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How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides.

ABSTRACT: Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods. We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates. Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification.

SUBMITTER: Seisel Q

PROVIDER: S-EPMC6689431 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides.

Seisel Quentin Q Pelletier François F Deshayes Sébastien S Boisguerin Prisca P

Biochimica et biophysica acta. Biomembranes 20190705 9

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a ser ...[more]

PMID: 31283917

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Project description:BackgroundPneumonitis is one of the most common adverse events induced by the use of immune checkpoint inhibitors (ICI), accounting for a 20% of all ICI-associated deaths. Despite numerous efforts to identify risk factors and develop predictive models, there is no clinically deployed risk prediction model for patient risk stratification or for guiding subsequent monitoring. We believe this is due to systemic suboptimal approaches in study designs and methodologies in the literature. The nature and prevalence of different methodological approaches has not been thoroughly examined in prior systematic reviews.MethodsThe PubMed, medRxiv and bioRxiv databases were used to identify studies that aimed at risk factor discovery and/or risk prediction model development for ICI-induced pneumonitis (ICI pneumonitis). Studies were then analysed to identify common methodological pitfalls and their contribution to the risk of bias, assessed using the QUIPS and PROBAST tools.ResultsThere were 51 manuscripts eligible for the review, with Japan-based studies over-represented, being nearly half (24/51) of all papers considered. Only 2/51 studies had a low risk of bias overall. Common bias-inducing practices included unclear diagnostic method or potential misdiagnosis, lack of multiple testing correction, the use of univariate analysis for selecting features for multivariable analysis, discretization of continuous variables, and inappropriate handling of missing values. Results from the risk model development studies were also likely to have been overoptimistic due to lack of holdout sets.ConclusionsStudies with low risk of bias in their methodology are lacking in the existing literature. High-quality risk factor identification and risk model development studies are urgently required by the community to give the best chance of them progressing into a clinically deployable risk prediction model. Recommendations and alternative approaches for reducing the risk of bias were also discussed to guide future studies.

Project description:Movement trajectories are usually recorded as a sequence of discrete movement events described by two parameters: step length (distance) and turning angle (bearing). One of the most widespread methods to record the geocoordinates of each step is by a GPS device. Such devices have limited suitability for recording fine movements of species with low dispersal ability including flightless carabid beetles at small spatio-temporal scales. As an alternative, the distance-bearing approach can avoid the measurement error of GPS units since it uses directly measured distances and compass azimuths. As no quantification of measurement error between distance-bearing and GPS approaches exists so far, we generated artificial fine-scale trajectories and in addition radio-tracked living carabids in a temperate forest and recorded each movement step by both methods. Trajectories obtained from distance-bearing were compared to those obtained by a GPS device in terms of movement parameters. Consequently, both types of trajectories were segmented by state-switching modeling into two distinct movement stages typical for carabids: random walk and directed movement. We found that the measurement error of GPS compared to distance-bearing was 1.878 m (SEM = 0.181 m) for distances and 31.330° (SEM = 2.066°) for bearings. Moreover, these errors increased under dense forest canopy and rainy weather. Distance error did not change with increasing distance recorded by distance-bearing but bearings were significantly more sensitive to error at short distances. State-switching models showed only slight, not significant, differences in movement states between the two methods in favor of the random walk in the distance-bearing approach. However, the shape of the GPS-measured trajectories considerably differed from those recorded by distance-bearing caused especially by bearing error at short distances. Our study showed that distance-bearing could be more appropriate for recording movement steps not only of ground-dwelling beetles but also other small animals at fine spatio-temporal scales.

Dataset Information

How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides.

Publications

How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides.

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