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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

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71801
10.1038/s41598-024-71801-9
Article
Singlet oxygen detection in vivo is hindered by nonspecific SOSG staining
Kadhem Zainab
Alkafeef Selma
Benov Ludmil ludmil.benov@ku.edu.kw

https://ror.org/021e5j056 grid.411196.a 0000 0001 1240 3921 Department of Biochemistry, Faculty of Medicine, Kuwait University, 13110 Kuwait, Kuwait
5 9 2024
5 9 2024
2024
14 2066913 6 2024
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Singlet oxygen is considered an important cell damaging agent due to its propensity to react with organic compounds. This drives the interest in developing methods for determination of 1O2. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo 1O2 detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of 1O2 involvement in cell and tissue damage. Our investigation, however, demonstrate that different microbial species and cancer cells become fluorescent when exposed to SOSG under conditions which exclude generation of 1O2. Cells, permeabilized with chlorhexidine or by heat exposure under anaerobic conditions, exhibited SOSG fluorescence. Permeabilized cells could be stained with SOSG even 24 h post-permeabilization. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was plasma membrane damage. Spectral analyses of different batches of SOSG revealed that SOSG endoperoxide (SOSG-EP) did not increase even after prolonged storage under the recommended conditions. The commercial preparations of SOSG, however, were not SOSG-EP free, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen production in vivo.

Keywords

Singlet oxygen
Fluorescent staining
Singlet oxygen sensor green
Photosensitizer
Photodynamic treatment
Candida albicans
Subject terms

Biophysical chemistry
Biochemistry
http://dx.doi.org/10.13039/501100004482 Kuwait University YM05/22 issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Singlet oxygen (1O2), an electronically excited form of molecular oxygen naturally produced in biological systems by metabolic and photochemical reactions, is a highly reactive molecular species that can rapidly oxidize compounds containing carbon–carbon double bonds to generate hydroperoxides and endoperoxides1. Protein cysteine, histidine, methionine, and tryptophan residues are particularly prone to oxidation by 1O2, as are membrane lipids, which can rapidly undergo peroxidation2. Due to the inevitable nature of 1O2 production in aerobic environments and its significantly damaging effects on biomolecules, the development of convenient and accurate methods to detect and quantify 1O2 in vivo is of keen interest.

Commercially introduced in 20043, singlet oxygen sensor green (SOSG) has been advertised as a cell-impermeable, fluorescent probe specific for the detection of singlet oxygen (1O2), the lowest state of electronically excited molecular oxygen. Since then, it has been used extensively to support claims of 1O2 production, both in vitro and in vivo4–8. Irrespective of its supposed cell-impermeability, SOSG has been successfully applied in fluorescent staining of mammalian cells5,9, microorganisms8,10, and plants2,11.

Singlet oxygen detection by SOSG is based on the 1O2-mediated conversion of SOSG to its strongly fluorescent endoperoxide adduct, SOSG-EP9. SOSG is composed of a fluorophore and an anthracene trapping moiety, which in the absence of 1O2, acts an electron donor to quench the fluorescence of the fluorochrome (an electron acceptor). When 1O2 is present, it is trapped by the anthracene moiety forming an endoperoxide that is no longer able to act as an intramolecular electron donor, allowing for green fluorescence with 488/530 nm excitation and emission wavelengths12. Therefore, fundamentally, oxygen is essential for SOSG activation, although non-specific activation by oxygen species other than 1O2, such as OH⋅, has been observed12,13. While SOSG has been widely used to support claims of 1O2 production in photodynamic therapy14–16, sonodynamic therapy, and other contexts17,18, it has been shown to display questionable specificity, with promiscuous activation by ultrasound12, ionizing radiation19, and short-wavelength light exposure9. Further, it has even been observed that upon illumination, SOSG is able to generate 1O2, resulting in self-activation and artificially augmenting fluorescent output3,9,20.

Here we demonstrate that cell envelope permeabilization is sufficient to induce fluorescent SOSG staining in fungal, bacterial, and mammalian cells even when treated in the absence of oxygen, a condition that precludes the participation of singlet oxygen and other reactive oxygen species in the activation of SOSG fluorescence.

Results and discussion

SOSG fluorescent staining of Candida

While using SOSG as a fluorescent probe to detect intracellular production of 1O2 by photosensitizers, a non-illuminated, heat-treated C. albicans control surprisingly stained positively with SOSG (Fig. 1a). No fluorescence was registered under the same conditions without heat treatment.Fig. 1 Fluorescent SOSG staining of heat-treated C. albicans. Two hundred µl aliquots of wild-type C. albicans (JYC5) cell suspensions were incubated in thermocycler at 95 °C for 5 min under (a) aerobic or (b) anaerobic atmosphere. The samples were cooled down, 10 µM SOSG was added to each sample, incubated 30 min in the dark, and images were captured by confocal fluorescent microscope.

It has been reported that exposure of Chlamydomonas to 40 °C resulted in fluorescent SOSG staining of the cells, attributed to generation of 1O2 by decomposition of lipid peroxides8. To exclude lipid peroxides-derived 1O2, the experiment was repeated in anaerobic Coy chamber. Heating the cells and incubation with SOSG was performed under anaerobic conditions. Results (Fig. 1b) demonstrate that treatment under anaerobic conditions did not prevent the fluorescent staining of the cells. Similar results were obtained with S. cerevisiae.

Adding SOSG 24 h after the heat treatment also resulted in fluorescent staining of the cells (Suppl. Fig. 1), which again points that it is not dependent on reactive species with short life or to short-lasting chemical reactions initiated during heating.

Since SOSG has been marketed as cell impermeable and heat is known to permeabilize microbial cells21–23, a possible explanation for the observed fluorescent staining of the heat-treated cells could be facilitated penetration of the fluorescent probe inside the cells.

To test if other membrane-permeabilizing agents cause SOSG staining, C. albicans cell suspension was incubated with chlorhexidine, an antiseptic with known cell envelope permeabilizing activity24–26. Direct interaction between chlorhexidine and SOSG has been avoided by thoroughly washing the cells before the addition of SOSG. As seen by the green fluorescence, chlorhexidine preincubation resulted in SOSG staining of the cells (Fig. 2a). Mechanical membrane disruption by sonication yielded similar results, with cells showing positive staining by SOSG (Suppl. Fig. 2).Fig. 2 Effect of pretreatment with chlorhexidine on SOSG fluorescent staining of C. albicans. Candida cell suspension in PBS-glucose were incubated for 30 min with 100 µg/ml chlorhexidine under (a) aerobic or (b) anaerobic atmosphere. The cells were washed with PBS, resuspended in PBS-glucose, 10 µM SOSG was added, incubated 30 min in the dark and images were obtained by confocal fluorescent microscope.

To rule out participation of ROS in the SOSG staining, incubation with chlorhexidine and SOSG staining were repeated in anerobic atmosphere. Results (Fig. 2b) show that anaerobiosis did not prevent the fluorescent staining of the cells. The conclusion that SOSG fluorescence was ROS-independent was strengthened by the fact that incubation of C. albicans with H2O2 at concentrations up to 10% did not result in fluorescent SOSG staining (Suppl. Fig. 3a). In contrast, addition of SOSG to cells preincubated with 3% tert-butyl hydroperoxide resulted in fluorescent staining (Suppl. Fig. 3b). This result can be explained by differences in the mechanisms of hydrogen peroxide and tert-butyl hydroperoxide induced cell damage27. While H2O2 kills cells mainly by causing DNA damage27–29, tert-butyl hydroperoxide disrupts membrane function resulting in loss of cell content27.

Gollmer et al.9 have pointed out that a small amount of SOSG-EP present as a contaminant could lead to misleading results. The fluorescent SOSG staining of cells without illumination and under anaerobic conditions raised a suspicion that the SOSG vials have been exposed to radiation during transportation, which increases the fluorescence of the probe13 or to improper storage leading to accumulation of SOSG-EP. To test such a possibility, we analyzed two different batches of SOSG received 13 years apart, one stored according to manufacturer’s instructions at − 24°C in unopened vials, the other analyzed immediately upon receipt. Analyses by UV–Vis and fluorescent spectroscopy revealed almost identical absorbance and fluorescent spectra. No indication of excessive SOSG-EP accumulation was found in the SOSG probe after prolonged storage. The absorption (Suppl. Fig. 4A) and fluorescence (Suppl. Fig. 4B) spectra of the two batches of SOSG closely resembled the spectra reported by other investigators9,30. A time-dependent increase of the intensity of SOSG florescence was observed when exposed to singlet oxygen generated by Rose Bengal (singlet oxygen quantum yield, ΦΔ = 0.7631) (Suppl. Fig. 4B). These results confirm that the two batches of SOSG can be used for detection of photochemically generated singlet oxygen. Illumination of SOSG with polychromatic light (115 mW/cm2 at 420 nm) in the absence of a photosensitizer increased the intensity of fluorescence in accordance with previous reports9,30 (Suppl. Fig. 4C). The spectral analyses, however, indicated that the two SOSG commercial preparations do not fulfil the requirement for zero SOSG-EP content, stipulated by Gollmer et al.9.

Since photogenerated 1O2 increases the intensity of SOSG fluorescence, we tested the effect of illumination of two photosensitizers, a commercial Rose Bengal and a previously investigated Zn(II) meso-tetrakis(N-hexylpyridinium-4-yl) porphyrin (ZnTnHex-4-PyP), which is known to disperse in membranes32, on the fluorescent SOSG staining of Candida albicans. Results demonstrated that illumination of cells in the absence of a photosensitizer did not result in fluorescent SOSG staining (Fig. 3a). A small number of cells were stained when illumination with Rose Bengal was performed (Fig. 3b) while almost all ZnTnHex-4-PyP loaded cells displayed fluorescence (Fig. 3c). Such differences in SOSG staining can be attributed to differences in the properties and cellular uptake of the two photosensitizers. Rose Bengal is anionic and hydrophilic and accumulates very inefficiently within cells33,34. In contrast, ZnTnHex-4-PyP is cationic and amphiphilic, which allows rapid uptake and accumulation in cells, particularly in membranes32. Irrespective of having comparable singlet oxygen quantum yields35, these two photosensitizers did not produce similar SOSG staining of Candida albicans.Fig. 3 SOSG fluorescent staining of C. albicans illuminated in the presence of photosensitizers. Candida suspensions in PBS-glucose were incubated 30 min in the dark without a photosensitizer (a), with 20 µM Rose Bengal (b) or with 10 µM ZnTnHex-4-PyP (c). After the incubation, the cells were washed to remove the unbound photosensitizers and were resuspended to the same density in PBS-glucose. SOSG was added and samples were illuminated for 30 min. Rose Bengal containing samples were illuminated with green light (540–550 nm) at a fluence of 155 mW/cm2, and ZnTnHex-2-PyP loaded cells were illuminated with polychromatic light (136 mW/cm2 at 420 nm). Respective controls were illuminated with either with green or polychromatic light respectively. Panel (a) shows cells illuminated with green light. After the illumination images were captured using confocal fluorescent microscope.

At the same time, no obvious difference could be observed between samples stained after heating or chlorhexidine treatment, and samples exposed to photo-generated singlet oxygen.

These results suggest that fluorescent staining of cells as a result of various treatments reflects loss of membrane integrity rather than generation of singlet oxygen. To further explore such a possibility, SOSG fluorescent staining of bacterial and cancer cells was investigated.

SOSG fluorescent staining of bacteria

Incorporation of SOSG in bacterial cells is restricted by the presence of a complex cell wall and in Gram-negative bacteria, an outer membrane. To prevent binding of SOSG to components of the medium9, incubation of bacteria with SOSG was performed in PBS glucose. Incubation of E. coli cell suspensions in PBS-glucose without any other treatment, however, did not result in fluorescent SOSG staining of the cells (Suppl. Fig. 5a). Exposure of E. coli suspensions to temperatures below 60 °C also did not result in fluorescent staining irrespective of substantial loss of cell viability. The cells displayed green fluorescence only after incubated at 60 °C or higher temperature (Suppl. Fig. 5c,d). Heat-induced cell permeabilization was time dependent. The number of fluorescent cells increased by the increase of the time of incubation reaching a maximum at 30 min (Suppl. Fig. 6).

Sodium azide (NaN3) is well-known physical quencher of singlet oxygen and is commonly used to prove singlet oxygen involvement36,37. Addition of NaN3 up to 10 mM to E. coli cell suspension before heating, however, did not affect the fluorescent staining by SOSG (Fig. 4a).Fig. 4 Fluorescent SOSG staining of heat-treated E. coli aerobically in the presence of azide or anaerobically. Stationary phase cultures grown in LB medium were washed with PBS and resuspended in PBS-glucose to OD600nm ~ 1.25. Aliquots were incubated for 15 min in a water bath at 95°–100° with or without NaN3. For anaerobic heat treatment, all manipulations were performed in a Coy anaerobic chamber and all solutions and samples were degassed. After the treatment, samples were cooled down and SOSG was added to a final concentration of 2.0 µM. Images were obtained 5–10 min. after the addition of SOSG. (a) Aerobically heat-treated control (no NaN3); (b) Aerobically heat-treated in the presence of 10 mM NaN3; (c) Heat treated anaerobically.

As in the case with Candida, heat treatment under anaerobic conditions did not abolish the fluorescent SOSG staining (Fig. 4b) and addition of SOSG 24 h after the heat treatment resulted in fluorescent staining similar to that observed immediately after cooling the samples (Suppl. Fig. 7).

Similar results were obtained when SOSG fluorescent staining of S. aureus was examined (Suppl. Fig. 8).

SOSG has been used to demonstrate intracellular singlet oxygen production by photosensitizers38, by photosynthesis2, and by various stress factors in plants11,39,40. Irrespective of the increase of fluorescence intensity by irradiation of SOSG in the presence of photosensitizers, it has been reported that no reproducible results could be obtained by irradiation of SOSG containing cells9. To test the effect of photosensitized production of 1O2 on SOSG fluorescent staining of E. coli, we used Zn–porphyrin based photosensitizers with progressively increasing lipophilicity. We have previously reported that illumination of E. coli suspensions in the presence of amphiphilic Zn–porphyrins with six and eight carbon chains caused loss of membrane integrity, while hydrophilic 2–4 carbon chains analogs did not41. When E. coli cells suspensions were illuminated in the presence of Zn–porphyrin photosensitizers, only samples containing six and eight carbon chains photosensitizers displayed fluorescence (Suppl. Fig. 9). Only a small fraction of E. coli cells became fluorescent when illuminated with the four-carbon chain-containing ZnTnBu-3-PyP (Zn(II) meso-tetrakis(N–n-butylpyridinium-3-yl)porphyrin), and no fluorescence was observed in samples illuminated in the presence of Zn-porphyrins with shorter aliphatic chain (Suppl. Fig. 9). Similar results were observed when SOSG was used to detect 1O2 photo-generated by TMPyP (H2TM-4-PyP) (100 µM) and protoporphyrin IX (PpIX) (4 µM) concentration in a human nasopharyngeal carcinoma cell line CNE25. The planar hydrophilic metal-free porphyrin para ligand, like its metalated ZnTM-4-PyP analog32, intercalates in DNA and accumulates in the nucleus. The more lipophilic PpIX, like the amphiphilic Zn(II) meso-tetrakis(N-hexylpyridinium-3-yl) (ZnTnHex-3-PyP) and Zn(II) meso-tetrakis(N-octylpyridinium-3-yl) (ZnTnOct-3-PyP) porphyrins32, disperses predominantly in biomembranes, including plasma membrane. Upon illumination, SOSG fluorescence was observed only in carcinoma cells treated with PpIX, but not in cells treated with TMPyP5. Based on the impermeability of the plasma membrane for SOSG, the authors concluded that 1O2 generated by PpIX in the plasma membrane diffused out of the cell interior and interacted with SOSG in the extracellular environment. No explanation was provided for fluorescent staining of the whole cells but not of the medium on Fig. 3 (90 s) of the same publication5.

SOSG fluorescent staining of cancer cells

It has been hypothesized that incorporation of SOSG in mammalian cells is prevented by affinity binding of the fluorescent marker to components of the medium9 and staining of mammalian cells has been achieved only in modified medium. To prevent such binding, immediately before adding SOSG, the medium was replaced with PBS-glucose. For the relatively short time of incubation applied in this study, SOSG did not stain the cancer cells (Fig. 5a). The cells displayed bright green fluorescence only after permeabilization of the plasma membrane by heating (Fig. 5b), after treatment with chlorhexidine (Fig. 5c), or after treatment with tert-butyl hydroperoxide (Fig. 5d). Chlorhexidine and tert-butyl hydroperoxide, however, lysed substantial fraction of the cells.Fig. 5 Fluorescent SOSG staining of pII cancer cells. Cells were grown in RPMI 1640 medium to a confluence of 70–90%. Immediately before treatment, growth medium was replaced with PBS-glucose. The cells were incubated 10 min either at 100 °C (b); at 25 °C with 10 µg/ml chlorhexidine (c); or with 3% tert-butyl hydroperoxide (d). Untreated controls (a) were kept at 25 °C during the duration of treatments.

Similar results were obtained when MCF-7 cancer cells were exposed to heat or to chlorhexidine (S. Fig. 10).

Fluorescent SOSG staining coinciding with loss of membrane integrity (propidium iodide staining) has been previously reported39, but has been attributed to generation of singlet oxygen.

This work did not investigate why SOSG became fluorescent when taken up by the cells. SOSG is made of two parts, anthracene and fluorescein, whose fluorescence is quenched by intramolecular electron transfer9. Reaction with singlet oxygen produces an endoperoxide which prevents the electron transfer and fluorescence is restored. Formation of endoperoxide, however, seems not to be the only mechanism preventing the electron transfer. Initial attempts to produce a singlet oxygen nanoprobe free from the pitfalls of SOSG resulted in a product with much higher fluorescence than SOSG42. The reason was SOSG microenvironment which prevented electron transfer, and as a result, quenching of the fluorescence was not efficient42. It is tempting to speculate that inside the cells, SOSG is exposed to natural agents which similar way suppress efficient electron transfer thus causing fluorescent staining of the cells even in the absence of singlet oxygen.

Conclusions

Detailed investigations of the SOSG fluorescent staining of different microbial species and cancer cell lines, demonstrated that cells become fluorescent under conditions which exclude generation of singlet oxygen or of any other of the so called ROS. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was cell envelope permeability.

Analyses of different batches of SOSG revealed that SOSG-EP does not increase even after prolonged storage, as long as manufacturer’s instruction are followed. The commercial preparations of SOSG, however, were not SOSG-EP free upon receipt, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen generation.

Reliable data in vivo could be obtained by using fluorescent probes free from the pitfalls of SOSG, like the NanoSOSG, which is cell permeable, does not interact with proteins, and whose fluorescence increases upon reaction with singlet oxygen42.

Materials and methods

Reagents

Two different batches of SOSG were investigated. One (Invitrogen / Molecular probes, Lot. No. 762624) was received in January 2011 and unopened vials were stored at − 24 °C according to manufacturer’s recommendations. The second (Invitrogen/Thermo Fisher Scientific, Lot. No. 2761161) was received in April 2024 and was used directly upon receipt. SOSG stock solution (500 µM) was prepared in deoxygenated methanol9. Work solutions were prepared immediately before use. For staining of cells, 2, 5, and 10 µM of SOSG were used. No differences in fluorescent staining were observed at this SOSG concentration range. Unless otherwise indicated, images show staining at 2 µM SOSG.

UV–Vis spectra were recorded in water at room temperature on a UV-2501PC Shimadzu spectrophotometer with 0.1 nm resolution in 1 cm quartz cuvette. Fluorescence emission spectra were recorded on a Shimadzu RF-6000 spectrofluorometer.

Strains and media

Candida albicans strain JYC5 was grown at 30 °C in YPD medium to a stationary phase (OD600nm ~ 10–14). The cells were washed twice with sterile phosphate-buffered saline (PBS) and resuspended in PBS-glucose (2% glucose) to OD600nm ~ 5.

The strains of Escherichia coli used in this study were GC4468 (F-Δlac U169 rpsL); AB1157 [F− thr-1 leuB6 proA2 his-4 thi-1 argE2 lacY1 galK2 rpsL surE44 ara-14 xyl-15 mtl-1 tsx-33]. E. coli was grown Luria Bertani (LB) medium to a stationary phase, the cells were washed with PBS and resuspended in PBS-glucose (0.2% glucose) to OD600nm ~ 1.2–1.3. Staphylococcus aureus strain ATCC2592343 was grown in brain heart infusion (BHI) medium to stationary phase, washed with PBS and resuspended in PBS-glucose to OD600nm ~ 1.2–1.3

MCF-7 and pII44 cancer cell cultures were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine and 1% penicillin/streptomycin as an antibacterial agent. Cultures were kept at 37 °C and 5% CO2 and used for experiments at 70—90% confluence. At the beginning of each experiment the growth medium was discarded, cells were gently washed with PBS and the medium was replaced with PBS-glucose.

Photosensitizers

Water soluble Zn(II) meso-tetrakis(N-alkylpyridinium-3(4)-yl) porphyrins, where alkyl was methyl to octyl, were used to demonstrate SOSG fluorescent staining of E. coli. Details about synthesis, characterization, cellular uptake, subcellular distribution, and photodynamic activities of the Zn-porphyrins can be found in earlier publications32,41,45,46.

Rose Bengal was obtained from Sigma.

Anaerobic experiments

An anaerobic chamber AC-855 (Plas Labs, Inc., MI, USA) was employed for hypoxic treatments. Hypoxic conditions in the chamber were confirmed by use of oxygen indicator strips BR0055 (Oxoid, UK) which detect oxygen down to < 0.3%.

Illumination

Samples were illuminated with green light form a Phillips 80 W E24 PAR30 bulb with emission maximum at 540–550 nm or where indicated, with polychromatic light from incandescent 300 W bulb41.

Confocal fluorescence microscopy

After 15–45 min incubation with SOSG, cell suspensions (2.5–4 µl) were transferred on microscopic slides and covered with cover slips. The slides were examined under oil immersion using a Zeiss LSM 980 META confocal fluorescence microscope. SOSG fluorescence was visualized by excitation 505 nm and emission at 658–669 nm range. Duration of incubation with SOSG had no observed effect on the cell fluorescent staining.

All experiments were repeated not less than three times with similar outcomes.

Supplementary Information

Supplementary Figures.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71801-9.

Acknowledgements

The authors are grateful to Ms. F. Sequeira for excellent technical assistance and to OMICSRU/RCF, projects SRUL02/13 & GM01/15, Kuwait University. This work was supported by Research Grant YM05/22 from Kuwait University and by the College of Graduate Studies, Kuwait University; results form part of Zainab Kadhem’s M.Sc. Thesis with co-supervisor Selma Alkafeef and supervisor L. Benov.

Author contributions

Conceptualization, S.A., L.B.; software, Z.K.; formal analysis, Z.K., S.A., L.B.; investigation, Z.K.; resources, S.A., L.B.; writing—original draft preparation, L.B.; writing—review and editing, Z.K., S.A., L.B.; supervision, S.A., L.B.; project administration, S.A., L.B.; funding acquisition, S.A., L.B. All authors have read and agreed to the published version of the manuscript.

Data availability

Publicly available datasets were analyzed in this study. This data can be found here: https://osf.io/3vs5m/.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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