Search

The ubiquitous presence of plastic brought on by the extensive use of plastic products calls for efficient and rapid plastic detection methods to detect and evaluate pollution. The commonly used Nile red dye takes many hours and is expensive while also not equally efficient across all the common plastic waste. To address this, we investigated the staining efficiency and optimized the ratio of a combined Coomassie brilliant blue and Methylene blue dye. In the optimisation process, Methanol-based Coomassie and Methylene blue dyes effectively stained the Polyethylene Terephthalate (PET), Polypropylene (PP), Polystyrene (PS), Linear Low-Density Polyethylene (LLDPE), Low-Density Polyethylene (LDPE), and Linear Low-Density Polyethylene (HDPE) plastics without compromising the plastic's integrity. Image analysis showed a generally better staining efficacy compared to Nile red. Through systematic experimentation, we identified specific optimal ratios of Coomassie (C) brilliant blue: Methylene (M) blue for various plastics: 5:5 (mass) for PVC, 7:3 (mass) for PET, and 8:2 (mass) for PP, LDPE, and HDPE. Additionally, the ratio of 10:0 (mass) was found suitable for PS and LLDPE. Given the cost-effectiveness, efficiency, and accessibility of the blue dyes in labs, the optimized ratio of the blue dyes makes it suitable for large-scale plastic staining across the six tested types of plastic, replacing Nile red. [ABSTRACT FROM AUTHOR]

As lipogenic yeasts are becoming increasingly harnessed as biofactories of oleochemicals, the availability of efficient protocols for the determination and optimization of lipid titers in these organisms is necessary. In this study, we optimized a quick, reliable, and high-throughput Nile red-based lipid fluorometry protocol adapted for oleaginous yeasts and validated it using different approaches, the most important of which is using gas chromatography coupled to flame ionization detection and mass spectrometry. This protocol was applied in the optimization of the concentrations of ammonium chloride and glycerol for attaining highest lipid titers in Rhodotorula toruloides NRRL Y-6987 and Yarrowia lipolytica W29 using response surface central composite design (CCD). Results of this optimization showed that the optimal concentration of ammonium chloride and glycerol is 4 and 123 g/L achieving a C/N ratio of 57 for R. toruloides, whereas for Y. lipolytica, concentrations are 4 and 139 g/L with a C/N ratio of 61 for Y. lipolytica. Outside the C/N of 33 to 74 and 45 to 75, respectively, for R. toruloides and Y. lipolytica, lipid productions decrease by more than 10%. The developed regression models and response surface plots show the importance of the careful selection of C/N ratio to attain maximal lipid production. Key points: • Nile red (NR)-based lipid fluorometry is efficient, rapid, cheap, high-throughput. • NR-based lipid fluorometry can be well used for large-scale experiments like DoE. • Optimal molar C/N ratio for maximum lipid production in lipogenic yeasts is ~60. [ABSTRACT FROM AUTHOR]

The calcifying cells of corals are responsible for skeleton formation, a process that is the basis for reef building. Their cell biology is therefore of primary interest, but current knowledge is limited because direct in vivo investigation of this cell type is challenging. Studies at the growing edge of laterally extending coral colonies allow for direct observations of actively calcifying cells in living samples. In the current study, we used this approach to study lipid droplets, which are considered storage organelles that fuel cells for physiological processes. Using the fluorescent lipid probe Nile Red and in vivo confocal microscopy, we observed lipid droplets in calcifying cells of the coral Stylophora pistillata and we suggest that they play a key role in coral calcification. [ABSTRACT FROM AUTHOR]

Large molecules of dyes or other substances can act as nano-adsorbents in aqueous surfactant solutions and, being coated with surfactant molecules, they resemble surfactant micelles with a solubilizate. These micelle-like particles were called protomicelles (or, more precisely, adsorption protomicelles, as in this paper). Their formation does not require a critical micelle concentration (CMC) and begins immediately when a surfactant is added to a solution. In this spectrophotometric study of Nile red (NR) in aqueous solutions of nonanoic acid (NOA), the first case where protomicelles were more important than micelles was demonstrated. It was shown that NR solubilization in both premicellar and micellar solutions proceeds through protomicelles rather than through NOA micelles. In the theoretical part of the paper, on the basis of the chemical potential of the nano-adsorbent, it was proved that the addition of a surfactant to a solution always increases the solubility of the nano-adsorbent. The dependence of adsorption on the curvature of the nano-adsorbent surface was considered, and it was shown that the adsorption equations for flat surfaces are applicable to curved surfaces with high accuracy. [ABSTRACT FROM AUTHOR]

Cysteine (Cys) is a significant biological mercaptan that achieves key roles in several important physiological processes, such as reversible redox homeostasis in living organisms. Abnormal levels of Cys in the human body are directly related to many diseases. In this work, we constructed a sensitive sensor (Cys-NR) by connecting a Cys recognition group to a Nile red derivative. Due to photo-induced electron transfer (PET), the Cys-NR probe showed little fluorescence at 650 nm. With the addition of Cys to the assay solution, the chlorine unit of the probe was substituted by the thiol group of Cys. Further, the amino and sulfhydryl groups in cysteine underwent an intramolecular rearrangement, which led to the Cys-NR probe water solution turning from colorless to pink with an enhancement in fluorescence. The red fluorescence at 650 nm increased about 20 times. Based on the turn-on signal, a selective Cys detection method is developed. The probe signal is not affected by various potential interferences or other competing biothiols and the limit of detection (LOD) is determined to be 0.44 μM. In addition, the probe is further employed for imaging of Cys in live cells, revealing good biological imaging ability that could provide a new way of intracellular Cys detection. [ABSTRACT FROM AUTHOR]

Environmental plastic contamination is a global problem, which is driven by the growing demand for plastics. Over time, plastics undergo fragmentation processes and become so-called microplastics, which have been shown to be a threat to the various environments where their presence occurs. The most accepted hypothesis is that, like macroplastics, microplastics have fragmented into smaller and smaller particles, with sub-micrometer sizes. However, little is known about fragmentation rates and which are the most determining factors in this process. The main objective of the present study was to investigate the biofragmentation capacity of microplastics by Daphnia similis, a microcrustacean from inland waters. The organisms were exposed to polystyrene spheres (PS; 24 µm), under two concentrations: 135 and 1350 items/50 mL, for a period of 6 days. The rate of biofragmentation was obtained by analyzing the size of the particles over time (48, 96, and 144 h); ingestion and survival rates were also investigated. At the end of the exposure, the microspheres had their size reduced by 32.8%. From the first 48 h, the organisms ingested particles, and the rate of ingestion was dose dependent. Mortality was shown to increase in the treatment of higher concentration. The results demonstrate the ability of this microcrustacean to induce the reduction of the dimensions of microplastics, evidencing a fragmentation pathway previously neglected. [ABSTRACT FROM AUTHOR]

To explore the cytotoxicity of chitosan-modified ploy lactic-co-glycolic acid (CS-PLGA) drug-loaded nanoparticle (NP) and its role in the treatment of inner ear disease, the CS-PLGA NP loaded with Nile red (CS-PLGA-NR NP) was constructed, and the safety of its transport in the inner ear was explored. First, PLGA NPs with different particle sizes were prepared and modified with CS to obtain CS-PLGA-NR NPs. After the CS-PLGA-NR NP suspension was incubated with HEI-OC1 cells, cytotoxicity and cellular uptake were detected. Subsequently, the CS-PLGA-NR NP suspension was injected into SD rat binaural tympanum, and healthy rats were taken as control. Morphological differences in the hair cells morphology and tissue morphology in rat vestibular sensory area and cochlea sensory area were measured via immunohistochemical staining and HE staining. It was found that the shape of the prepared PLGA NP was relatively round and evenly distributed, and there was no adhesion. In addition, different NPs had similar infrared spectrum characteristics. Verified by X-ray photoelectron spectroscopy (XPS), CS-PLGA-NR NPs were successfully prepared. In vitro test results showed that the viability of HEI-OC1 cells after being incubated with 1–10 mg/mL CS-PLGA-NR NP was higher than 90%, and the cell uptake also increased with the increase of concentration of CS-PLGA-NR NP (within the range of 0–150 μg/mL). In vivo results showed that after CS-PLGA-NR NP treatment, the rat hair cells in vestibule and cochlea sensory areas were arranged regularly, without deficient. In addition, the morphology and size of rat vestibule and cochlea tissue cells were normal, and no inflammatory cells such as granulocytes appeared. In summary, the prepared CS-PLGA-NR NP had low cytotoxicity and favorable inner ear tissue biocompatibility. [ABSTRACT FROM AUTHOR]

Microplastic (MP) is yet another form of chronic anthropogenic contribution to the environment. MPs are plastic particles (<5 mm) that have been widely found in the most diverse natural environments, but their real impacts on ecosystems are still under investigation. Here, we studied the toxicity of naturally aged secondary polypropylene (PP) MPs after constant exposure to ultraviolet radiation (26 µm) to the third instar larvae of Chironomus sancticaroli, a dipteran species. The concentrations tested were 13.5; 67.5; and 135 items g−1 of dry sediment. C. sancticaroli organisms were investigated for fragment ingestion, mortality and changes in enzymatic biomarkers after 144 h of exposure. The organisms were able to ingest MPs from the first 48 h, and the amount of items internalized was dose-dependent and time-dependent. Overall, the results show that mortality was low, being significant at the lowest and highest concentrations (13.5 and 135 items g−1). Regarding changes in biochemical markers, after 144 h MDA and CAT activities were both significantly altered (increased and reduced, respectively), while SOD and GST levels were unchanged. In the present study, naturally aged polypropylene MPs induced biochemical toxicity in C. sancticaroli larvae, with toxicity being higher according to exposure time and particle concentration. [ABSTRACT FROM AUTHOR]

Traditional stereomicroscopy (SM) is limited for the identification of microplastics of less than 500 µm in wastewater treatment plants (WWTPs). Accordingly, novel methods for the accurate quantification of these microplastics are needed. In this study, we investigated the polymer type, morphology, size distribution, and abundance of microplastics in each unit of three selected WWTPs by SM and a fluorescence-based protocol (FR) combined with FTIR. Using the FR method, most microplastics detected in the three WWTPs were 50–200 µm in size. Polyethylene, polypropylene, and polyamide were the main polymer types, and the distributions of fibers, films, and debris were determined. Despite highly similar microplastic removal rates (78.6‒95.2% (SM) and 77.4‒94.2% (FR)) in the WWTPs by the two methods, the microplastic abundances obtained by FR (405‒6987 items/L) were approximately 2 orders of magnitude higher than the corresponding results by SM (1‒21 items/L). In addition, a considerable number of small-sized microplastics (< 500 µm) were detected in the effluents (405‒947 items/L) using FR. These results clearly reveal that microplastics in WWTPs have been seriously underestimated in most previous studies based on SM. Further research should focus on the environmental risks of small-sized microplastics from WWTPs. [ABSTRACT FROM AUTHOR]

Investigating the role of lipids in mammalian preimplantation development is an important task in modern embryology. The aim of this study was to evaluate the amount of intracellular lipids in preimplantation mouse embryos during in vivo and in vitro development using confocal laser scanning microscopy. The development of preimplantation embryos was accompanied by a decrease in total lipid content, which was observed in vivo at the blastocyst stage and at the morula stage in vitro. Besides, total lipid content was lower during in vitro embryo development as compared to in vivo development. Thus, lipid metabolism during the preimplantation period depends on the conditions in which development occurs (in vivo or in vitro); at the blastocyst stage, the amount of intracellular lipids in preimplantation mouse embryos decreases significantly. [ABSTRACT FROM AUTHOR]

At present, due to rapid urbanization and increasing anthropogenic activities, the occurrence of microplastics (MPs) in riverine fishes is becoming an emerging area of food safety concern worldwide. This study was conducted to determine the abundance of MPs in three riverine fish species (Amblypharyngodon mola, Puntius ticto, and Mystustengara) collected from six major rivers of Chhattisgarh, India. The Nile Red staining method was used for the identification and quantification of MPs in fish samples. Out of 140 fish samples examined, 87.9% of the fishes had ingested MPs. The abundance of MPs per gram of fish and per fish was 3.25 ± 2.58 and 14.9 ± 8.12, respectively. Most of the MPs identified were of fragment type and with their size ranging between 20and 300 μm. The demersal fishes had higher levels of MP contamination as compared to surface feeder fishes. Fish samples collected from Mahanadi River in Janjgir-Champa district showed the highest abundance of MPs both in terms of MPs per gram (5.93 ± 3.29) and per fish (22.7 ± 6.59), and this could be attributed to the high level of human activity with municipal waste dumping in the area. The results further suggest the possibility of human ingestion of MPs through the consumption of riverine fishes in this region. This study shows the abundance of MPs in riverine fishes which raises health concerns for the fish consumers and further research is required to assess the overall extent of the impact of MPs on the environment and human health. [ABSTRACT FROM AUTHOR]

Solubilization of nile red (NR) dye in aqueous solutions of tetradecyltrimethylammonium bromide (TB) was studied by spectrophotometric method. Experiments were carried out both with NR saturated solutions under conditions of thermodynamic equilibrium of the solution with a dye precipitate, and at an NR constant weight. The solubility of NR in water (about 2 μM) has been refined. Extinction coefficients of NR monomers in water and micellar surfactant solutions were found. The micellization critical concentration (CMC) of TB in the presence of NR was determined, and the theoretical conclusion about the decrease in the surfactant CMC under the action of solubilizate was confirmed. The solubilization thermodynamic parameters were calculated on the basis of the following experimental data: the solubilization capacity of micelles, the coefficient of the solubilizate distribution between micelles and the surrounding solution, and also the standard affinity of NR solubilization. The problem of molecular NR aggregation in water and aqueous solutions of TB was considered. It has been found that the rate of reaching aggregative equilibrium is much lower than the rate of NR dissolution, therefore published spectral data based on the presence of only NR monomers mostly refer to the insufficiently equilibrium cases. It is shown that the process of NR solubilization begins in the prеmicellar region of TB, but is most effective in the presence of micelles. [ABSTRACT FROM AUTHOR]

The mixotrophic growth of microalgae offers great potential as an efficient strategy for increasing biomass production. In this study, Scenedesmus obliquus was used to investigate the biomass growth conditions and biochemical parameters of algae cells in autotrophic and mixotrophic conditions with and without aeration. It was found that mixotrophic cultivation of algae cells by 50 mM acetate had a strong effect on increasing fresh weight, chlorophyll a, b, carotenoid, astaxanthin, protein, and lipid content (with 20, 4.6, 8.7, 60, 4.5, 4.8, and 18.4-fold, respectively) in comparison to the control cultures. Mixotrophy by pyruvate also increased the amount of all mentioned parameters. The best concentration of pyruvate was found to be 50 mM, but the effects of acetate were stronger. Aeration in a short time (2 h) had a different effect on mixotrophic cultures with acetate and pyruvate. It declined the effect of acetate on increasing the fresh weight and the amount of other biochemical parameters. Unlike the acetate treatment, aeration had an enhancement effect on increasing the fresh weight and biochemical parameters content in 50 and 100 mM pyruvate treatments. In the control cultures, the saturated acid, caprylic acid (C8:0) (40%), and the polyunsaturated eicosapentaenoic acid (EPA; C20:5 n3) (17.5%) showed higher percentages. Mixotrophy caused a decrease in the percentage of saturated fatty acids (SFAs) and, on the other hand, an increase in the percentage of unsaturated fatty acids (UFAs). EPA was the main fatty acid in the mixotrophic condition. Aeration in the phototrophic condition decreased the percentage of SFAs, while increasing that of UFAs. Aeration under mixotrophic conditions increased UFAs in both acetate and pyruvate-treated cultures. The results suggest that aeration could be an important factor in interaction with other algae growth conditions. It should be noted that acetate had generally better effects on the biomass quality of S. obliquus without aeration. [ABSTRACT FROM AUTHOR]

A recently reported Nile red (NR) dye conjugated with benzothiadiazole species paves the way for the development of novel organic-based sensitizers used in solar cells whose structures are susceptible to modifications. Thus, six novel NR structures were derived from two previously developed structures in laboratories. In this study, density functional theory (DFT) calculations and time-dependent DFT (TD-DFT) were used to determine the optoelectronic properties of the NR-derived moieties such as absorption spectra. Various linkers were investigated in an attempt to understand the impact of π-linkers on the optoelectronic properties. According to the findings, the presence of furan species led to the planarity of the molecule and a reduction in the band gap between the LUMO and the HOMO. Each one of the aforementioned molecules exhibited great delocalization of π-electrons. Based on the TD-DFT calculations, two furans had the highest value for the red-shift. There is an excellent correlation observed between the computed optoelectronic properties and calculated HOMO-LUMO gaps. In conclusion, the current work aimed at clarifying the impact of π-linkers on the photophysical properties of the NR-derived moieties. Also, the current study provided useful insights into the development of novel species used in optoelectronic devices. [ABSTRACT FROM AUTHOR]

The study devised a detection process combining Nile red-containing media, polymerase chain reaction (PCR), and gas chromatography (GC) to evaluate the possibility of microbes becoming polyhydroxyalkanoate (PHA) producers. The Nile red and PCR detection steps of designating PHA producers had true positive rates of 39.4% and 40%, respectively, and the use of GC analysis as the final step yielded accurate results for the production types and yields of PHAs. When the number of screening samples was up to 102, connecting all three inspection methods in tandem generated economic benefits. When up to 105 samples were screened, the use of all three detection methods reduced the cost to 3% of the cost and the time consumed 6% of using just Nile red plus GC or PCR plus GC. However, when the sum of samples exceeded 108, the cost of combining the three methods exceeds 1 million US dollars and was excessive; here, the combination of Nile red plus PCR could be considered, even though the true positive rate was only 30.7%. [ABSTRACT FROM AUTHOR]

The micellization processes in the systems tetraethylene glycol monododecyl ether (C12E4)–heptane (1) and C12E4–Nile red–heptane (2) have been studied by electron absorption spectroscopy. In system 1, reverse micelles of small size appear, which makes it difficult to find the critical micelle concentration (CMC). In this regard, a method for determining CMC based on spectroscopic data was tested. The same one was done for system 2, where the appearance of Nile red as a C12E4 nano-adsorbent leads to the formation of protomicelles below the CMC. The CMC itself in system 2 turns out to be lower than the CMC in system 1, which corresponds to the predictions of the theory. The water role in the formation of reverse micelles was studied using a commercial analogue of C12E4 preparation Brij 30 (with a water content of 1%). The results obtained are consistent with theoretical predictions. [ABSTRACT FROM AUTHOR]

The widespread presence of microplastics in multiple environmental compartments has largely been demonstrated. Assessing the ecological risk that microplastics pose is, at the present stage, hindered due to methodical differences. Moreover, different methods hamper meaningful comparisons between studies and data on microplastics <300 μm is scarce. Therefore, we focused on microplastics >20 μm in freshwater and sampling-related aspects in this concern. Sampling was conducted between 2018 and 2020 in the Tollense catchment in northeastern Germany and was carried out by in situ pump filtration. Two different sampling systems (cutoff sizes 20 μm and 63 μm) were applied to filter water volumes of 0.075–1.836 m3. Retained particles were analyzed by a combination of Nile red staining and micro-Raman spectroscopy. Thereby, we found microplastic concentrations between 123 and 1728 particles m−3 using the 63-μm cut-off size and between 1357 and 2146 particles m−3 using the 20-μm cut-off size. Local hydrodynamics (discharge and flow velocity) and land cover are likely influencing the observed microplastic concentrations and fluxes. The variability between both sampling systems cannot fully be explained by the different mesh sizes used. We argue that differentiation between a theoretical cut-off size (finest mesh) and a factual cut-off size (reliable quantification) can help to understand sampling related differences between studies. [ABSTRACT FROM AUTHOR]

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00–40.00 mg L−1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00 mg L−1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00 mg L−1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points: • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis). [ABSTRACT FROM AUTHOR]

Contamination from microplastics is a growing concern around the globe that materialized over the last few decades. Road dust is one of the main contributors of microplastics in an urban environment and presence of microplastics can be a major detriment to health of urban population. The present study focuses on the presence, quantification, and identification of microplastics in the ambient air. Coarse (PM10) and fine (PM2.5) particulates from urban and rural sites of Nagpur, India is collected using high volume air samplers for weekdays and weekends for a period of 24 h. Samples are collected in Polytetrafluoroethylene filter papers and are analyzed using a stereomicroscope, fluorescence microscopic study, and Fourier transform infrared spectroscopy (FTIR) analysis. Fluorescence microscopy is conducted using Nile Red staining for analyzing the presence of microplastics and studying its shapes and colors. An average total abundance ranging from 50 to 120 particles/day is observed. Fibers are the dominant shape, and transparent/white is the dominant color observed in all four sites. FTIR analysis is conducted for identification of the chemical composition of particles in Attenuated Reflectance mode. The polymers identified in the region are low-density polyethylene, rayon, rubber fiber, polystyrene, polyaniline, polyolefin, and chlorinated polyvinyl chloride. This research identified significant contamination of the study region from microplastics and highlights the necessity of performing a comprehensive health risk analysis of the study region to examine the impact of microplastics on urban health. Article Highlights: PM10 and PM2.5 samples are collected from urban and rural environment of Nagpur, India Microplastic particles collected from particulate samples are classified based on shape and colour White/transparent and Fibrous microplastics are found to contribute most to the study region Least contribution is from spherical and coloured microplastics Polyethylene is the dominant polymer identified in the study region [ABSTRACT FROM AUTHOR]

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 530/575 nm 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. [ABSTRACT FROM AUTHOR]

Despite the exponential increase of studies on plastic debris in recent years, there are still few works focusing on the problem as it relates to inland waters: little is known about the accumulation and dispersion dynamics on lake shores, and there are no standardized sampling methods for monitoring purposes. The accumulation of plastic litter in natural habitats also threatens the resident organisms. In this paper, we investigated the abundance and accumulation of plastic particles, ranging in size from 1 to 50 mm, from the beach sediment of three volcanic lakes in Central Italy: Albano, Bracciano, and Vico. The collection was designed to define the most important variables that one must consider in order to obtain a representative sample of plastic litter in a lake environment. In view of the high heterogeneity of sampling protocols used, comparison among the obtained results is limited and sometimes impossible. By using one of the proposed sampling methodologies, and critically analyzing the results, we aimed to highlight a possible monitoring criterion and to identify specific elements that can be meaningful and representative. The samples were collected in May and September 2017. For each lake, we sampled plastic items and sediments from two beaches. Albano contained the largest amount of plastic (in weight), while Bracciano had the largest number of particles. Our observations lead us to infer that the number of particles is the parameter most sensitive to environmental variations, as well as the more suitable for monitoring with greater definition the differences between sites. Moreover, sampling should be taken in different seasons, following a sampling pattern that includes at least two beaches placed in strategic positions with respect to wind and waves. In order to identify new indicators to evaluate the entry points of plastic into the food web, we collected, from the same sites analyzed, some specimens of the Talitrid Amphipod Cryptorchestia garbinii, a detritivorous species having a critical role in debris turnover of these environments. To investigate the microplastic (MP) ingestion in natural conditions, we analyzed their digestive tracts with both Nile red staining method and micro-FTIR spectroscopy. The analyses confirmed that C. garbinii was able to ingest plastics in natural conditions. Therefore, it can signify one of the entry points for microplastics (MPs) in the trophic chain. This observation constitutes the first evidence of MP ingestion in this species. [ABSTRACT FROM AUTHOR]

The release of nanoplastics (NP) from the weathering of microplastics is a major concern for the environment. Methods for the detection of NP in biological tissues are urgently needed because of their ability to penetrate not only in tissues but also in cells. A simple fluorescence-based methodology for the detection of polystyrene NP in biological tissues is proposed using the solvatochromic properties of Nile red. Although NPs alone increased somewhat Nile red fluorescence, a characteristic hypsochromic shift in the emission spectra was found when the dye and NP were incubated with subcellular tissue fraction. To explain this, the probe and NPs (50 and 100 nm) were prepared in the presence of increasing concentrations of two detergents (Tween-20, Triton X-100) as a proxy to phospholipids. The data revealed that both detergents readily increased fluorescence values when added to the NP and Nile red. The addition of NPs in tissue extracts blue-shifted further the emission spectra to 623 nm from the normal Nile red-lipid peak at 660 nm. The fluorescence intensity was proportional to the NP concentration. A methodology is thus proposed for the detection of NPs in laboratory-exposed organisms based on the solvatochromic properties of Nile red. The methodology was used to detect the presence of NP and changes in polar lipid contents in Hydra attenuata exposed to polystyrene NP. [ABSTRACT FROM AUTHOR]

One-pot synthesis of a photostable, hydrophobic and green fluorescent probe remains a challenge to develop luminescent biomaterials as well as biomedia technology. Semiconducting polymer dots (PDs) hold a great promise as fluorescence nanoprobes due to their photostability, biocompatibility and high quantum yield. Here, the synthesis and characterization of Nile red-doped PFBT PDs as a highly sensitive hydrophobicity probe has been reported. Nile red, a phenoxazone dye, fluoresces intensely in organic solvents and hydrophobic lipids and in varying color; however its fluorescence is totally quenched in water. First, Nile red-doped poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1′,3)-thiadiazole)] (PFBT) PDs were synthesized through a nano-precipitation technique. Energy transfer from PFBT to Nile red happens through FRET mechanism. The PDs exhibited a red fluorescence with a peak centered at 620 nm (photo-excited at 460 nm). In aqueous media, the fluorescence strongly quenched, but by increasing the hydrophobicity the fluorescence of PDs significantly enhances as the polarity of the medium decreases. This can happen by adding organic solvents or nonpolar species to the medium. The Nile red@PFBT PDs have remarkable specification over Nile red dye. Compared to Nile red, this probe is soluble in water, more photostable, and more brightness due to light-harvesting ability of PDs and less wavelength shift in different medium polarity. We used this probe to investigate polarity of different organic solvents, to study surface hydrophobicity changes of BSA and also Ca2+ determination due to conformational changes of calmodulin. Also we used this probe for imaging of lipid bilayers and cellular membrane. [ABSTRACT FROM AUTHOR]

Algal oils, which can be converted into biofuels, are one of the few possible substitutes for fossil fuels that can be used in conventional transportation systems. The algae have the distinctive advantage of having an exceptionally high productivity much higher than that of terrestrial plant-based oil production. However, the commercial viability of algal oils can only be achieved if the algal lipid content and consequently their productivity are still further enhanced. In this study, bicarbonate ions were investigated for their potential as a lipid-inducing chemical because they are one of the first key substrates in triacylglycerol (TAG) biosynthesis in microalgae. To examine this hypothesis, Chlamydomonas reinhardtii CC-124 was chosen as a model microalgae species, and the TAG content in CC-124 was semi-quantitatively detected by the Nile Red staining method. The highest TAG accumulation was observed for 12.5 g L−1 sodium bicarbonate. At this concentration, the light intensity was found to have a more important role than the cultivation temperature. This result suggests that the simple addition of sodium bicarbonate to a harvested and concentrated algae solution could serve as a facile and effective method for boosting the biofuel productivity of algae. [ABSTRACT FROM AUTHOR]

The potential of lipid accumulation by oleaginous yeast Cryptococcus vishniaccii grown on amaranth seed aqueous extract (AAE) media was assessed. Maximum cell biomass productivity of 104 mg/L/h, lipid productivity of 54 mg/L/h, and lipid content of 52.31% were recorded on AAE when carbon to nitrogen (C:N) ratio increased from 134 to 147 after removal of ammonia nitrogen. The lipid droplet (LD) size (2.32 ± 0.38 μm) was visualized by fluorescence microscopy using Nile red stain indicating maximum accumulated triacylglycerol (TAG) at C:N 147. Fatty acid methyl ester (FAME) profile obtained after transesterification of extracted lipid revealed the presence of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Data showed the presence of high monounsaturated fatty acid (MUFA) content (68.17%) depicting improved winter operating conditions of biodiesel. Various quality parameters of biodiesel were evaluated and compared to the American and European biodiesel standards specifications. Based on the lipid productivity, distribution of fatty acids, and evaluated properties obtained; the lipid accumulation by C. vishniaccii utilizing amaranth seeds as substrate could serve as a feasible feedstock for biodiesel production. [ABSTRACT FROM AUTHOR]

Micelles are of increasing importance as versatile carriers for hydrophobic substances and nanoprobes for a wide range of pharmaceutical, diagnostic, medical, and therapeutic applications. A key parameter indicating the formation and stability of micelles is the critical micelle concentration (CMC). In this respect, we determined the CMC of common anionic, cationic, and non-ionic surfactants fluorometrically using different fluorescent probes and fluorescence parameters for signal detection and compared the results with conductometric and surface tension measurements. Based upon these results, requirements, advantages, and pitfalls of each method are discussed. Our study underlines the versatility of fluorometric methods that do not impose specific requirements on surfactants and are especially suited for the quantification of very low CMC values. Conductivity and surface tension measurements yield smaller uncertainties particularly for high CMC values, yet are more time- and substance consuming and not suitable for every surfactant. [ABSTRACT FROM AUTHOR]

Encapsulation of drugs in nanoparticles can enhance the accumulation of drugs in tumours, reduce toxicity toward healthy tissue, and improve pharmacokinetics compared to administration of free drug. To achieve efficient delivery and release of drugs at the target site, mechanisms of interaction between the nanoparticles and cells and the mechanism of delivery of the encapsulated drug are crucial to understand. Our aim was to determine the mechanisms for cellular uptake of a fluorescent hydrophobic model drug from poly(butylcyanoacrylate) nanoparticles. Prostate adenocarcinoma cells were incubated with Nile Red-loaded nanoparticles or free Nile Red. Uptake and intracellular distribution were evaluated by flow cytometry and confocal laser scanning microscopy. The nanoparticles mediated a higher intracellular level and more rapid uptake of encapsulated Nile Red compared to model drug administered alone. The main mechanism for delivery was not by endocytosis of nanoparticles but by nanoparticle-cell contact-mediated transfer directly to the cytosol and, to a smaller extent, release of payload from nanoparticles into the medium followed by diffusion into cells. The payload thus avoids entering the endocytic pathway, evading lysosomal degradation and instead gains direct access to intracellular targets. The nanoparticles are promising tools for efficient intracellular delivery of hydrophobic anticancer drugs; therefore, they are clinically relevant for improved cancer therapy. [ABSTRACT FROM AUTHOR]

Microalgae are considered a promising source of oil for biodiesel production. This work reports an estimation method of oil content inside living microalgal cells by visualization and image processing techniques. This approach was used to analyze the time course of oil accumulation patterns in Nile Red-stained microalgal cells of Scenedesmus sp. cultivated in nitrogen-deficient medium used to induce oil accumulation in microalgal cells. Nile Red staining is a widely used technique for studying oil content of microalgal cells. The intracellular oil content was estimated by mathematically evaluating the oil volume inside the stained cell. This novel visualization approach has the potential to be used in ex vivo studies of oil content at the level of single microalgal cells. This method can also be applied to other types of oil-producing microorganisms because of its accuracy, precision, and reduction in the time and effort required for optimization. [ABSTRACT FROM AUTHOR]

A process has been developed for the production of poly(3-hydroxybutyrate) (PHB) with bacterium Rhodococcus equi using crude palm kernel oil (CPKO) as a carbon source. Such process will enable the production of biodegradable biopolymers to overcome some of the limitations associated with the use of petroleum-based plastics. A pure isolate was obtained from a fertile soil and was screened using the Nile Red stain for its ability for polyhydroxyalkanoates production. The isolate was identified by morphological characterization and biochemical tests. Identification by 16S rRNA has confirmed the species as Rhodococcus equi. Different carbon sources were used in an attempt to find the best one for the biosynthesis of PHB. The microscopic observations of PHB inside the bacteria were checked by the phase-contrast light microscope which showed a bright appearance. Moreover, the fluorescent microscope showed a bright orange fluorescence, and the transmission electron microscope showed white PHB granules with different sizes and different numbers within the bacteria cells. Rhodococcus equi gave the maximum cell dry mass as 1.43 g/L with the maximum PHB content as 38.1 % by weight when CPKO (1 %) was used as a carbon source. [ABSTRACT FROM AUTHOR]

The present work is focused on the long-term stability and in vitro cellular internalization of newly designed biocompatible polyester nanocapsules prepared via nanoprecipitation approach with mean diameter <165 nm and narrow size distribution, dedicated to theranostic applications. We monitored the optical, morphological, and biological properties of the nanocarriers loaded by multifunctional cargo, i.e., paclitaxel (PTX) and a fluorescent marker: coumarin-6 (CR-6) or Nile Red (NR), by fluorescence and UV-vis spectroscopy (encapsulation efficiency), dynamic light scattering (average size expressed as hydrodynamic diameter, D), zeta potential ( ζ, colloidal stability), atomic force microscopy (AFM, imaging), and confocal laser scanning microscopy (CLSM, nanocapsule visualization, and cellular internalization in vitro by human breast cancer MCF-7/WT cells). The fabricated nanocapsules with optimal composition of oleic phase, i.e., coconut oil, palm oil, and Capmul MCM, as well as polymeric shell, i.e., polylactic acid (PLA), poly ( ε-caprolactone) (PCL), and poly (lactide-co-glycolide) (PLGA), showed high loading capacity, long-term stability, and improved localization of the active cargo in studied tumor cells. Therefore, our results prove that the studied polyester oil core nanocapsules provide lifelong and biocompatible nanocarriers suitable for in vivo administration and for diagnostic applications. [ABSTRACT FROM AUTHOR]

Rapid determination of the specific lipid content of microalgae can accelerate screening and development of algae fuel application. Until recently, lipid content was commonly measured using gravimetric solvent extraction. Such methodology is time consuming and requires relatively large sample volumes. The Nile Red lipid assay has recently gained acceptance as it requires minute sample sizes. However, the current methodology is highly biased through interference of chlorophyll. Chlorophyll content can vary significantly between species, cultivation conditions, and culture age, remaining a serious but accepted problem. The assay was improved in this study in two ways. The cell dry weight determination was generalized by chemical chlorophyll removal prior to optical cell density measurements. Algae samples with different chlorophyll content were used to correlate optical measurements to cell dry weight and the correlation was substantially improved compared to non-treated samples (97 % decrease in sum of squared residuals). Degradation of chlorophyll was also found to increase the emission intensity of Nile Red, presumably by removing a source of molecular absorption of red light, potentially increasing the detection threshold. Secondly, milk fat was used as a standard for correlating lipid concentration to Nile Red fluorescence. It was cheaper, more precise, easier to accurately prepare, and linearly correlated to lipid concentration over a broader range than triolein, largely due to the fact that it forms a stable lipid/water micro-emulsion. The improved methodology for the Nile Red lipid assay was found to significantly increase the working range and accuracy of the assay. [ABSTRACT FROM AUTHOR]

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure. [ABSTRACT FROM AUTHOR]

Due to the increasing environmental problems caused by the use of fossil fuels, microalgae have been spotlighted as renewable resources to produce biomass and biofuels. Therefore, the investigation of the optimum culture conditions of microalgae in a short time is one of the important factors for improving growth and lipid productivity. Herein, we developed a PDMS-based high-throughput screening system to rapidly and easily determine the optimum conditions for high-density culture and lipid accumulation of Neochloris oleoabundans. Using the microreactor, we were able to find the optimal culture conditions of N. oleoabundans within 5 days by rapid and parallel monitoring growth and lipid induction under diverse conditions of light intensity, pH, CO and nitrate concentration. We found that the maximum growth rate ( µ = 2.13 day) achieved in the microreactor was 1.58-fold higher than that in a flask ( µ = 1.34 day) at the light intensity of 40 µmol photons m s, 5 % CO (v/v), pH 7.5 and 7 mM nitrate. In addition, we observed that the accumulation of lipid in the microreactor was 1.5-fold faster than in a flask under optimum culture condition. These results show that the microscale approach has the great potential for improving growth and lipid productivity by high-throughput screening of diverse optimum conditions. [ABSTRACT FROM AUTHOR]

Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, ), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses. [ABSTRACT FROM AUTHOR]

Nile Red (NR) staining potentially offers a simple method for monitoring lipid accumulation in microalgal cultivation. However, variable staining efficiencies and methods have been reported. The effect of dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol on NR penetration with four different phytoplankton species representing different taxonomical groups was studied. Treatment with the solvents enhanced the NR fluorescence of the diatom Phaeodactylum tricornutum during kinetic fluorescence measurements, but high concentrations of solvents were needed. None of the solvents improved NR staining of the green alga Chlorella pyrenoidosa and Scenedesmus obliquus, which are known to be difficult to stain due to their thick and rigid cell walls. The naked Isochrysis sp. cells stained best without solvents. The results confirm that NR staining protocol needs to be optimized for each species. [ABSTRACT FROM AUTHOR]

The present study documented the potential of two lignocellulosic wastewaters viz. corncob waste liquor (CWL) and paper mill effluent (PME) and a cellulosic waste (deoiled algae extract (DAE)) as feedstock for microbial oil production by Aspergillus awamori (MTCC 11639). Using cellulosic waste as substrate/feedstock for single cell oil (SCO) production reduced the overall cost of production and also accounts for simultaneous waste remediation. DAE showed improvement in biomass production with time, in terms of dry weight (12.8 g/l), followed by CWL (10.2 g/l) and PME (9.83 g/l). Total/neutral (T/N) lipid productivity recorded the same trend where higher T/N was obtained with DAE (35.4 and 18 %), followed by CWL (33.3 and 16.3 %) and PME (32.2 and 15 %). During the growth of fungi, efficient colour (78 %) along with good carbohydrate/chemical oxygen demand (COD) removal was registered, depicting the potential of A. awamori in utilizing the available carbon in waste. The presence of extracellular laccase enzyme in the fermentation medium is also an indicator that validates the observed colour removal and lignin degradation. Higher number of saturated fatty acid (SFA) than unsaturated fatty acid (USFA) in the fatty acid methyl esters (FAME) analysis indicates that the fungal oil obtained has properties similar to those of biodiesel. This study establishes a basis for fungal-based oil production utilizing cellulosic waste as primary feedstock. Integrating waste remediation with microbial oil production also forms an essential element in the present work, especially in the context of biorefinery. [ABSTRACT FROM AUTHOR]

Nitzschia ZJU1, which originated from the diatom Nitzschia sp. (ash free dry weight of biomass was 0.12 g L and its lipid content was 13.34 %) after Co-γ ray irradiation and domestication at high-salinity, was re-mutated by Cs-γ irradiation to increase lipid productivity. The lipid yields of the new mutants Nitzschia ZJU2 and Nitzschia ZJU3, which were selected by Nile Red fluorescence, were increased from 209.9 mg L (for the original Nitzschia ZJU1) to 245.5 mg L and 311.6 mg L, respectively. The lipid content of the two strains increased to 64.42 and 62.61 % of ash free dry weight, respectively, when the cells were cultured with nitrogen and silicon deprivation. It was found that 3,063 reads of genetic expression including Acetyl-CoA carboxylase and other important genes in lipid synthesis pathway were up-regulated and 4,598 reads of genetic expression were down-regulated in mutant Nitzschia ZJU2, when the cells were cultured in optimized growth medium without nitrogen and silicon. The gene expression levels of ATP-binding cassette transporters, arginine and proline metabolism, and proteasome in metabolic pathways were up-regulated to different degrees in mutant Nitzschia ZJU2 under the same cultivation condition. [ABSTRACT FROM AUTHOR]

Encapsulation of drugs in nanoparticles can enhance the accumulation of drugs in tumours, reduce toxicity toward healthy tissue, and improve pharmacokinetics compared to administration of free drug. To achieve efficient delivery and release of drugs at the target site, mechanisms of interaction between the nanoparticles and cells and the mechanism of delivery of the encapsulated drug are crucial to understand. Our aim was to determine the mechanisms for cellular uptake of a fluorescent hydrophobic model drug from poly(butylcyanoacrylate) nanoparticles. Prostate adenocarcinoma cells were incubated with Nile Red-loaded nanoparticles or free Nile Red. Uptake and intracellular distribution were evaluated by flow cytometry and confocal laser scanning microscopy. The nanoparticles mediated a higher intracellular level and more rapid uptake of encapsulated Nile Red compared to model drug administered alone. The main mechanism for delivery was not by endocytosis of nanoparticles but by nanoparticle-cell contact-mediated transfer directly to the cytosol and, to a smaller extent, release of payload from nanoparticles into the medium followed by diffusion into cells. The payload thus avoids entering the endocytic pathway, evading lysosomal degradation and instead gains direct access to intracellular targets. The nanoparticles are promising tools for efficient intracellular delivery of hydrophobic anticancer drugs; therefore, they are clinically relevant for improved cancer therapy. [ABSTRACT FROM AUTHOR]

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL, respectively. High correlation coefficients of R = 0.990 and R = 0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R = 0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates. [ABSTRACT FROM AUTHOR]

Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the SPTLC1 gene. The SPTLC1 protein is part of the SPT enzyme which is a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer's and Parkinson's disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Inducing lipid accumulation in microalgae cells without suppressing cell growth is vital to the economical production of biodiesel from microalgae. In two experiments, we demonstrate that the cell concentration and lipid content of marine microalgae Isochrysis galbana depend upon the iron concentration in the growth media. In Experiment I, adding chelated FeCl to the medium at the late exponential growth phase prolonged this phase and increased the lipid content in I. galbana cells. The final cell density and lipid content of I. galbana supplemented with chelated FeCl was approximately 2 and 1.65 times higher than that of non-supplemented cultures, respectively. In Experiment II, I. galbana cells in the late exponential phase were collected and re-inoculated into new media containing Fe at various concentrations. The final cell concentration and lipid content were maximized at the highest iron concentration (38% biomass by dry weight at 1.2×10 mol/L Fe). In this study, intracellular neutral lipid storage was evaluated by fluorescent spectrophotometry using fluorochrome Nile red, and the measurement conditions were optimized. [ABSTRACT FROM AUTHOR]

The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL −1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8 × 10 6 cells mL −1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production. [ABSTRACT FROM AUTHOR]

Nile Red solvatochromism is used to monitor phase separation in concentrated poly( N-isopropyl acrylamide) (PNIPAm) aqueous solutions. Below the lower critical solution temperature (LCST), Nile Red molecules are in a polar environment and thus exhibit negligible fluorescence. Above the LCST, the aggregation of the PNIPAm chains into hydrophobic mesoglobules provides a nonpolar environment, causing a strong increase of fluorescence. The spectra show two emission bands, which can be related to the partitioning of Nile Red molecules between the core and the surface of the mesoglobules. More generally, the technique appears as a new and promising tool to probe microheterogeneities in polymer solutions or mixtures. [ABSTRACT FROM AUTHOR]

The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL −1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8 × 10 6 cells mL −1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production. [ABSTRACT FROM AUTHOR]

Here, we report on a highly sensitive method for the detection of P(3HB) accumulation in Escherichia coli cells based on the automated flow cytometry system using fluorescent dyes. E. coli containing P(3HB) were stained with either BODIPY or Nile red fluorescent dye, and their staining properties were analyzed under a variety of conditions. Compared with Nile red, BODIPY was much more sensitive in staining P(3HB) and overall demonstrated a more rapid staining of cells, a greater resistance to photobleaching, and greater cell viability. In addition, we also successfully monitored heterogeneity in P(3HB) accumulation within a cell population using BODIPY staining and flow cytometry. We believe this optimized staining method using BODIPY in combination with screening by high-speed flow cytometer will be helpful in the engineering of host cells toward an enhanced production of bioplastics. [ABSTRACT FROM AUTHOR]

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry. [ABSTRACT FROM AUTHOR]

This study shows how relative fatty acid content and unsaturation index of Dunaliella salina strains can be estimated rapidly by analysis with flow cytometry and Nile Red staining. This technique is valid for analysing intraspecific variability and shows no significant distortion phenomena, such as variability in the staining of strains not associated with different fatty acid composition. These phenomena have been related to variations in the permeability of the cell membrane and cell wall to Nile Red in a number of microalgae strains and species. The saturated fatty acids/monounsaturated fatty acids/polyunsaturated fatty acids ratio was estimated indirectly from the relative polar and neutral lipid composition. High intraspecific variability was noted in the fatty acid composition of D. salina, which makes it an ideal species for conducting screening tests of new strains for oil production, using the strategic advantages associated with this species such as the possibility of mass culturing in inexpensive, open systems and high biomass processability due to its lack of cell wall. [ABSTRACT FROM AUTHOR]

The yeast Rhodosporidium toruloides NCYC 921 was grown on carbon or nitrogen limited batch cultures. The fermentations were monitored using traditional techniques and multi-parameter flow cytometry. The lipid content was assessed by flow cytometry in association with the fluorocrome Nile Red which emits yellow gold fluorescence when dissolved in neutral lipids and red fluorescence when dissolved in polar lipids. In this way, it was possible to at-line monitor the yeast lipid composition in terms of polarity classes throughout the batch growths. It was found that the neutral lipids decreased during the carbon-limited stationary phase, and increased during the nitrogen-limited batch growth. The maximum lipid content was obtained for the nitrogen-limited yeast culture (24% w/w lipids). The yeast cells with permeabilised membranes profile remained almost unchanged during the time course of both fermentations. The scatter light measurements (forward and side scatter signals) provided information on the yeast growth phase. The multi-parameter flow cytometric approach here reported represents a better control system based on measurements made at the single cell level for optimization of the yeast lipid production bioprocess performance. [ABSTRACT FROM AUTHOR]