Nile Red Staining Research Articles

Chaperonin counteracts diet-induced non-alcoholic fatty liver disease by aiding sirtuin 3 in the control of fatty acid oxidation.

The mitochondrial chaperonin heat shock protein (HSP) 60 is indispensable in protein folding and the mitochondrial stress response; however, its role in nutrient metabolism remains uncertain. This study investigated the role of HSP60 in diet-induced non-alcoholic fatty liver disease (NAFLD). We studied human biopsies from individuals with NAFLD, murine high-fat-diet (HFD; a diet with 60% energy from fat)-induced obesity (DIO), transgenic (Tg) mice overexpressing Hsp60 (Hsp60-Tg), and human HepG2 cells transfected with HSP60 cDNA or with HSP60 siRNA. Histomorphometry was used to assess hepatic steatosis, biochemistry kits were used to measure insulin resistance and glucose tolerance, and an automated home cage phenotyping system was used to assess energy expenditure. Body fat was assessed using MRI. Macrophage infiltration, the lipid oxidation marker 4-hydroxy-2-nonenal (4-HNE) and the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were detected using immunohistochemistry. Intracellular lipid droplets were evaluated by Nile red staining. Expression of HSP60, and markers of lipogenesis and fatty acid oxidation were quantified using RT-PCR and immunoblotting. Investigations were analysed using the two-way ANOVA test. Decreased HSP60 expression correlated with severe steatosis in human NAFLD biopsies and murine DIO. Hsp60-Tg mice developed less body fat, had reduced serum triglyceride levels, lower levels of insulin resistance and higher serum adiponectin levels than wild-type mice upon HFD feeding. Respiratory quotient profile indicated that fat in Hsp60-Tg mice may be metabolised to meet energy demands. Hsp60-Tg mice showed amelioration of HFD-mediated hepatic steatosis, M1/M2 macrophage dysregulation, and 4-HNE and 8-OHdG overproduction. Forced HSP60 expression reduced the mitochondrial unfolded protein response, while preserving mitochondrial respiratory complex activity and enhancing fatty acid oxidation. Furthermore, HSP60 knockdown enhanced intracellular lipid formation and loss of sirtuin 3 (SIRT3) signalling in HepG2 cells upon incubation with palmitic acid (PA). Forced HSP60 expression improved SIRT3 signalling and repressed PA-mediated intracellular lipid formation. SIRT3 inhibition compromised HSP60-induced promotion of AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor α (PPARα levels), while also decreasing levels of fatty acid oxidation markers. Mitochondrial HSP60 promotes fatty acid oxidation while repressing mitochondrial stress and inflammation to ameliorate the development of NAFLD by preserving SIRT3 signalling. This study reveals the hepatoprotective effects of HSP60 and indicates that HSP60 could play a fundamental role in the development of therapeutics for NAFLD or type 2 diabetes.

A spatial and temporal assessment of microplastics in seafloor sediments: A case study for the UK

Seafloor sediments have been defined as sinks for microplastics in the marine environment and could therefore represent suitable matrices for their long-term monitoring. On a regional aspect, the adoption of a common indicator for microplastics in seafloor sediments would allow regional monitoring and assessments through international frameworks such as OSPAR. This study presents the findings of a research and development project monitoring the occurrence and abundance of microplastics in UK seafloor sediments for the period 2013-2021, supporting the development of a national monitoring programme for microplastics in seafloor sediments for the UK (England and Wales). A fast-screening technique based on Nile Red staining of polymers coupled with µ-FTIR was applied and validated using µ-FTIR-FPA. Microplastic particles were detected in all 189 sediment samples from 15 stations selected around the UK for each investigated year (2013 - 2021). Microplastic concentrations (20 – 5000 µm) ranged from 133 – 6,933 particles kg-1 dry weight sediment for 2020-2021. Microplastics mainly consisted of fragments (73%), fibres (19%) and spheres (8%). Plastic fragments ranged from 46 to 3276 µm and plastic fibres from 300 to 1731 µm in length with a mean diameter of 16 µm. Single particle analysis using µ-FTIR indicated a prevalence of Rayon, PP, Rayon/Nylon/Polyester blends, Rubber, PS, PE, PA, PVC, Acrylic, EMAA and PET. Fragments were mainly white in colour (46%) followed by brown (38%), black (1%), blue (1%) and orange (1%). Fibres were mainly white in colour (57%) followed by black (14%), orange (14%) and red (14%). A trend assessment at the UK regional seas levels indicated little visual evidence of any trends in the abundance of microplastics over time in any of the regional seas – with the possible exception of the Northern North Sea with a reduction in abundance over time for the time-period 2013-2021.

Microplastic pollution in the food web: observation of ingestion by the talitrid amphipod Cryptorchestia garbinii on the shores of Lake Garda

Plastic pollution in the environment has become a much-discussed issue worldwide. In recent decades, the contamination of all environments has become increasingly evident, in particular, that of water is highly concerning. Ingestion by different animal species under natural conditions has also been demonstrated. Among these is the Talitrid Amphipod Cryptorchestia garbinii, which lives on the banks of the internal waterways and lakes’ shorelines. As detritivores species, it is very exposed to microplastics that can be ingested, probably mistaking them for food. Aiming to highlight the microplastic ingestion and the role of this species as an entry point for the food web, we analyzed 80 specimens from 4 sites along the shores of Garda Lake, one of the first lakes in Italy to be studied for this type of contamination. The microplastics ingested were observed and quantified through the Nile Red staining method. We were able to verify the presence of ingested microplastics in all the samples analyzed and, therefore, in the food web. This species could serve as valuable natural models of plastic exposure. Microplastic sentinel species can be used as a proxy for environmental exposure and ecosystem monitoring tools to quantify and assess the impacts of microplastic contamination.

Regulatory Effect and Mechanism of Oenothein B in Reducing Fat Accumulation in Caenorhabditis Elegans

AbstractOenothein B (OEB) displays various biological activities, including immunomodulatory, antioxidant, and anticancer effects. However, the underlying mechanisms remain unclear. Therefore, in this study, the effect of OEB isolated from Eucalyptus leaves on fat deposition and its underlying mechanism are explored in Caenorhabditis elegans （C.elegans). OEB shows remarkable antioxidative capacity by reducing reactive oxygen species (ROS) and enhancing the activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px). Oil Red O (ORO) and Nile Red staining show that OEB significantly inhibits fat accumulation. Furthermore, the size and number of lipid droplets in transgenic DHS‐3::GFP worms decrease, and the ratios of unsaturated fatty acids in worms are increased by OEB. Furthermore, quantitative reverse transcription‐polymerase chain reaction （qRT‐PCR） analysis shows that the expression of mdt‐15, fat‐5, fat‐7, fasn‐1, pod‐2, vit‐2, and tub‐1 is remarkably changed by OEB. Fat inhibition by OEB is abolished in the tested mutants. These results suggest that the mechanism underlying the inhibitory effects of OEB on fat deposition is dependent on dietary restriction as well as fatty acid synthesis, β‐oxidation, and storage. This study provides new insights into the mechanisms underlying lipid metabolism regulation by OEB.Practical Applications: Obesity is a global health problem and a risk factor for cardiovascular disease, diabetes, and many forms of cancer. Evidence in the scientific literature shows that some polyphenols play an important role in inhibiting obesity. OEB has a surprisingly broad spectrum of biological activities, including immunomodulatory, antioxidant, and anticancer activities. In this study, the effect of OEB on fat metabolism and its related mechanisms in Caenorhabditis elegans are investigated. The findings outlined in the current study show that OEB is effective in reducing fat accumulation, enhancing the ratios of unsaturated fatty acids, and affecting fat metabolism pathways in C. elegans. This study will be helpful in elucidating the characteristics and underlying mechanisms of lipid metabolism by OEB.

Microplastics in road dust: A practical guide for identification and characterisation

The contamination of the environment by microplastics (MPs) in road dust poses a serious ecological and health concern. MPs have been detected in road dust worldwide and their presence has been mainly attributed to plastic litter fragmentation and vehicle tyre abrasion. Although current technologies such as Raman and Fourier Transform InfraRed spectroscopy as well as Scanning Electron Microscopy are capable of detecting MPs in road dust, the analysis of MPs shape and MPs smaller than 20 μm is limited and often labour demanding. More accurate, cost-effective and rapid techniques have now become necessary to analyse MPs in road dust, particularly since the development of large infrastructure projects that incorporate recycled plastic into road assets and roadside furniture. Nile red (NR) staining is a promising technique to identify MPs in environmental samples; however, it has not yet been applied to road dust. This study investigates the use of NR fluorescence microscopy to detect MPs in road dust and provides information about MP amount, shape and size distribution. The staining duration and temperature, solvent selection and NR concentration were optimised considering 33 different road dust materials, including 13 types of plastic. The NR staining procedure developed in this work is capable of successfully differentiating between MPs down to 1 μm and other non-plastic road dust materials. Future applications include assessing the contribution of plastic-modified roads to MP pollution, comparing the level of MP pollution in urban and rural areas and providing a rapid, simple, inexpensive and reliable monitoring approach for further studies to compare MP using a singular optimised methodology.

Potential anti-adipogenic activity of Calligonum comosum cuminaldehyde on mouse 3T3-pre-adipocytes

Background: Obesity is a medical condition characterized by augmented body fat mass that can adversely affect human health. Several regimens were suggested to counteract obesity and fat accumulation with limited success. As plants are well-known source of medicinal products, we studied the potential anti-adipogenic activity of the essential oil extracted from Calligonum comosum plants growing in the desert of the United Arab Emirates. Methods: C. comosum essential oil was extracted and fractionated on thin layer chromatography. The effect of total oil extract, the major compound-rich fraction, and the pure compound (cuminaldehyde) were tested on the viability, lipid content, and glucose uptake of 3T3-L1 cells. The capability of cuminaldehyde to reduce the formation of 3D-adipocyte pellets and expression of related transcripts was also tested. Results: The results showed that C. comosum essential oil, particularly its major component cuminaldehyde, caused a significant reduction in the viability of 3T3-L1 cells when compared with fibroblasts, employed as controls. Furthermore, cuminaldehyde caused a significant reduction in the lipid content of 3T3 cells, as determined by Nile red stain, reduction in the glucose uptake, and reduction in the levels of both triglycerides and cholesterol. Moreover, cuminaldehyde significantly reduced the formation of 3D-adipocyte pellets and the expression of adipocyte-specific transcripts, CAAT-enhancer binding protein-alpha, and peroxisome proliferator-activated receptor-gamma. Conclusion: Taken together, these results demonstrated a potential inhibition of lipid accumulation in 3T3 adipocytes after treatment with cuminaldehyde extracted from C. comosum oil. Thus, cuminaldehyde can be considered as a new potential anti-adipogenic agent for the prevention and treatment of obesity.

Cold Atmospheric Plasma Inhibits Lipogenesis and Proliferation of Human Sebocytes and Decreases Sebum Production in Human Facial Skin

Background. Although several energy devices targeting sebaceous glands have been developed, an effective and safe therapeutic tool for hyperseborrhea is still needed. Nonthermal atmospheric‐pressure plasma (NTAPP) induces microscopic tissue reactions in sebaceous glands of rat skin. Objective. The aim of the study is to investigate the effects of NTAPP on sebum production in human skin in vivo followed by an experimental study of human sebocytes. Methods. Fourteen healthy volunteers with oily facial skin underwent three sessions of argon‐ and nitrogen‐NTAPP treatment at a 1‐week interval and were followed up for 8 weeks. The casual sebum level, sebum excretion rate, and porphyrin index were evaluated. Histological analysis was performed using skin biopsy specimens taken from two subjects at the baseline and week 2. SZ95 sebocytes were stimulated with testosterone and linoleic acid (T/LA) with or without treatment with NTAPP. BODIPY and Nile red staining were used for qualitative lipids analysis. Proliferation and differentiation markers were also assessed. Results. Casual sebum levels and sebum excretion rates in facial skin decreased by 26 and 24%, respectively, at week 4 compared to those of the baseline. Porphyrin index also decreased by 38% at week 2. Histologically, NTAPP‐treated human skin showed no obvious thermal injury, but the number of Ki67+ cells in the sebaceous glands decreased at week 2. Argon‐ and nitrogen‐NTAPP attenuated T/LA‐induced increases in neutral lipid accumulation, Ki67+ cells, and peroxisome proliferator‐activated receptor‐ɣ transcription in human sebocytes at energy settings that did not induce apoptosis. Conclusion. Argon‐ and nitrogen‐NTAPP can be a safe and effective therapeutic tool for hyperseborrhea‐associated diseases such as acne. This trial is registered with NCT04917835.

Activation of β-Adrenoceptors Promotes Lipid Droplet Accumulation in MCF-7 Breast Cancer Cells via cAMP/PKA/EPAC Pathways

Physiologically, β-adrenoceptors are major regulators of lipid metabolism, which may be reflected in alterations in lipid droplet dynamics. β-adrenoceptors have also been shown to participate in breast cancer carcinogenesis. Since lipid droplets may be seen as a hallmark of cancer, the present study aimed to investigate the role of β-adrenoceptors in the regulation of lipid droplet dynamics in MCF-7 breast cancer cells. Cells were treated for up to 72 h with adrenaline (an endogenous adrenoceptor agonist), isoprenaline (a non-selective β-adrenoceptor agonist) and salbutamol (a selective β2-selective agonist), and their effects on lipid droplets were evaluated using Nile Red staining. Adrenaline or isoprenaline, but not salbutamol, caused a lipid-accumulating phenotype in the MCF-7 cells. These effects were significantly reduced by selective β1- and β3-antagonists (10 nM atenolol and 100 nM L-748,337, respectively), indicating a dependence on both β1- and β3-adrenoceptors. These effects were dependent on the cAMP signalling pathway, involving both protein kinase A (PKA) and cAMP-dependent guanine-nucleotide-exchange (EPAC) proteins: treatment with cAMP-elevating agents (forskolin or 8-Br-cAMP) induced lipid droplet accumulation, whereas either 1 µM H-89 or 1 µM ESI-09 (PKA or EPAC inhibitors, respectively) abrogated this effect. Taken together, the present results demonstrate the existence of a β-adrenoceptor-mediated regulation of lipid droplet dynamics in breast cancer cells, likely involving β1- and β3-adrenoceptors, revealing a new mechanism by which adrenergic stimulation may influence cancer cell metabolism.

Microplastics Release from Conventional Plastics during Real Open Windrow Composting

The recycling of bio-waste plays an important role in a circular economy as it transforms bio-waste into a valuable resource (organic fertilizer). However, even separately collected bio-waste can contain some plastic waste, which is usually separated after composting and not before it. Primary studies have confirmed the degradation of plastic during composting, but the release of microplastics from them has not been studied. This article presents a quantification and comparison of the release of microplastics from commonly used plastics during green waste composting. Microplastics were identified by Nile red staining and examination under a fluorescent microscope. Plastic degradation was assessed by weight loss calculation, scanning electron microscope (SEM), and Fourier-transform infrared spectroscopy (FTIR) analysis. On average, 17 to 52 microplastics’ are released from 5-by-5 cm pieces of conventional plastics during composting. The control polylactic acid sample showed the smallest amount of released microplastics: four particles on average. The number of released microplastics depended on the polymer type and thickness of the samples. The results of the current article can be further used for the prediction of microplastic generation and setting a limit on the plastic content in bio-waste

A Comparison of Primary Human Hepatocytes and Hepatoma Cell Lines to Model the Effects of Fatty Acids, Fructose and Glucose on Liver Cell Lipid Accumulation.

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma-derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Sesquiterpenoids from the Florets of Carthamus tinctorius (Safflower) and Their Anti-Atherosclerotic Activity.

(1) Background: The florets of Carthamus tinctorius L. are traditionally used as a blood-activating drug and can be used for the treatment of atherosclerosis, but no compounds with anti-atherosclerotic activity have been reported. (2) Methods: This study investigated the chemical compounds from the florets of C. tinctorius. Comprehensive spectroscopic techniques revealed their structures, and ECD calculations established their absolute configurations. Nile Red staining, Oil Red O staining, and cholesterol assessment were performed on these compounds and their aglycones for the inhibitory activity against the formation of foam cells induced by oxidized low-density lipoprotein (ox-LDL) in RAW264.7 macrophages. In addition, RAW264.7 macrophages were tested for their anti-inflammatory activity by measuring the inhibition of NO production caused by LPS. (3) Results: Five new sesquiterpenoids (1-5) isolated from the florets of C. tinctorius were identified as (-)-(1R,4S,9S,11R)-caryophyll-8(13)-en-14-ol-5-one (1), (+)-(1R,4R,9S,11R)-caryophyll-8(13)-en-14-ol-5-one (2), (-)-(3Z,1R,5S,8S,9S,11R)-5,8-epoxycaryophyll-3-en-14-O-β-D-glucopyranoside (3), (+)-(1S,7R,10S)-guai-4-en-3-one-11-O-β-D-fucopyranoside (4), and (-)-(2R,5R,10R)-vetispir-6-en-8-one-11-O-β-D-fucopyranoside (5). All compounds except for compound 3 reduced the lipid content in ox-LDL-treated RAW264.7 cells. Compounds 3 and 4 and their aglycones were found to reduce the level of total cholesterol (TC) and free cholesterol (FC) in ox-LDL-treated RAW264.7 cells. However, no compounds showed anti-inflammatory activity. (4) Conclusion: Sesquiterpenoids from C. tinctorius help to decrease the content of lipids, TC and FC in RAW264.7 cells, but they cannot inhibit NO production, which implies that their anti-atherogenic effects do not involve the inhibition of inflammation.

Nimbin analogs N5 and N7 regulate the expression of lipid metabolic genes and inhibit lipid accumulation in high-fat diet-induced zebrafish larvae: An antihyperlipidemic study

BackgroundExcess accumulation of lipids leads to obesity. Triterpenoids are a group of plant compounds which poses various biological activities. The biological activities of Nimbin analogs N5 and N7 were addressed in this study on inhibiting lipid aggregation and underlying the derivatives molecular mechanisms for a therapeutical approach. AimThis study aims to evaluate the anti-adipogenic activity of semi-natural Nimbin analogs, N5 and N7, on zebrafish larvae induced with oxidative stress due to a high-fat diet (HFD) and adipogenesis using specific fluorescent stains. Materials and methodsZebrafish at 4 days post fertilized (dpf) larvae were divided into groups for the HFD diet along with exposure to various concentrations of N5 and N7. HFD induced accumulation of neutral lipids and triglycerides (Oil Red O and Nile red staining, respectively) with weight gain, which generated intracellular ROS (DCFH-DA staining) and superoxide anion production (DHE staining) with depleted glutathione levels (NDA staining) were assayed. HFD exposure promoted the accumulation of inflammatory macrophages (Neutral red staining) and impaired glucose metabolism (2NBDG staining). The ability of N5 and N7 to reduce total regulating lipogenic specific genes C/EBP-α, SREBP-1 and FAS were evaluated using relative gene expression. Key findingsThe Nimbin analogues N5 and N7 suppressed adipogenesis, forming intracellular ROS and superoxide anion while simultaneously restoring glutathione levels. The analogues significantly lowered total TC and TG levels, prevented inflammatory macrophage build-up and boosted glucose absorption. Also, N5 and N7 down-regulate the lipogenic-specific genes. SignificanceNimbin analogs N5 and N7 enhance lipolysis and inhibit adipogenesis in in-vivo zebrafish larvae model.

A novel small compound TOIDC suppresses lipogenesis via SREBP1-dependent signaling to curb MAFLD

BackgroundInhibition of hepatic lipogenesis is widely regarded as an effective treatment for metabolic-associated fatty liver disease (MAFLD), although numerous related drugs have failed to reach clinical application. The goal of this study is to identify a novel small compound that can effectively treat MAFLD.MethodsPrimary hepatocytes were first exposed to palmitic acid and oleic acid, then treated with compounds prior to high through screening for cellular lipid content. The efficacy of these compounds was measured by Nile Red staining and triglyceride analysis. The potential cellular toxicity caused by these compounds was evaluated by CCK8 assay. qPCR and Western blot were used to determine expression of RNAs and proteins, respectively. The compound was intraperitoneally injected into diet-induced obese (DIO) mice to examine its efficacy in vivo.ResultsWe identified the dimethyl 1-methyl-2-thioxoindoline-3,3-dicarboxylate (TOIDC) as a powerful chemical to reduce cellular lipid with minimal cellular toxicity. When injected intraperitoneally, TOIDC effectively ameliorates MAFLD in DIO mice. Mechanically, TOIDC suppresses de novo lipogenesis through inhibiting sterol regulatory element-binding protein 1 (SREBP1).ConclusionsOur findings indicate that TOIDC could be a promising lead compound to develop new drugs to treat MAFLD.Graphical

Cucurbitacin B-, E-, and I-Induced Browning of White Adipocytes Is Promoted by the Inhibition of Phospholipase D2.

The mechanism of white adipose tissue browning is not well understood; however, naturally occurring compounds are known to play a positive role. The effects of cucurbitacins B, E, and I on the browning of mature white adipocytes were investigated. First, the cell viability exhibited by cucurbitacins B, E, and I in pre- and mature adipocytes was verified. Cucurbitacins B, E, and I had no effect on cell viability in pre- and mature adipocytes at concentrations up to 300 nM. To investigate the characteristics of representative beige adipocytes, the formation and morphology of cucurbitacin B, E, and I lipid droplets were verified. The total lipid droplet surface area, maximum Feret diameter, and total Nile red staining intensity of cucurbitacin B-, E-, and I-treated adipocytes were lower than those of mature white adipocytes. Furthermore, treatment of white mature adipocytes with cucurbitacin B, E, and I led to the formation of several small lipid droplets that are readily available for energy expenditure. We evaluated the effect of cucurbitacins B, E, and I on the expression of representative browning markers UCP1, PGC1a, and PRDM16, which participate in the browning of white adipose tissue. Cucurbitacins B, E, and I increased the mRNA and protein expression levels of UCP1, PGC1a, and PRDM16 in a concentration-dependent manner. To promote energy consumption by beige adipocytes, active mitochondrial biogenesis is essential. Next, we investigated the effects of cucurbitacin B, E, and I on mitochondrial biogenesis in mature adipocytes. Mitochondrial mass increased when mature adipocytes were treated with cucurbitacin B, E, and I. The degree of cucurbitacin B-, E- and I-induced transformation of white adipocytes into beige adipocytes was in the order of Cu E > Cu B > Cu I. To verify the effect of phospholipase D2 on the browning of white adipocytes, CAY10594—a PLD2 pharmacological inhibitor, and a knockdown system were used. PLD2 inhibition and knockdown improved the expression levels of UCP1, PGC1a, and PRDM16. In addition, PLD2 inhibition and knockdown in mature white adipocytes promoted mitochondrial biosynthesis. The effect of PLD2 inhibition and knockdown on promoting browning of white adipocytes significantly increased when Cu B, Cu E, and Cu I were co-treated. These data indicate that mature white adipocytes’ beige properties were induced by cucurbitacins B, E, and I. These effects became more potent by the inhibition of PLD2. These findings provide a model for determining anti-obesity agents that induce browning and increase energy expenditure in mature white adipocytes.

Rapid detection of nanoplastics and small microplastics by Nile-Red staining and flow cytometry

Microplastics are of rising health concerns because they have been detected even in remote and pristine environments, from the Artic snow to the Marianne Trench. The occurrence and impact of nanoplastics in ecosystems is almost unknown, in particular due to analytical limitations such as very small sizes that fall below detection limits of current techniques. Here we take advantage of a common interference in analytical flow cytometry to develop a method for the quantification of the number of plastic particles in the 0.6–15 µm size range. Plastic particles are stained with the lipophilic dye Nile-Red then detected by flow cytometry, a method regularly used in biology for rapid quantification of fluorescent cells. We found that sample analysis lasts 90 s, which is hundreds of times faster than the analysis of filter portions by micro-Raman and other spectroscopic techniques. Our method is highly efficient in detecting polyethylene, with staining efficiency higher than 70% and signal linearity with concentration. Staining efficiency up to 96% was observed for polyvinylchloride and for polystyrene.

A six lipid metabolism related gene signature for predicting the prognosis of hepatocellular carcinoma

Globally, hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors. Studies have shown that alterations in the tumor immune microenvironment (TIME) play a significant role in the pathogenesis and progression of HCC, and notably, lipid metabolism has been shown to regulate TIME. Therefore, in predicting the prognosis and efficacy of immunotherapy in patients with HCC, lipid metabolism-related prognostic factors are highly relevant. mRNA expression data of HCC were obtained from the Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and gene expression omnibus (GEO) databases. and lipid metabolism-related genes were also obtained from the GSEA databases. Least absolute shrinkage and selection operator regression analysis, univariate and multivariate Cox proportional hazards analysis were used to explore lipid metabolism-related prognostic genes and further construct a prognostic signature in the training set, ICGC and GSE54236 were used to validate the accuracy of the signature. qRT-PCR was used to detect the mRNA levels of lipid metabolism-related prognostic genes in HCC tissues and their paired adjacent tissues. Nile red staining was used to demonstrate lipid content in HCC tissues. Immunofluores-cence and ELISA were used to detect immune cells and immune responses in HCC tissues and serum. Six lipid metabolism-related genes (ADH1C, APEX1, ME1, S100A10, ACACA and CYP2C9) were identified as independent prognostic factors, which were used for risk model construction for HCC patients. The areas under the 1-, 2-, and 3-year ROC curves for the TCGA cohort were 0.758, 0.701 and 0.671, respectively. Compared with paired paracancerous tissues, qRT-PCR revealed that APEX1, ME1, S100A10 and ACACA were up-regulated in HCC tissues, whereas ADH1C and CYP2C9 were down-regulated in HCC tissues. Nile red staining indicated that this study showed that both the HCC tissue and serum of patients in the high-risk group exhibited lipid accumulation. Our identified prognostic model comprising six lipid metabolism-related genes could provide survival prediction. Moreover, HCC drug therapy target selection and molecular marker research can be guided by our predictive model.

First Evidence of Microplastic Presence in Bed Load Sediments of a Small Urban Stream in Warsaw

Microplastics (MPs) are considered as a contaminant of widespread global concern. Due to their small size, MPs become bioavailable to many types of organisms and affect them. However, there is still little known about MP release on land, storage in soils and sediments, or their transport by runoff in rivers. Thus, the aim of this work was to present the results of the first, initial investigation on microplastic presence in an urban stream located in Warsaw, Poland. A simple and relatively inexpensive procedure that leads step-by-step to the detection of microplastics in bed load sediment is presented. It consists of sampling, sieving, density separation, organic matter reduction, and Nile Red staining. The presence of MP in the channel of Służew Creek was confirmed. The estimated amount of particles ranged from 191 to 279 pieces per 30 g of bed load sediment for the selected sampling sites. The number of particles seemed to increase with the catchment area. There is a need for further broad research focusing, among others, on the standardization of methods and laboratory procedures leading to microplastic detection.

Lack of Low-affinity Phosphate Transporter Pho91 Alters Lipid Metabolism in Yeast Saccharomyces cerevisiae

Objective: The current study tries to elucidate the impact of low-affinity Pi transporter Pho91 (using pho91Δ cells) on lipid metabolism and cellular organelles morphology (mitochondria and vacuole) in yeast Saccharomyces cerevisiae. Methods: The lipid profile was performed using thin layer chromatography (TLC), and the membrane defect was determined using DiOC6 staining, lipid droplets (LDs) were observed by Nile red staining, mitochondrial morphology was observed using aconitase 1 GFP, vacuolar morphology was studied by FM4-64 staining and were performed with the aid of laser scanning fluorescent microscope. Findings: Pho91 is a low-affinity phosphate (Pi) transporter in the vacuoles that functions under Pi-rich conditions, but its role in lipid metabolism is largely unknown. In this study, we used defined synthetic complete media (SC). The deletion of Pho91 depicted a moderate growth defect but increased the major phospholipids PC, PE, and PI. Alterations in the membrane phospholipids resulted in defective membrane morphology. In pho91 mutant (pho91Δ), the neutral lipids TAG and SE were increased and stored as LDs. The LD numbers were increased in pho91Δ cells than in WT cells. Altered phospholipids also defective mitochondrial morphology and enlarged vacuoles in pho91 deletion. Novelty: In the absence of low-affinity Pi transporter pho91, it increases phospholipid, neutral lipid levels, LD numbers, and impacted mitochondria along with vacuolar structures. Keywords: Phosphate transporter; Low affinity; Phospholipids; Neutral lipids; Lipid droplets

Isolation and Characterization of Two PHA Producing Strains Isolated from Soil Sample of Nalgonda District

Two whitish-colored gram-positive strains producing Polyhydroxyalkanoates (PHAs) were isolated from a soil sample from Nalgonda district in the Telangana state of India. With the help of Sudan black B staining, ten isolates with lipids, which showed bluish black color were selected from twenty-six bacterial strains which were selected randomly and purified from the serial diluted plate. Among ten isolates, 3D1 and 3D10 isolates were confirmed with Nile blue A and Nile Red staining for their PHA granules producing capacity. These two isolates grew optimally at a temperature of 37°C and a pH of 9. Furthermore, these strains were able to resist NaCl up to 10%, whereas, optimum NaCl required for the growth of 3D1 was 2%, but optimum NaCl required for the growth of 3D10 was shown to be 4%. PHAs produced by the two strains, 3D1 and 3D10, were extracted and quantified, which produced 68% PHA with a polymer concentration of 4.902 g/L and 61% PHA with a polymer concentration of 4.023 g/L, respectively. Biochemical, Morphological and Molecular characterization were performed on these two isolates. These two strains, 3D1 and 3D10, were closely related to Bacillus sonorensis with similarity of 99.51% and Bacillus safensis subsp. safensis with similarity of 99.66%, respectively. The 16S rDNA gene sequences of these two isolates were submitted to the NCBI Gene bank and the accession numbers were also sought.

Automated method for routine microplastic detection and quantification

Microplastics (MPs) are a heterogeneous group of solid polymers with dimensions <5 mm, which are a widespread contaminant of the environment. Their ubiquitous presence grabbed researchers' attention in the last decade, and the problem of MPs detection and quantification is currently a topic of utmost importance. Most identification and quantification protocols are still based on the visual count, which is an extremely time-consuming and error-prone task due to operator subjectivity. To address such an issue, different software analysis procedures are available, but they mainly rely either on the use of optical microscopy, covering a minimal area for each sample (mm2 size), or they allow only the identification of the largest particles (>1 mm). Here, a semi-automatic innovative image processing method for quantifying and measuring microplastics on filter membrane substrates is presented and validated, comparing results with data obtained using visual counting performed by an experienced operator. The algorithm was tested with artificially generated microplastic images and samples taken from natural environments. Samples of Borgio Verezzi show cave sediment and Po River water were filtered on a glass filter membrane, and photographs were taken under 365 nm illumination, both without and with Nile Red staining. The proposed image analysis method, implemented in an easy-to-use Python script, was quite accurate and fast (about 10 s/image average processing time), showing an average deviation below 10 %, which is further reduced to about 8 % if the samples are stained with Nile Red.