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Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification.

ABSTRACT: High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

SUBMITTER: Ramirez-Castrillon M

PROVIDER: S-EPMC7969498 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification.

Ramírez-Castrillón Mauricio M Jaramillo-Garcia Victoria P VP Lopes Barros Helio H Pegas Henriques João A JA Stefani Valter V Valente Patricia P

Frontiers in microbiology 20210304

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleagin ...[more]

PMID: 33746916

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Project description:The intracellular unbound drug concentration (Cu,cell) drives pharmacological and toxicological responses for targets inside cells. We have previously shown that Cu,cell is not always equal to extracellular unbound drug concentrations (e.g. plasma concentrations). However, the underlying mechanisms that drive cellular drug binding are poorly understood. The aim of this study was to evaluate drug binding to individual cellular components, such as neutral lipids (NL) and phospholipids (PL). The fibroblastic 3T3-L1 cell line was used as a cellular model. The cells were either used as wild-type (WT cells) or were treated with a differentiation cocktail that induced an adipocyte-like phenotype with large NL droplets (NL+PL+ cells), or treated with propranolol to induce accumulation of PL (PL+ cells). The three cell types were analysed for morphology, global protein expression, lipid content, cellular volume and lysosomal appearance. The cells were exposed to 23 drugs with diverse physicochemical properties, e.g., with regard to lipophilicity, charge, molecular weight and polar surface area. The cellular binding (fu,cell) and the total accumulation ratio (Kp) were measured and used to obtain the intracellular bioavailability (Fic)—defined as the unbound fraction of the drug concentration added to the cells that is available for intracellular target interaction. The NL+PL+ cells had an adipocyte-like morphology, whereas the PL+ were not visually distinguishable from WT cells. Analysis of the global proteome combined with pathway analysis supported the morphological appearances and confirmed the adipocyte-like phenotype of NL+PL+ cells and the normal morphology of PL+ cells. Fic was significantly altered in both treated cell types as compared to WT. A strong negative correlation between fu,cell and PL content in the cell homogenates was observed, whereas the increased NL content in the NL+PL+ cells did not increase binding further. The importance of PLs for drug binding was confirmed by affinities to beads coated with purified PLs. The NL+PL+ cells lacked acidic subcellular compartments (i.e., endo-lysosomal space), which further influenced the subcellular distribution of cationic drugs. In conclusion, our results suggest that PL content, but not NL content, is a major determinant of drug binding in cells, and that PL beads may constitute a simple alternative to more cumbersome cell distribution studies.

Project description:BackgroundReading is important for children's success in school and beyond, yet many adolescents fail to reach expected levels of proficiency. This highlights the need to better understand the factors that influence reading effectiveness over time, including genes and environment. Greater expression of genetic influence on first- and second-grade reading fluency has been observed in higher quality classroom reading environments. To what degree this early environment continues to influence genetic and other environmental influences on later reading is unknown and was tested in this study.MethodsThe quality of the early classroom reading environment was approximated by gains in oral reading fluency (ORF) across the school year among first- or second-grade classmates of 546 MZ and 1,016 DZ twin children (mean age = 7.13 years; SD = 0.45) who had reading comprehension scores from a state-wide mandatory test in school year 2013-2014 when most twin pairs were in seventh to tenth grade (mean age = 14.41; SD = 1.13) in a variable called Class ORF Gain. Biometrical models were fit to the data to assess whether Class ORF Gain moderated the genetic, shared environmental and/or nonshared environmental variance associated with adolescent reading comprehension.ResultsClass ORF Gain moderated shared environmental influences on reading comprehension 6-9 years later. When early classroom reading gains were poor, variability in reading comprehension in adolescence was high and was associated largely with shared environmental influences. When early classroom reading gains were good, overall and shared environmentally influenced variability in adolescent reading comprehension was lower so that genetic influences were most relevant in explaining that variability.ConclusionsOur findings suggested that classroom reading environment experienced when children were learning to read had a lasting influence on the factors underlying variability in later reading effectiveness.

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Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification.

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Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification.

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