to be understood. The present study was to study the occurrence of microplastics in five species used as seafood and in zooplankton, representing different trophic levels and feeding habits caught within coastal waters off Negombo. The Negombo coastal environs are known for high ecological diversity still subjected to massive anthropogenic activities as a major fishing and tourism hub in Sri Lanka. The same area was subjected to contaminating microplastics after the MV X-Press Pearl ship disaster which caught fire and spilled the largest point source of microplastic nurdles pollution in the world. Field sampling was carried out from October to-December 2021 in Negombo, Sri Lanka. The study included zooplankton, some fish (Sardinella gibbosa (n=10), Hemiramphus archipelagicus (n=10), and Ephipphus orbis (n=10)), crustaceans (Scylla serrata, (n=10)) and a bivalve (Perna perna, (n=10)). The samples were subjected to wet peroxide oxidation and acid digestion. Microplastic identification was conducted microscopically and confirmed with Fourier-Transform Infrared (FTIR) spectroscopy, Raman spectroscopy, and Nile red staining techniques. Microplastics were present in all the species studied except zooplankton samples. Four polymer types were identified and confirmed as Polyamide (PA), Polystyrene (PS), Polypropylene (PP), and Polycaprolactone (PCL). According to the literature, this is the first recorded PCL contamination in marine species in Sri Lanka. Fibers were the most abundant microplastic type recorded followed by fragments, films, spheres, and flakes. Red, green, blue, yellow, black, and transparent microplastics were recorded while blue colour was the most prominent. The mean number of microplastics recorded was 0.93±0.29 for S. gibbosa, 0.97±0.17 for H. archipelagicus, 0.53±0.09 for E. orbis, 0.36±0.16 for S. serrata and 1.13±0.29 items/g for P. perna. Investigation of microplastic concentrations at different trophic levels suggests that organisms at lower trophic levels are at greater risk of microplastics. 
 Keywords: Microplastics, Trophic transfer, Marine organisms, Seafood, Negombo 

Analytical Methods for Extraction of Microplastics from Compost in Sri Lanka

Microplastics (MP) are the plastics with 1 μm-5 mm dimensions. Meanwhile, compost has been identified as a major source of MP in the soil, although compost is a great plant nutrient source, and it provides tremendous benefits for crops. This is primarily ascribed to most of the compost being produced through Municipal Solid Waste Management projects. However, due to the detrimental impacts on soil’s biological, physical, and chemical health, together with the high accumulation probability of MP caused by repeated application of compost, regulations should be implemented on the limitation of MP in compost. Even though there are a few different methods available in the literature for MP extraction and quantification in organic matter (OM)-rich matrices, like compost, sludge, and sediments, a properly validated, routine analytical procedure is missing in Sri Lanka Standards (SLS). Therefore, in this project, the effectiveness of four different MP extraction methods in the literature were compared and validated for compost, to implement an MP analysis method suitable for compost specifications in the SLS. The methodologies included MP extraction by density separation using NaCl or ZnCl2 solutions followed by digestion of OM using 30% KOH: NaOCl solution or Fenton’s reagent. The common MP analysis method for the four protocols was staining with the hydrophobic, fluorescent stain Nile Red, and visual observation. Digestion efficiency of OM and spike recovery of Polystyrene (PS), Polyvinyl Chloride (PVC), Polyethylene terephthalate (PET), High-Density Polyethylene (HDPE), and Polypropylene (PP) microplastics in 1-2 mm dimension by the four MP extraction methods were compared for the validation. T-tests (confidence level=95%) were carried out to determine the significant difference between the OM digestion efficiency in each of the four methods. Each of the combinations compared was significantly different from the other except for ZnCl2/Fenton and NaCl/30% KOH:NaOCl comparison. Meanwhile, the OM removal by ZnCl2/Fenton and NaCl/30% KOH:NaOCl excluding the density separation step also was not significantly different. The spike recovery analysis showed ZnCl2/Fenton combination as the best out of the tested combinations with above 95% recovery percentages for PS, PVC, PP and 100 for HDPE and PET by particle count. However, precipitate formations in the ZnCl2/Fenton combination might cause false negative or positive results for spike recovery when applied to the real samples with smaller MP. The extraction reagents, NaCl was not an ideal solution and ZnCl2 was a good solution for the MP types used. Moreover, in this project, double digestion of OM with NaCl/30% KOH:NaOCl before and after the density separation with ZnCl2 is suggested as a novel and more effective OM digestion protocol before Nile Red staining, and further validations are suggested prior to its final implementation.
  
 Keywords: Microplastics, Compost, Extraction, Organic matter

Efficacy of Jiuzao polysaccharides in ameliorating alcoholic fatty liver disease and modulating gut microbiota

Jiuzao, the residue from Baijiu production, has shown radical scavenging properties in prior investigations, suggesting its potential as a hepatoprotective agent against acute liver damage. This study reveals that Jiuzao polysaccharides ameliorated liver morphological damage in zebrafish larvae afflicted with alcoholic fatty liver disease (AFLD), as evidenced by Oil red O, H&E, and Nile red staining. These polysaccharides notably modulated antioxidant enzyme levels and lipid peroxidation components. The real-time quantitative polymerase chain reactions analyses illustrated the significant impact of Jiuzao polysaccharides on genes integral to ethanol and lipid metabolism. The 16 S rRNA results showed that Jiuzao polysaccharides could improve the intestinal flora in zebrafish larvae exposed to ethanol. In summary, Jiuzao polysaccharides efficaciously mitigate liver lipid accumulation, enhance ethanol metabolism, and reduce oxidative stress by downregulating genes involved in AFLD development. They also regulate the changes in gut microbiota, providing further protection against acute alcoholic liver insult in zebrafish larvae.

The Endoplasmic Reticulum-Plasma Membrane Tethering Protein Ice2 Controls Lipid Droplet Size via the Regulation of Phosphatidylcholine in Candida albicans.

Lipid droplets (LDs) are intracellular organelles that play important roles in cellular lipid metabolism; they change their sizes and numbers in response to both intracellular and extracellular signals. Changes in LD size reflect lipid synthesis and degradation and affect many cellular activities, including energy supply and membrane synthesis. Here, we focused on the function of the endoplasmic reticulum-plasma membrane tethering protein Ice2 in LD dynamics in the fungal pathogen Candida albicans (C. albicans). Nile red staining and size quantification showed that the LD size increased in the ice2Δ/Δ mutant, indicating the critical role of Ice2 in the regulation of LD dynamics. A lipid content analysis further demonstrated that the mutant had lower phosphatidylcholine levels. As revealed with GFP labeling and fluorescence microscopy, the methyltransferase Cho2, which is involved in phosphatidylcholine synthesis, had poorer localization in the plasma membrane in the mutant than in the wild-type strain. Interestingly, the addition of the phosphatidylcholine precursor choline led to the recovery of normal-sized LDs in the mutant. These results indicated that Ice2 regulates LD size by controlling intracellular phosphatidylcholine levels and that endoplasmic reticulum-plasma membrane tethering proteins play a role in lipid metabolism regulation in C. albicans. This study provides significant findings for further investigation of the lipid metabolism in fungi.

Distribution, screening and biochemical characterization of indigenous microalgae for bio-mass and bio-energy production potential from three districts of Pakistan.

Trend of biofuel production from microalgal triacylglycerols is enhancing, because this substrate is a good sustainable and advantageous alternative to oil and gas fuel. In the present study, indigenous micro algal isolates were screened from water (n=30) and soil (n=30) samples collected from three districts of Punjab, Pakistan to evaluate their biofuel production potential. The samples were inoculated on BG - 11 agar medium plates by incubating at room temperature of 25°C providing 1000 lux for 16h light cycle followed by 8h of dark cycle for 15 d. Water samples were found to be rich in microalgae and 65.33% microalgae (49 isolates) were isolated from Faisalabad district. On the basis of microscopic morphology microalgal isolates (n=180) were selected and subjected to lipid detection by Nile red staining assay. Nile red positive isolates (n=23) were processed for biochemical (lipid, protein and carbohydrates) characterization. AIN63 isolate showed higher lipids (17.4%) content as detected by micro vanillin assay. Algal isolate AIN128 showed best protein contents (42.91%) detected by Bradford assay and AIN172 isolate showed higher carbohydrate contents (73.83%) as detected by anthrone assay. The selected algal isolates were also analyzed by Fourier transform infrared (FTIR) spectroscopy for confirmation of carbohydrate, protein and lipid analysis. These indigenous algae have the potential for in-vitro biofuel production from agricultural waste.

Effects of stearic acid on the embryo cryopreservation in mouse

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 μM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation.

Nile red staining of polyethylene and polystyrene in Daphnia magna tests

Microplastics pollution represents a stringent global issue. Their accumulation in the aquatic environment remains complicated due to the abundance, distribution, and resulting environmental effects they induce. Recently, assessing the toxicity level of aquatic organisms has gained scientific interest. Due to the diverse types and sizes of polymers present in the environment, the detection and evaluation of their effects are still a challenging issue. Polyethylene (PE) and polystyrene (PS) are the most commonly used polymers and are therefore predominantly detected in both marine and freshwater aquatic environments. This paper aims to evaluate the effects of Red Nile-stained PE and PS, tested in different particle sizes (PE 40-48, 125, >125 µm, and PS 20, 200, 430 µm) on Daphnia magna, and highlight them through microscopic analysis. Acute toxicity tests conducted over a 48-hours exposure did not reveal significant toxicity effects in terms of mortalities compared to the controls. Red Nile staining allowed the visualization of the shapes and sizes of the tested microplastics and their entry pathways into the Daphnia bodies. PS of 20 µm size was detected in the digestive tract of Daphnia, indicating as primary pathway of entry into the body of aquatic organisms. No acute toxic effects were recorded as a result of direct exposure to PE and PS particles. However, sub-lethal effects such as feeding and growth disturbances, which could affect organisms in the long term, are suspected.

Organophosphate ester cresyl diphenyl phosphate disrupts lipid homeostasis in zebrafish embryos

As a new class of organophosphate ester, cresyl diphenyl phosphate (CDP) has been widely monitored in environmental matrices and human samples, nonetheless, its toxicity is not fully understood. Here we described an in-depth analysis of the disruptions in lipid homeostasis of zebrafish following exposure to CDP concentrations ranging from 2.0 to 313.0 μg/L. Nile red staining revealed significant alterations in lipid contents in 72 hpf zebrafish embryos at CDP concentrations of 5.3 μg/L and above. Lipidomic analysis unveiled substantial disruptions in lipid homeostasis. Notably, disruptive effects were detected in various lipid classes, including phospholipids (i.e. cardiolipin, lysophosphatidylcholine, and phosphatidylethanolamine), glycerolipids (triglycerides), and fatty acids (fatty acids (FA) and wax esters (WE)). These alterations were further supported by transcriptional changes, with remarkable shifts observed in genes associated with lipid synthesis, transport, and metabolism, encompassing phospholipids, glycerolipids, fatty acids, and sphingolipids. Furthermore, CDP exposure elicited a significant elevation in ATP content and swimming activity in embryos, signifying perturbed energy homeostasis. Taken together, the present findings underscore the disruptive effects of CDP on lipid homeostasis, thereby providing novel insights essential for advancing the health risk assessment of organophosphate flame retardants.