Nile Red Staining Research Articles

Detection limits are central to improve reporting standards when using Nile red for microplastic quantification

Beyond simple identification of either the presence or absence of microplastic particles in the environment, quantitative accuracy has been criticised as being neither comparable nor reproducible. This is, in part, due to difficulties in the identification of synthetic particles amidst naturally occurring organic and inorganic components. The fluorescent stain Nile red has been proposed as a tool to overcome this issue, but to date, has been used without consideration of polymer specific fluorescent variability. The aim of this study was to evaluate the efficacy of Nile red for microplastic detection by systematically investigating what drives variations in particle pixel brightness (PPB). The results showed that PPB varied between polymer type, shape, size, colour and by staining procedure. Sand, an inorganic component of the sample matrix does not fluoresce when stained with Nile red. In contrast the organic components, wood and chitin, fluoresce between 1.40 and 12 arbitrary units (a.u.) and 32 and 74 a.u. after Nile red staining, respectively. These data informed the use of a PPB threshold limit of 100 a.u., which improved the detection of EPS, HDPE, PP and PA-6 from the 6 polymers tested and reduced analysis time by 30–58% compared to unstained samples. Conversely, as with traditional illumination, PET and PVC were not accurately estimated using this approach. This study shows that picking a threshold limit is not arbitrary but rather must be informed by polymer specific fluorescent variability and matrix considerations. This is an essential step needed to facilitate comparability and reproducibility between individual studies.

The Influence of Light and Nutrient Starvation on Morphology, Biomass and Lipid Content in Seven Strains of Green Microalgae as a Source of Biodiesel

The development of clean and renewable energy sources is currently one of the most important challenges facing the world. Although research interests in algae-based energy have been increasing in the last decade, only a small percentage of the bewildering diversity exhibited by microalgae has been investigated for biodiesel production. In this work, seven strains of green microalgae belonging to the genera Scenedesmus, Tetradesmus and Desmodesmus were grown in liquid medium with or without a nitrogen (N) source—at two different irradiances (120 ± 20 and 200 ± 20 μmol photons m−2 s−1)—to evaluate biomass production and FAME (fatty acid methyl esters) content for biodiesel production. The strains of Tetradesmus obliquus and Desmodesmus abundans grown in N-deprived medium showed the highest FAME content (22.0% and 34.6%, respectively); lipid profile characterization highlighted the abundance of saturated FAME (as C16:0 and C18:0) that favors better viscosity (flow properties) and applicability of biodiesel at low temperatures. Light microscopy and confocal laser scanning microscopy observations were employed as a fast method to monitor the vital status of cells and lipid droplet accumulation after Nile red staining in different culture conditions.

Abstract 2562: Causes and consequences of adiposomogenesis in breast cancer cells

Abstract Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and thrive and to out-compete the stroma into which they invade. Acid adaptation has been associated with chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. In addition to these survival mechanisms, tumor cells under acidic conditions accumulate cytoplasmic lipid droplets (adiposomes); dynamic organelles that store neutral lipids surrounded by a shell of perilipin (PLIN) proteins and a phospholipid monolayer. Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins (PLIN2) and a phospholipid monolayer. High expression of PLIN2 was observed to be strongly associated with poor overall survival in breast cancer patients. In vitro, breast cancer cells rapidly and robustly accumulated adiposomes when grown in acidic media revealed by nile red and PLIN-2 staining. The acid-induced lipogenic phenotype persists even when the cells are grown in de-lipidated serum, indicating that the source of lipids is de-novo and endogenous. Adiposome formation at low pH was attenuated when cells were treated with inhibitors of fatty acid synthesis, FAS; such as TOFA or C75. Further, these inhibitors were selectively cytotoxic under acidic conditions indicating that adiposomogenesis is a survival mechanism. Consistent with increased FAS we also observed using 13C isotopomer analysis a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, which results in increased production of NADPH, necessary for de novo lipid synthesis. In addition, we observed that cells at low pH had higher rates of oxygen consumption (OCR) compared to controls using Seahorse profiling. The major source of the lipid precursors was identified to be autophagy-derived ketogenic amino acids using LC-LS/MS following 13C tracer pre-incubation. Further, we tested the hypothesis that adiposomogenesis is induced by acid signal and this signaling is mediated by one (or more) acid sensing G-protein coupled receptors. Both OGR1 and TDAG8 were strongly expressed in our systems. CRISPR/Cas9 mediated depletion of these receptors showed that TDAG8 KO had no effect, but that KO of OGR1 abrogated adiposome accumulation under acidic conditions in MCF7 and T47D cells. Further, OGR1 knockout cells were defective in acid induced autophagy. In xenograft models, OGR1 knockout MCF7 cells formed significantly smaller tumors compared to control cells. Hence, accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. This increased dependence on lipid metabolism revealed novel therapeutic vulnerabilities Citation Format: Smitha R. Pillai, Iqbal Mahmud, Michael Langsen, Jonathan Wojtkowiak, Jonathan Nguyen, Marylin Bui, Robert Gatenby, Timothy Garrett, Robert Gillies. Causes and consequences of adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2562.

Spectroscopic analysis of microplastic contaminants in an urban wastewater treatment plant from Seoul, South Korea

In this study, a systematic multi-spectroscopic analysis of microplastics (MPs) sampled from a metropolitan area of Seoul was undertaken to elevate understanding of the role of wastewater treatment plants (WWTPs) in eliminating suspended contaminants including MPs before releasing the effluent water into the environment. We analyzed pollutants in influent and effluent samples from a WWTP in Seoul, South Korea. Spectroscopic and microscopic methods were used to analyze MPs. Fourier-transform infrared (FT-IR) spectroscopy in the wavenumber region between 4000 and 715 cm−1 was employed to estimate the abundance of MPs in wastewater. Stereomicroscope images and Nile red staining were used to facilely identify MPs in both influents and effluents to compare the results with those of FT-IR data. Hyperspectral imaging could identify MPs in the influent sample with the reflection method at 400–900 nm. Our preliminary results indicate that the most observed MPs after the wastewater were filtered by a 45 μm stainless steel mesh filter were polyethylene (PE) and polypropylene (PP). The total number of the prevalent MPs in influent samples decreased significantly. Nanostructure particles could be found by field-emission scanning electron microscopy (FE-SEM). Our combined multi-spectroscopic study should be helpful to provide a guideline for the rapid spectroscopic analysis of freshwater in the Han River, Seoul, South Korea.

A robust and miniaturized screening platform to study natural products affecting metabolism and survival in Caenorhabditis elegans

In this study a robust, whole organism screening based on Caenorhabditis elegans is presented for the discovery of natural products (NP) with beneficial effects against obesity and age-related diseases. Several parameters of the elaborated workflow were optimized to be adapted for probing multicomponent mixtures combining knowledge from traditional medicine and NP chemistry by generating optimized small-scale extracts considering scarcity of the natural source, solubility issues, and potential assay interferences. The established miniaturized assay protocol allows for in vivo probing of small amounts of even complex samples (~ 1 mg) to test their ability to increase the nematodes’ survival time and the suppression of fat accumulation assessed by Nile red staining as hall marks of “healthy aging”. The workflow was applied on 24 herbal and fungal materials traditionally used against symptoms of the metabolic syndrome and revealed promising results for the extracts of Gardenia jasminoides fruits and the sclerotia from Inonotus obliquus. Tested at 100 µg/mL they were able to significantly reduce the Nile red fluorescence and extend the 50% survival rate (DT50) compared to the control groups. This phenotype-directed in vivo approach opens up new horizons for the selection of natural starting materials and the investigation of their active principles as fast drug discovery tool with predictive value for human diseases.

Naringin ameliorates type 2 diabetes mellitus-induced steatohepatitis by inhibiting RAGE/NF-κB mediated mitochondrial apoptosis

AimsRecent findings have instituted the role of hyperglycemia-related AGE/RAGE and NF-κB in instigating reactive oxygen species (ROS) mediated mitochondrial dysfunction and apoptosis of hepatocyte, which leads to steatohepatitis. Naringin, a flavanone glycoside found to possess myriads of pharmacological benefits along with its antioxidant and anti-inflammatory properties. Consequently, we aimed to decipher the effect of naringin on RAGE/NF-κB mediated mitochondrial apoptosis in type 2 diabetes mellitus (T2DM)-induced steatohepatitis. Main methodsHepatic HepG2 cells were cultured in palmitic acid medium with and without naringin. Lipid content was examined by Oil Red O and Nile Red staining. Cellular apoptosis was determined by Annexin V-FITC/PI staining. An experimental T2DM-induced steatohepatitis was developed in Sprague Dawley rats by high-fat diet (HFD) for 12 weeks. The naringin was administrated orally at a dose of 100 mg/kg, daily for eight weeks. Glucose and insulin tolerance test was performed. Liver sections were stained by hematoxylin-eosin and picrosirius red. The mRNA and protein expression of RAGE and NF-κB were determined by qPCR, Immunofluorescence, and Immunoblotting. Mitochondrial membrane potential (MMP), cellular and mitochondrial ROS were measured by FACS. Key findingsPalmitic acid encountered HepG2 cells and HFD fed rats exhibited hyperlipidemia, insulin resistance, abnormal aminotransferases, steatosis, and fibrosis. Besides, the level of AGEs, RAGE, NF-κB, and oxidative stress were exacerbated. Moreover, MMP, cellular and mitochondrial ROS were altered in diabetic rats. Nevertheless, the naringin treatment ameliorated the steatohepatitis by improving the levels of aforementioned parameters. SignificanceCollectively, these findings suggested anti-steatohepatitis potential of naringin in diabetics.

Exploring the potential of photoluminescence spectroscopy in combination with Nile Red staining for microplastic detection

The significant amount of plastic litter in the form of microplastics (size <5 mm) is garnering attention owing to its potential threat to marine life. Reliable, cost- and time-efficient analysis methods for monitoring microplastic abundance globally are still missing. Several studies proposed a fast detection method by binding the solvatochromic dye Nile Red on the surface of microplastics and using fluorescence microscopy for their detection. All the staining approaches reported so far differ in terms of Nile Red concentration, solvents, and staining procedure. Here, we compare the staining protocols published prior to 2019 and propose an optimized staining protocol. Furthermore, we explore the potential of Nile Red staining in combination with photoluminescence spectroscopy to identify the polymer type and to distinguish plastics from non-plastics.

Bacterial degradation of distillery wastewater pollutants and their metabolites characterization and its toxicity evaluation by using Caenorhabditis elegans as terrestrial test models

Distillery wastewater has significant amount of coloring compounds and organic substances even after the secondary treatment process, which poses many severe environmental and health threats. However, the recalcitrant coloured compounds have not yet been clearly identified. In this study, two bacterial strains DS3 and DS5 capable to decolorize distillery wastewater (DWW) pollutants were isolated and characterized as Staphylococcus saprophyticus (MF182113) and Alcaligenaceae sp. (MF182114), respectively. Results showed that mixed bacterial culture was found more effective decolorizing 71.83% DWW compared to axenic culture DS3 and DS5 resulting only 47.94% and 50.67% decolorization, respectively. The FT-IR and LC-MS/MS analysis of untreated DWW showed the presence of many recalcitrant compounds having different functional groups, but after bacterial treatment, most of compounds get diminished and the toxicity of DWW was reduced significantly. Further, the Nile red staining of Caenorhabditis elegans exposed to untreated and bacteria treated DWW for evaluation of toxicity assay and results revealed that the worms exposed to untreated DWW showed sharp reduction in total fat content having more profound effects, suggesting the diminished nAchR signaling as compare to bacterial treated DWW. Hence, this study revealed that inadequate disposal of untreated DWW may cause transfer of toxic substances into the environment and receiving water bodies.

Genetic Variation in Complex Traits in Transgenic α-Synuclein Strains of Caenorhabditis elegans.

Different genetic backgrounds can modify the effect of mutated genes. Human α-synuclein (SNCA) gene encodes α-synuclein, and its oligomeric complexes accumulate with age and mediate the disruption of cellular homeostasis, resulting in the neuronal death that is characteristic of Parkinson’s Disease. Polymorphic variants modulate this complex pathologic mechanism. Previously, we constructed five transgenic introgression lines of a Caenorhabditis elegans model of α-synuclein using genetic backgrounds that are genetically diverse from the canonical wild-type Bristol N2. A gene expression analysis revealed that the α-synuclein transgene differentially affects genome-wide transcription due to background modifiers. To further investigate how complex traits are affected in these transgenic lines, we measured the α-synuclein transgene expression, the overall accumulation of the fusion protein of α-synuclein and yellow fluorescent protein (YFP), the lysosome-related organelles, and the body size. By using quantitative PCR (qPCR), we demonstrated stable and similar expression levels of the α-synuclein transgene in different genetic backgrounds. Strikingly, we observed that the levels of the a-synuclein:YFP fusion protein vary in different genetic backgrounds by using the COPAS™ biosorter. The quantification of the Nile Red staining assay demonstrates that α-synuclein also affects lysosome-related organelles and body size. Our results show that the same α-synuclein introgression in different C. elegans backgrounds can produces differing effects on complex traits due to background modifiers.

Chlorin e6-mediated photodynamic therapy modulates adipocyte differentiation and lipogenesis in 3T3-L1 cells.

Chlorin e6-mediated photodynamic therapy (Ce6-mediated PDT) is a therapeutic modality that consists of a photosensitizer, source of light at a suitable wavelength, and molecular oxygen. PDT has been widely used for the treatment of cancers and non-neoplastic disorders. However, information on the inhibitory activity of Ce6-mediated PDT on adipogenesis and lipogenesis in 3T3-L1 cells is not available to date. MTT assay, Oil Red O staining, and Nile red staining were used to investigate lipid accumulation in 3T3-L1 cells to clarify adipocyte differentiation. The expression levels of various molecular signals associated with adipogenesis and lipogenesis were examined by RT-PCR and western blot analysis. Adipocyte differentiation and lipid accumulation were reduced by Ce6-mediated PDT in 3T3-L1 cells. Inhibition of the expression of PPAR-γ, C/EBPα, β, δ, SREBP-1, FAS, and LPL and the activation of AMPK, revealed concise modulation of adipogenic and lipogenic molecules by Ce6-mediated PDT. The anti-adipogenic effect of Ce6-mediated PDT was reversed by AMPKα siRNA. Ce6-mediated PDT inhibits adipocyte differentiation and lipogenesis via regulating AMPK. Ce6-mediated PDT might serve as a potential therapy for the treatment of obesity and obesity-associated diseases.

Pilot scale wastewater treatment, CO2 sequestration and lipid production using microalga, Neochloris aquatica RDS02

In present investigation carried out large-scale treatment of tannery effluent by the cultivation of microalgae, Neochloris aquatica RDS02. The tannery effluent treatment revealed that significant reduction heavy metals were chromium-3.59, lead-2.85, nickel-1.9, cadmium-10.68, zinc-4.49, copper-0.95 and cobalt-1.86 mg/L on 15th day of treatment using N. aquatica RDS02. The microalgal biosorption capacity q max rate was Cr-88.66, Pb-75.87, Ni-87.61, Cd-60.44, Co-52.86, Zn-84.90 and Cu-54.39, and isotherm model emphasized that the higher R 2 value 0.99 by Langmuir and Freundlich kinetics model. The microalga utilized highest CO2 (90%) analyzed by CO2 biofixation and utilization kinetics, biomass (3.9 mg/mL), lipid (210 mg mL−1), carbohydrate (102.75 mg mL−1), biodiesel (4.9 mL g−1) and bioethanol (4.1 mL g−1). The microalgal-lipid content was analyzed through Nile red staining. Gas chromatography mass spectrometric (GCMS) analysis confirmed that the presence of a biodiesel and major fatty acid methyl ester (FAME) profiling viz., tridecanoic acid methyl ester, pentadecanoic acid methyl ester, octadecanoic acid methyl ester, myristic acid methyl ester, palmitic acid methyl ester and oleic acid methyl ester. Fourier transform infrared (FTIR) analysis confirmed that the presence of a functional groups viz., phenols, alcohols, alkynes, carboxylic acids, ketones, carbonyl and ester groups. The bioethanol production was confirmed by high-performance liquid chromatography (HPLC) analyze.

Pollution of Microplastics in Coastal Plain of the Huangshui River Basin

Marine microplastic pollution is one of the hot spots in current marine environment research. The coastal zone has unique micro-plastic pollution characteristics at the junction of land and sea, and the micro-plastic pollution in the coastal zone soil is still unclear. In this study, Huangshui river basin was selected as the research area in Laizhou Bay, China. Soil samples were collected from the depth of 0-3, 3-6 and 6-9 cm at each location. Microplastics were separated by flotation. Microplastic characteristics were investigated and analyzed by Nile red staining and Fourier infrared spectrometer. The results showed that there was slight microplastic pollution in the coastal plain. 92.9% of the detected microplastics were granular, 6.1% were fibers, and only 1.0% were films. Microplastics with a particle size of 20-100 μm accounted for 57.9% of the total, particles with the size of 100-1000 μm accounted for 38.6%, and particles with the size of 1000-5000 μm accounted for 3.5%. The presence of film-like PE was detected, indicating that microplastic contamination in the soil was likely to come from the utilization and mulching of agricultural mulch. The amount of microplastics in soil was related to the size of microplastics. Abundance of microplastics in soil increased with decreasing of the size of soil. In addition, the microplastic content in soil decreased, and particles with a smaller size occupied a larger proportion, with the increasing of the depth of soil.

Bacteria from industrial waste: potential producers of polyhydroxyalkanoates (PHAs) in Manizales, Colombia.

Polymers are currently used in the industry as raw material, yet they are rapidly eliminated and largely contaminate the environment. To address this issue, there is a special interest in biodegradable polymers, namely, polyhydroxyalkanoates (PHAs), produced by microorganisms. This study identifies PHA-producing bacteria from two industrial wastewaters of Manizales, Colombia. The samples were cultured in mineral salt medium with glucose as the carbon source in the presence of Nile red stain. The fluorescent colonies were independently transferred to another medium and assessed through fluorescence microscopy with Nile blue stain. The fluorescent strains under Nile blue staining were purified in Nutrient Agar, and their morphological and microbiological characteristics were determined. The bacteria positive for red-orange fluorescence were purified in Nutrient Agar medium, and molecular analyses were performed by PCR amplification of a 650-bp fragment of the 16S ribosomal DNA gene. The bacteria were also assessed in terms of PHA production. We confirmed the identity of 12 out of 14 PHA-positive strains, which belonged to the following genera: Bacillus, Lactococcus, Citrobacter, Enterobacter, and Acinetobacter. Five of the isolates (Enterobacter cloacae, Enterobacter sp., Enterobacter ludwigii, Bacillus thuringiensis, and Bacillus safensis) are promising strains for PHA production, with production values ranging from 0.360 to 0.9960g/L. Bacteria that produce more than 0.3g/L are considered useful for the industrial manufacture of bioplastic. We recommend performing large-scale studies on these strains to assess their use for the industrial production of biopolymers, allowing to generate high-impact bioconversion processes of industrial interest.

Exploring the distinctiveness of biomass and biomolecules from limnic microalgae of unexplored waters of Noyyal River, Western Ghats, for exploitation.

Oleaginous microalgae with high biomass productivity, lipid content, and lipid productivity are desirable for sustainable biofuel production. Rapid and accurate quantification of lipid content facilitates the identification of promising microalgae candidates. In the present study, 23 freshwater microalgae species from river Noyyal were isolated and identified based on their morphological and molecular (18S rRNA) features and recorded as Karunya Algae Culture Collection (KACC). Their biomass and lipid content were characterized and screened using FT-IR, Nile red staining, and gravimetric method. Results generated from FT-IR spectra differentiated KACC microalgae based on their biochemical contents with Scenedesmus rubescens KACC 2 and Chlorococcum sp. KACC 13 possessed high total protein and lipid content, respectively. Nile red fluorescence at 530/575nm showed the yellow fluorescence under a fluorescent microscope giving the evidence of high neutral lipids in 10 KACC microalgae isolates. Total lipid content showed prominent variation between the KACC isolates and found in the range of 4 to 32% of DW. Lipid productivity and biomass productivity showed a similar pattern among KACC strains. Thus, our findings serve as a baseline data on the bioprospecting potential of KACC isolates from river Noyyal, an unexplored area of Western Ghats.

Process optimization and characterization of polyhydroxyalkanoate copolymers produced by marine Pichia kudriavzevii VIT-NN02 using banana peels and chicken feather hydrolysate

Abstract The present study was focused on the production of polyhydroxyalkanoates (PHA) by halophilic yeast using biowaste materials. The yeast strain was isolated from marine seaweeds and identified as Pichia kudriavzevii VIT-NN02 based on partial 18SrDNA sequencing and phylogenetic analysis. PHA granules were observed by Sudan Black B and Nile Red staining methods. Banana peels and chicken feathers hydrolysate were screened as potential carbon and nitrogen sources for PHA content in biomass (%) by P. kudriavzevii. Process parameters were optimized using 3-level, six variables, Box-Behnken design showing significant improvement in PHA content in biomass from 40.00 ± 0.1 to 79.68 ± 0.2% under optimized growth conditions viz. banana peel extract (40%), salt (16.6gL-1), incubation time (96 h), pH (10), temperature (37 °C) and chicken feather hydrolysate (0.80%). The statistical significance of the model equation was calculated by F-test and R2 values of the respective responses were found to be 0.9904 and 0.9926, indicating a realistic fit of the model to the experimental data. The actual values of the PHA content in biomass (79.68 ± 0.2%) was in close agreement to the predicted value (79.68 ± 1.5%), confirmed the validity of the model. The biopolymer was identified as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer based on Gas Chromatography-Mass Spectrometry (GC-MS), Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance spectroscopy (NMR) analysis. The [P(3HB-co-3HV)] copolymer was found to be highly crystalline and thermally stable through X-Ray Diffraction (XRD), Thermogravimetric analysis (TGA) and Differential scanning calorimetric (DSC) analysis. The Scanning Electron Microscopic (SEM) images of [P(3HB-co-3HV)] copolymer showed porous, smooth and spherical surface morphology.

Autophagy regulates lipid production and contributes to the sebosuppressive effect of retinoic acid in human SZ95 sebocytes

BackgroundAutophagy is a catabolic process for eliminating damaged organelles or proteins to maintain cellular homeostasis. Recently, lipids have been demonstrated to be targets for autophagosomal degradation. Therefore, autophagy might be involved in sebaceous gland homeostasis, however, relevant data are lacking. ObjectivesWe investigated the role of autophagy in sebaceous lipogenesis and its regulatory mechanisms in human SZ95 sebocytes. We also examined the possible role of autophagy in 13-cis-retinoic acid (13-cis-RA)-mediated sebosuppression. MethodsAutophagy markers expression was examined by immunohistochemistry in normal and acne lesional skin. SZ95 sebocytes were treated with autophagy inhibitors under starvation or treated with a combination of testosterone and linoleic acid (testosterone/LA), with or without autophagy inducer rapamycin or 13-cis-RA. Lipids were assessed by BODIPY and quantitative Nile Red staining. Autophagy‐related gene 7 small interference RNA was used to confirm the role of autophagy on the sebosuppressive effect of rapamycin or 13-cis-RA. ResultsAutophagy markers were strongly expressed in the maturing sebaceous gland cells in healthy skin, whereas downregulated in the acne-involved sebaceous glands. Testosterone/LA or insulin-like growth factor-1 inhibited starvation-induced sebocyte autophagy. Pharmacological inhibition of autophagy led to increased sebaceous lipid accumulation. Contrary, rapamycin inhibited the testosterone/LA-induced lipogenesis and expression of fatty acid synthesis genes via activating the autophagy pathway. 13-cis-RA increased autophagy in SZ95 sebocytes, partly via FoxO1 activation, and inhibition of autophagy abolished the sebosuppressive effect of 13-cis-RA. ConclusionsAutophagy plays an important role in the modulation of lipogenesis in human sebocytes and is involved in the sebostatic effect of 13-cis-RA.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism

BackgroundOrotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena.MethodsIn our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography–mass spectrometry.ResultsOur data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles.ConclusionsThe present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

Fatty-Acid Uptake in Prostate Cancer Cells Using Dynamic Microfluidic Raman Technology.

It is known that intake of dietary fatty acid (FA) is strongly correlated with prostate cancer progression but is highly dependent on the type of FAs. High levels of palmitic acid (PA) or arachidonic acid (AA) can stimulate the progression of cancer. In this study, a unique experimental set-up consisting of a Raman microscope, coupled with a commercial shear-flow microfluidic system is used to monitor fatty acid uptake by prostate cancer (PC-3) cells in real-time at the single cell level. Uptake of deuterated PA, deuterated AA, and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were monitored using this new system, while complementary flow cytometry experiments using Nile red staining, were also conducted for the validation of the cellular lipid uptake. Using this novel experimental system, we show that DHA and EPA have inhibitory effects on the uptake of PA and AA by PC-3 cells.

Design and in vitro evaluation of a novel synergic intra-articular treatment for post-traumatic osteoarthritis

Purpose: Post-traumatic (PT) OA, subsequent to joint injury, accounts for over 12% of the overall disease burden of OA. In the development of PTOA, the chondrocytes activity is progressively impaired with upregulation of proteolytic enzymes and downregulation of the most important growth factors involved in promoting chondrocyte anabolism. Based on the literature, we selected three promising target for PTOA treatment: (1) MMP3 is one of the metalloproteases mainly involved in collagen degradation, since several clinical studies reported an increase of synovial fluid MMP3 level in OA patients. In our previous work a Hyaluronic Acid (HA) partial hexadecylamide (HYADD®4) was selected as the strongest MMP inhibitor (MMPI) among a series of glycosaminoglycans. (2) TRPV4 channel is a Ca2+-permeable osmomechano-TRP channel, highly expressed in articular chondrocytes. It plays a pivotal role in chondrocyte mechanotransduction; if activated after a trauma, it is able to induce anabolic responses in vitro and chondroprotection in vivo. RN-1747, a small synthetic hydrophobic molecule was already reported as selective TRPV4 agonist. (3) mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase that plays a vital role in cartilage development and homeostasis as well as contributing to the process of cartilage degeneration associated with OA. Rapamycin is an mTOR inhibitor already FDA-approved for the prevention of organ transplant rejection. The objective of these studies are: (1) to optimize the HYADD®4 chemical structures in order to increase the MMPI potency and selectivity; (2) to develop a stable formulation of the MMPI with an active molecule (RN-1747 or Rapamycin) in order to obtain a novel synergic intra-articular (i.a.) treatment for PTOA. Methods: A series of HA derivatives were synthesized, varying the polymer backbone and the hydrophobic side chain, chemically characterized by 1H-NMR and tested in vitro as MMP3 inhibitors. The inhibitor selected, FID-145 (a partial oleylamide of sulphated HA; patent pending), was screened in vitro against 10 different human MMPs and other targets. The efficacy of FID-145 was assessed in an in vitro model of inflammatory arthritis, where bovine cartilage biopsies were exposed to IL-1β/Oncostatin M (OSM) for 3 weeks; at the same time, cartilage explants were treated with FID-145. Collagen release in the surnatants was quantified and compared to controls. The formulation of FID-145 in PBS was characterized by TEM analysis and a Nile Red staining, where increasing concentrations of FID-145 were mixed with the same concentration of Nile Red and the fluorescence emission at 600 nm was measured. Micellar formulations of FID-145 with either RN-1747 or Rapamycin were obtained through mixing, sonication and sterilizing filtration. The loading degree in either RN-1747 or Rapamycin and the stability of both formulations were analyzed by HPLC-MS. The mTOR inhibition activity and the TRPV4 activation potency were assessed by two specific assays (K-Elisa mTOR Activity Kit, Merck, and Fluo 8 AM calcium assay, AAT Bioquest, in primary chondrocyte, respectively). Kinetics of RN-1747 release from FID-145 formulation was determined in vitro, in human synovial fluid, at 37°C. Results: Among the tested HA derivatives, FID-145 showed the highest inhibition potency against MMP3 and no activity against ADAMTS4 and elastase in vitro. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed when FID-145 was added to the cartilage biopsies and collagen release decreased compared to control. FID-145 is an amphiphilic polymer and its ability to form micelles in water was confirmed by: 1) the TEM images showing the presence of spherical aggregates with diameter around 40 nm and 2) a Critical Micelle Concentration (CMC) of 0.2±0.1 mg/mL, measured with the Nile Red staining. RN-1747 and Rapamycin were successfully loaded at 0.15±0.05 mg/mL in 12 mg/mL FID-145 in PBS; both formulations proved to be stable, sterile and suitable for i.a. injection for OA treatment. The mTOR inhibition and the TRPV4 activation effect of the Rapamycin and TRPV4 formulations, respectively, was showed to be maintained in vitro. The controlled release of RN-1747 from the FID-145 micellar formulation showed that only about 50% of total loaded RN-1747 was released within 7 days. Conclusions: FID-145, a new amphiphilic HA derivative, was screened as a relatively potent and selective MMP3 inhibitor. When formulated in water, FID-145 yielded a stable micellar formulation easily loaded with hydrophobic, pharmacologically active molecules such as RN-1747 or Rapamycin, of promising relevance in the development of a novel synergic i.a. treatment for PT-OA.

Long noncoding RNA lncARSR promotes nonalcoholic fatty liver disease and hepatocellular carcinoma by promoting YAP1 and activating the IRS2/AKT pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) is the main cause for hepatocellular carcinoma (HCC). This study was intended to identify the function of long non-coding RNA (lncRNA) lncARSR in NAFLD and its role in human HCC cells (HepG2) proliferation and invasion.MethodsLncARSR expression was detected both in high fatty acid-treated HepG2 cells and NAFLD mouse model. After gain- and loss-of-function approaches in high fatty acid-treated HepG2 cells and NAFLD mice, lipid accumulation in livers from NAFLD mice and high fatty acid-treated cells was determined by H&E staining, Oil Red-O staining or Nile Red staining respectively. Expression of YAP1, adipogenesis- (Fasn, Scd1 and GPA) and IRS2/AKT pathway-related genes was measured. Cell proliferation was monitored by MTT and soft-agar colony formation assays, cell cycle was analyzed by flow cytometry, and cell invasion was examined by transwell assay. The tumor weight and volume were then measured through in vivo xenograft tumor model after silencing lncARSR.ResultsLncARSR was highly expressed in high fatty diet (HFD)-fed mice and high fatty acid-treated HepG2 cells. LncARSR was observed to bind to YAP1, which inhibited phosphorylation nuclear translocation. LncARSR activated the IRS2/AKT pathway by reducing YAP1 phosphorylation, and further increased lipid accumulation, cell proliferation, invasion and cell cycle. Silencing lncARSR in HFD-fed mice alleviated NAFLD by regulating YAP1/IRS2/AKT axis.ConclusionSilencing lncARSR suppressed the IRS2/AKT pathway, consequently reducing HCC cell proliferation and invasion and inhibiting lipid accumulation in NAFLD mice by downregulating YAP1, which suggests a clinical application in treating NAFLD.