scholarly journals Label-free characterisation of amyloids and alpha-Synuclein polymorphs by exploiting their intrinsic fluorescence property

2021 ◽  
Author(s):  
Chyi Wei Chung ◽  
Amberley D Stephens ◽  
Edward Ward ◽  
Yuqing Feng ◽  
Molly Jo Davis ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Alpha Synuclein ◽  
Fluorescence Lifetimes ◽  
Label Free ◽  
Model Free ◽  
Inhibitory Compounds ◽  
Β Sheets ◽  
Β Lactoglobulin

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime represented by model-free phasor plots, as a label-free assay to characterise amyloid structure. Intrinsic amyloid fluorescence arises from structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids (i.e., α-Synuclein (αS), β-Lactoglobulin and TasA) and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of pre-formed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct to those of their fibrillar counterpart. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathology), and for testing small molecule inhibitory compounds.

scholarly journals CHARACTERIZING FLUORESCENCE LIFETIME OF NAD(P)H IN HUMAN LEUKEMIC MYELOID CELLS AND MONONUCLEAR CELLS

2013 ◽  
Vol 06 (04) ◽  
pp. 1350042
Author(s):  
LI-SHENG LIN ◽  
LI-NA LIU ◽  
HUI-FANG HUANG ◽  
YUAN-ZHONG CHEN ◽  
BU-HONG LI ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mononuclear Cells ◽  
Relative Concentration ◽  
Myeloid Cells ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Fluorescence Lifetimes ◽  
Label Free ◽  
Time Resolved ◽  
Significant Difference ◽  
The Mean

The aim of this ex vivo study was to explore the potential of using the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as a label-free indicator to characterize the differences between human leukemic myeloid cells and normal mononuclear cells (MNC). The steady-state and time-resolved autofluorescence of two human leukemic myeloid cell lines (K562, HL60) and MNC were measured by a spectrofluorimeter. According to excitation–emission matrix (EEM) analysis, the optimal emission of NAD(P)H in these cells suspensions occurred at 445 nm. Furthermore, the fluorescence lifetimes of NAD(P)H in leukemic myeloid cells and MNC were determined by fitting the time-resolved autofluorescence data. The mean fluorescence lifetimes of NAD(P)H in K562, HL60, and MNC cells were 5.57 ± 1.19, 4.45 ± 0.71, and 7.31 ± 0.60 ns, respectively. There was a significant difference in the mean lifetime of NAD(P)H between leukemic myeloid cells and MNC (p < 0.05). The difference was essentially caused by the change in relative concentration of free and protein-bound NAD(P)H. This study suggests that the mean fluorescence lifetime of NAD(P)H might be a potential label-free indicator for differentiating leukemic myeloid cells from MNC.

scholarly journals The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 855 ◽  
Author(s):  
Tomas Sneideris ◽  
Andrius Sakalauskas ◽  
Rebecca Sternke-Hoffmann ◽  
Alessia Peduzzo ◽  
Mantas Ziaunys ◽  
...  
Keyword(s):  
Ph Value ◽  
Experimental Conditions ◽  
Disease Modifying Drugs ◽  
Amyloid Fibril Formation ◽  
Inhibitory Compounds ◽  
Parkinson’S Diseases ◽  
Key Factor ◽  
Small Change

Millions of people around the world suffer from amyloid-related disorders, including Alzheimer’s and Parkinson’s diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clinical trials is very high, even for compounds that show promising anti-amyloid activity in vitro. In this study, we demonstrate that even small changes in the chemical environment can strongly modulate the inhibitory effects of anti-amyloid compounds. Using one of the best-established amyloid inhibitory compounds, epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson’s disease-related α -synuclein, we shed light on the previously unexplored sensitivity to solution conditions of the action of this compound on amyloid fibril formation. In the case of insulin, we show that the classification of EGCG as an amyloid inhibitor depends on the experimental conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the experiment. For α -synuclein, we show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behaviour by the increased stability of EGCG against oxidation at pH 6.

Discrimination of normal and malignant mouse ovarian surface epithelial cells in vitro using Raman microspectroscopy

2015 ◽  
Vol 7 (22) ◽  
pp. 9520-9528 ◽  
Author(s):  
S. Borel ◽  
E. A. Prikryl ◽  
N. H. Vuong ◽  
J. Jonkman ◽  
B. Vanderhyden ◽  
...  
Keyword(s):  
Statistical Analysis ◽  
Multivariate Statistical Analysis ◽  
Raman Microspectroscopy ◽  
Live Cells ◽  
Label Free ◽  
Multivariate Statistical ◽  
Ovarian Surface Epithelial ◽  
Free Classification

Raman microspectroscopy in conjunction with multivariate statistical analysis is a powerful technique for label-free classification of live cells based on their molecular composition, which can be correlated to variations in protein, DNA/RNA, and lipid macromolecules.

scholarly journals In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

2007 ◽  
Vol 74 (1) ◽  
pp. 294-299 ◽  
Author(s):  
Petr Walczysko ◽  
Ute Kuhlicke ◽  
Sabine Knappe ◽  
Christiana Cordes ◽  
Thomas R. Neu
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Bacterial Cells ◽  
Fluorescence Lifetimes ◽  
Fast Growing ◽  
Lifetime Imaging ◽  
Rna:Dna Ratio ◽  
Syto 13

ABSTRACT In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

Label-free metabolic clustering through unsupervised pixel classification of multiparametric fluorescent images

2020 ◽  
Author(s):  
Giada Bianchetti ◽  
Fabio Ciccarone ◽  
Maria Rosa Ciriolo ◽  
Marco De Spirito ◽  
Giovambattista Pani ◽  
...  
Keyword(s):  
Label Free ◽  
Pixel Classification

In Vitro Toxicology Methods in Risk Assessment

1996 ◽  
Vol 24 (3) ◽  
pp. 325-331
Author(s):  
Iain F. H. Purchase
Keyword(s):  
Risk Assessment ◽  
Hazard Assessment ◽  
Human Health Risk ◽  
In Vitro Test ◽  
In Vitro Methods ◽  
New Concepts ◽  
Vitro Test

The title of this paper is challenging, because the question of how in vitro methods and results contribute to human health risk assessment is rarely considered. The process of risk assessment usually begins with hazard assessment, which provides a description of the inherent toxicological properties of the chemical. The next step is to assess the relevance of this to humans, i.e. the human hazard assessment. Finally, information on exposure is examined, and risk can then be assessed. In vitro methods have a limited, but important, role to play in risk assessment. The results can be used for classification and labelling; these are methods of controlling exposure, analogous to risk assessment, but without considering exposure. The Ames Salmonella test is the only in vitro method which is incorporated into regulations and used widely. Data from this test can, at best, lead to classification of a chemical with regard to genotoxicity, but cannot be used for classification and labelling on their own. Several in vitro test systems which assess the topical irritancy and corrosivity of chemicals have been reasonably well validated, and the results from these tests can be used for classification. The future development of in vitro methods is likely to be slow, as it depends on the development of new concepts and ideas. The in vivo methods which currently have reasonably developed in vitro alternatives will be the easiest to replace. The remaining in vivo methods, which provide toxicological information from repeated chronic dosing, with varied endpoints and by mechanisms which are not understood, will be more difficult to replace.

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