Volume 12 - 2024 | https://doi.org/10.3389/fchem.2024.1436389
Bioluminescent high-throughput screening (HTS) assays
based largely on the activity of firefly (FLuc)
are widely utilised in research and drug discovery
we quantify the luciferase-based real-life HTS assay interference from biologically and environmentally relevant metal ions ubiquitously present in buffers
and as contaminants in plastics and compound libraries
We also provide insights into the cross-effects of metal ions and other key experimental and biological reagents (e.g.
and glutathione) to inform HTS assay design
A total of 21 ions were screened in three robust HTS assays (“SC” assays) based on the luminescence of FLuc
Three newly optimised HEPES buffer variants (“H” assays) were developed for direct luciferase comparison
Interference in bioluminescent signal generation was quantified by calculating the IC50 values from concentration-dependent experiments for selected highly active and relevant metal ions
Metal ion inhibition mechanisms were probed by variations in specific reagents
and the sequence of addition and buffer composition
we revealed a significant impact of metal ions’ salts on luciferase-mediated bioluminescence
even at biologically and environmentally relevant concentrations
The extent of signal interference largely aligned with the Irving–Williams series of metal ion–ligand affinities (Cu > Zn > Fe > Mn > Ca > Mg)
supporting previous reports on metal ion-dependent FLuc inhibition
the absolute magnitude and relative extent of signal reduction by metal ions’ salts differed between SC and H assays and between luciferases
suggesting a complex network of metal ions’ interactions with enzymes
The diversity of the tested conditions and variability of responses provided insights into potential interference mechanisms and synergies that may exacerbate or alleviate interference
The beneficial influence of EDTA and the impact of glutathione
on bioluminescence readout were pinpointed
Given the ubiquity of metal ions in analysed samples
the causative role in false-positive generation in drug discovery
and the wide breadth of luciferase-based assays used in screening
awareness and quantification of metal influence are crucial for developing assay validation protocols and ensuring reliable screening data
ultimately increasing the critical robustness of bioluminescence-based HTS assays
Scheme 1. Mechanism of bioluminescence for D-luciferin (FLuc substrate), coelenterazine (RLuc substrate), and furimazine (NLuc substrate). Adopted from the study by Kaskova et al. (2016)
Despite some work on the ionic interference with FLuc-mediated bioluminescence
the studies regarding the impact of metal ions on their bioluminescence are limited
no comprehensive bioluminescence interference studies in HTS-format assays in the context of screening interference were performed for any of the three enzymatic detection systems
it is crucial to determine the influence of metal ions on bioluminescence-based assays using FLuc
In this work, various metal ion salts were tested in specific concentration ranges and underwent experimental bioluminescence-based HTS assay conditions established previously (https://www.eu-openscreen.eu/services/bioprofiling-assays.html); three new HEPES buffer variants of those assays were also included to enable more direct comparison between luciferases
The main objective of this research was to pinpoint possible interfering metal ion salts that could impair bioluminescence-based HTS assays involving FLuc
Identifying these interfering metal ions will enable research workers to prevent compromised interpretations and optimise the design of bioluminescence-based HTS assays
ultimately increasing their robustness and saving time and resources by minimising false-positives in early stages of drug discovery
Milli-Q water was used to prepare all aqueous solutions
All salts available from commercial sources were of analytical grade
Iron(III) chloride hexahydrate (# 236489)
iron(II) sulphate heptahydrate (#215422)
manganese(II) sulphate monohydrate (#M7899)
copper(I) tetrafluoroborate (# 677892)
and Trizma base (# T6066) were obtained from Sigma-Aldrich
Sodium chloride (# 54447) was from Warchem
Calcium nitrate tetrahydrate was from Chmes
Caesium carbonate (# C2160) was from TCI
Potassium tetrachloroplatinate(II) (# 11396315)
zinc nitrate hexahydrate (# 10036103)
aluminium nitrate nonahydrate (# 15269686)
copper(II) nitrate trihydrate (#10124790)
nickel(II) nitrate hexahydrate (# 10401651)
and sodium nitrate (# 11904281) were from ThermoScientific Acros
Potassium carbonate (# 427465708) and sodium sulphate (# 118078707) were from Chempur
Ammonium iron(II) sulphate hexahydrate (# 10087291)
cobalt(II) nitrate hexahydrate (# 10529380)
and lithium nitrate (# 10568620) were from Fisher Chemical
Cadmium nitrate tetrahydrate (# 11351579) and gallium nitrate monohydrate (# ALF-032116-14) were from Alfa Aesar
PBS (# 10010-015) and EDTA (# AM9260G) were from Thermo Fisher Scientific
Glycine (# GLN001) and ATP (# ATP007) were from BioShop Canada
BSA (# 160069) was from MP Biomedicals
NaOH (# 810981424) was obtained from POCH
Positive controls for the luciferases activity assays are as follows: PT-C124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid} (VWR # BIOV9421-10)
and N-benzyl-p-toluenesulfonamide BTS (VWR # J64910
Luciferase enzymes for the activity assays were as follows: the firefly enzyme (Promega # E1701)
and Renilla enzyme (RayBiotech # RB-15-0003P-50)
The substrates for the activity assays were beetle luciferin (Promega # E1603)
and coelenterazine (Promega # S2001)
All bioluminescent signal generation experiments were performed in 384-well plate formats
The assay plates used were the LUMITRAC 384 (#781075) white from Greiner Bio-One
Multidrop Combi (Thermo Fisher) non-contact dispenser was used to add solutions of enzymes and substrate to the assay multi-well plates
Measurements of bioluminescent signal generation in the corresponding buffers were recorded with a CLARIOStar (BMG Labtech) plate reader
The pH measurements were carried out in vials with a Mettler Toledo™ FiveEasy FP20 pH metre
Abbreviations used throughout the text are listed alphabetically: N-benzyl-p-toluenesulfonamide (BTS)
half maximal inhibitory concentration (IC50)
Ataluren (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (PTC-124)
All metal salts were abbreviated to their corresponding metal ion (e.g.
For several salts containing both the same metal ion and oxidation state (e.g.
The following abbreviations have been used for the corresponding salts: AgNO3
The negative controls had the same %DMSO as the corresponding positive controls (0.04% for FLuc
luminescent signal was measured using a CLARIOStar plate reader
Final experimental conditions for each luciferase activity assay
the luminescent signal was measured using a CLARIOStar plate reader
and RLuc activity assays were carried out in screening conditions (SC) and in HEPES conditions (H) for 0.01
and 5 mM of the final metal ion salt concentrations
The data were analysed with KNIME software
and RLuc activity assays were carried out both in SC and in H with at least 11 concentrations of metal ion salts in the range 0.16–20 mM as the final concentrations
Every concentration of each metal was tested in triplicates on the same plate
if no top or bottom plateau was observed in the IC50 curve
additional experiments were performed for extended concentration ranges
and relative %INHIBITION values were combined on one figure for IC50 curve fitting
The data were analysed with KNIME software (see below)
and bottom of plateau) and 1 mM were chosen as the final concentrations
Several pre-incubation tests were carried out with the same SC final conditions but with different orders of addition and longer incubation time (30 min instead of 10 min)
ATP + M then S: FLuc assay with the subsequent addition of substrate and enzyme to a 30-min pre-incubated solution of ATP + metal
E + M then S: addition of a solution of substrate (+ATP in the case of FLuc) to a 30-min pre-incubated solution of metal + enzyme
S + M then E: subsequent addition of enzyme (and ATP in the case of FLuc assay) to a 30-min pre-incubated solution of substrate + metal (grey columns)
EDTA testing was carried out in SC for NLuc with the final concentrations 0 and 0.5 mM and for RLuc with 0.25 and 0.5 mM
Corresponding EDTA concentrations for the EDTA–enzyme and EDTA–substrate solutions to be added to the well for NLuc were 4x (5 µL) and 2x (10 µL)
whereas the corresponding EDTA concentrations for the EDTA–enzyme and EDTA–substrate solutions to be added to the well for RLuc were 2x (5 µL) and 1x (10 µL)
GSH testing was carried out with the final concentrations of 0
Corresponding GSH concentrations for the GSH–enzyme and GSH–substrate solutions to be added to the well were 4x (5 µL) and 2x (10 µL)
KNIME pipeline was generated and applied for the analysis of the results
The raw data files (one txt file per plate) were read and matched with plate template followed by calculation of averages and standard deviation of controls and compound wells
and Z′ factor values for each plate
generating one dataset for all the tested plates
Automatic outlier removal was used for detecting control outliers for plates with Z′ factor values below 0.5
The plates for which removal of the outliers did not lead to improvement of the Z′ factor (higher than 0.5) or the algorithm was detecting more than two outliers were retested
The raw data values were normalised to positive and negative controls for each plate to calculate percent inhibition (% INHIBITION = (1-(x- μp)/(μn–μp))* 100%)
Z' = 1- (3*(SDp + SDn)/(μn - μp))
signal to background ratio (S/B = μn/μp)
variance coefficient (CV = SD of population/mean *100%)
percent normalised bioluminescent signal (% N_VALUE = (x/μn)*100)
and normalised bioluminescence signal change (% N_SCh = (x - μn)/μn*100)
where x represents the readout VALUE of the well
μp represents the mean of the positive controls
μn represents the mean of the negative controls
SDn represents the standard deviation of the negative controls
and SDp represents the standard deviation of the positive controls for each tested assay plate
The heatmap of % INHIBITION was then generated for each plate in order to identify plate effects or signal distribution patterns
a validation plate (no metal ions added) was run for each assay under the screening conditions
All raw data as well as platemaps and calculated QC parameters are provided in Supplementary Tables 1, 2 spreadsheet files attached in the Supplementary Material
second sets of aliquots were added to the same vials to reach a final concentration of 5 mM for all salts (except Zn in SC RLuc buffer
These experiments were performed in triplicates
Concentrations (mM) of each metal ion at their respective addition steps (x
To 5 mL of 100 mM HEPES buffer, aqueous stock solutions of metal ion salts (0.2 M, Ag, Cu(II), Fe(II)-1, Fe(II)-2, Fe(III), and Zn) were added in four incremental steps into the same vial. The volume added at each step was equal to the volume required to achieve the concentration as indicated in Table 2 (x1
with the final addition leading to the concentration of 5 mM
The pH was measured upon stirring before and after each addition
and the experiment was repeated in triplicates
The highest Z’ factor value of 0.88 was observed for the NLuc assay in HEPES (H) conditions vs
the assays in SC exhibited slightly higher Z’ factor values (respectively
Each condition for a given type of experiment was performed in triplicate on the same plate
but every sample is prepared independently and each experiment type was performed on a different day
All of this confirms the robustness of the assays and the lack of position-dependent artefacts
enabling reliable data generation independent of the plate design
26 salts of 21 different biologically and environmentally relevant metal ions were selected
Most of the salts were in the form of highly soluble nitrates
and tin(II) (Sn (II)) as chlorides; iron(II) (Fe(II)-1 and 3)
and sodium (Na-3) as sulphates; potassium (K-2) and cesium (Cs(I)) as carbonates; and copper(I) (Cu(I)) as a tetrafluoroborate
In order to get insight into the ionic strength and/or anionic effects
three different salts of sodium (sodium nitrate,Na-1; sodium chloride
two different salts of potassium (potassium nitrate
and three different salts of iron(II) (ammonium iron(II) sulphate hexahydrate
The aqueous solutions of three of the selected metal ions (Fe(II)
and they needed to be prepared freshly every time and used immediately after dissolution in water/buffer
Fe(III) tends to form various water insoluble oxides that precipitate out of the solution in time and/or at higher concentrations
leading to a change in effective metal concentration that cannot be easily estimated
Fe(II) and Cu(I) are also particularly susceptible to oxidation even in air
and so they needed to be handled with care and just for short periods of time
In addition to having high oxidation susceptibility
most of the widely available Cu(I) salts remain rather insoluble in aqueous solutions
the acetonitrile Cu(I) complex was used as a source of Cu(I)
The salt is relatively stable in the solid form and is highly soluble in acetonitrile that stabilizes the Cu(I) state
After subsequent dilution of acetonitrile stock solutions of Cu(I) in water
the ion becomes highly susceptible to oxidation (and partial or total ligand exchange to water can occur); therefore
Cu(I) aqueous solutions were freshly prepared every time and measured immediately after addition to the reaction buffer
with lower but statistically significant quenching effect (between 71.2% and 90.8% of residual signal) noticeable for Al
Normalised bioluminescent signal in the presence of three different concentrations (0.01
white) of metal ion salts in optimised luciferase activity assays of (A) FLuc SC (10 µM substrate
and pH 7.5) (C) NLuc SC (10 µM substrate
PBS pH 7.4 0.75% 0.1% BSA) (D) NLuc H (100 mM HEPES
Positive controls (red) used are PTC-124 (1 µM)
The negative controls (green) had the same %DMSO as the corresponding positive controls.Each condition for a given type of experiment was performed n = 3 on the same plate
but every sample was prepared independently and each experiment type was performed on a different day
Normalisation of the bioluminescent signal was carried out against the negative control output of the corresponding bioluminescent assay plate
Statistical two-tailed Student’s t-test (95% confidence) was carried out between a given sample and the corresponding negative control
The samples were deemed statistically different from the negative control for p < 0.05 and were marked with black “*.”
Summary of the effect of metal ion salts on bioluminescent signal (residual % signal after the addition of metal
0%: full quenching) in screening buffer and in HEPES buffer for FLuc
For NLuc and RLuc assays in SC (Table 3; Figures 2C, E respectively)
little to no effect was observed for any of the metal ions at 0.01 mM
with the exception of Ag in the NLuc assay
Very similar trends (and similar types of metal ions) were observed for both at higher concentrations
with almost complete quenching observed for Ag
and Pt(II) at 1 mM and 5 mM concentrations and Ga(II)
quenching in the NLuc SC assay was less pronounced than that in the RLuc one
and the effect was even more clearly visible at 5 mM concentration for Pb(II) (only 6% residual signal in RLuc vs
44.2% in NLuc) and especially Ca(II) (19.5% in RLuc vs
In analogous assays with NLuc and RLuc, but in HEPES buffer conditions (Table 4; Figure 2C vs. Figure 2D for NLuc; Figure 2E vs. Figure 2F for RLuc)
SC at even 5 mM concentrations is observed for Sn(II) on RLuc and NLuc and for Ca(II) in the RLuc H assay
is observed in the RLuc assay for Ga at 1 mM (no quenching in SC vs
only 10.6% residual signal remaining in H)
the opposite effects of changing the buffer from SC to H when comparing both luciferases are observed for Cd and Co(II) (weakened for NLuc but more pronounced for RLuc) as well as Pb(II) (weakened for RLuc but exacerbated for NLuc)
IC50 values from the curve fitting for each luciferase activity assay in SC and in H
including Sn(II) in particular for all assays but FLuc in SC
100% inhibition was not achieved even at the highest concentrations
leading to an inadequate fit and no reliable IC50 estimation
In the case of Ag and Cu(I) salts in the FLuc H assay
the inhibition was very significant even at the highest concentration
leading to the lack of reliable IC50 fit but clearly indicating that the value of that parameter lies below 0.1 µM
Further examination of the IC50 and the curves largely confirms qualitative observations from the initial screen
the influence of metal ions is more pronounced in FLuc assays
with the following order of the strength of inhibition according to IC50 values (the lower the IC50 value
Cu(I) (0.01 µM) > Cu(II) (0.07 µM) > Zn (0.88 µM) > Cd (1.21 µM) > Fe(II)-3 (2.73 µM) and Pb(II) (2.74 µM) > Ga (11 µM) > Fe(II)-1
and Fe(III) (all between 22–24 µM) > Co(II) (31 µM) > Pt(II) (40 µM)
The IC50 values in the SC conditions for the FLuc assay are in the same order of magnitudes for Fe(III)
and Pt(II); only slightly higher (weaker inhibition) for Ag (0.24 µM in SC); one order of magnitude higher for Co(II)
and Fe(II)-2; two orders of magnitude higher for Fe(II)-3
and Cd; more than three orders of magnitude larger for Zn and Cu(II); and over 10,000 times larger for Cu(I)
only Ag in the SC buffer has an IC50 value below 10 µM (4.5 µM); Ag in the H buffer and Cu(I) and Cu(II) in both H and SC buffers exhibit IC50 values between 22 µM and 56 μM
with the rest of the metal ions’ IC50 being above 100 µM in both H and SC
and Sn(II) in both SC and H and Pb(II) in SC conditions
the strongest inhibition is seen for Cu(II) ions in the H condition (7.79 µM)
with Cu(I) and all iron salts in the H buffer and Ag in both H and SC being within the 18–68 µM range
and Sn(II) in both buffers have all shown IC50 values of above 1 mM
higher inhibition can be found generally in the H conditions
with Ag being approximately 2 times less inhibiting in SC than in H
and Zn salts having one order of magnitude higher IC50 values in SC than H
Co(II) (688 µM in SC) and Pt(II) (350 µM in SC vs
723 µM in H) are the only metal ions with higher inhibitory effect in SC than in H for RLuc assays
a handful of the most potent and biologically relevant metal ions were selected to further investigate the nature of the potential interference of metal ions with bioluminescent signal generation in luciferase activity assays
This included Ag (highest potency across the board)
and high abundance in environment as well as impurity)
and Zn due to its biological and environmental ubiquity
All of the experiments below will therefore involve only these selected metal ions
Result of pH titration experiments (with standard deviations
n = 3) of various metal ions in SC conditions and in H conditions for different concentrations (see Table 2 for concentrations of x
the addition of the first aliquot did not change the pH significantly apart from Zn (2.5 mM led to a decrease in pH from 6.89 to 5.39)
whereas 5-mM solutions of all the metals led to a comparable drop of 1–1.5 pH units in the case of Ag
and Fe(II)-2 (to 5.97–5.75); almost two pH units drop for Zn (to 5.05); and a dramatic drop to pH 3.53 mM for Cu(II) and 3.29 mM for Fe(III)
Similarly, little to no decrease in the pH was observed with the addition of the first metal aliquot to the SC RLuc buffer (50 mM Tris-HCl, 12.5 mM KH2PO4, 125 mM NaCl, 0.25 mM EDTA, 0.1% BSA, and 0.3 M sodium ascorbate; Table 1)
apart from Zn (drop from 7.03 to 6.54 for 5 mM solution and then 5.84 for 10 mM solution)
the pH of the solution for all of the metals ranged between 6.37 (for Fe(III)) and 6.92 (for Ag)
In case of the NLuc screening buffer (Table 1; PBS 0.75% × 0.1% BSA)
no change in the pH was observed even for 5-mM concentration of Ag
Initial addition of Cu(II) (20 µM) did not change the pH
the pH dropped to 4.89.5 mM of Fe(II)-1
whereas Fe(II)-2 solution had a pH of 6.73 at 2.5-mM concentration and 6.28 at 5-mM
Fe(III) induced most dramatic changes with pH 5.74 (2.5 mM) and 2.71 (5 mM)
the addition of 5 mM of Zn to NLuc SC led to a pH of 5.55
metal ion salts were also pre-incubated with ATP as a potentially reactive and yet critical element of the buffer
Four different concentrations of the salts (for Ag(I) in FLuc
only three concentrations) were selected based on IC50 experiments
including 1 mM (same for all the salts)
the concentration near the IC50 value itself
and one lower and one higher than that (respectively
closer to concentrations inducing the lower and upper limit responses in the IC50 curve for a given assay)
Normalised percentage of bioluminescent signal for (A) FLuc
and (C) RLuc assays for different pre-incubations
FLuc assay with subsequent addition of substrate and enzyme to a 30-min pre-incubated solution of ATP + metal salt (white dashed)
Addition of a solution of substrate (+ATP
in the case of FLuc) to a 30-min pre-incubated solution of metal salt + enzyme (black)
Subsequent addition of enzyme (and ATP in the case of FLuc assay) to a 30-min pre-incubated solution of substrate + metal (grey)
Positive controls used are PTC-124 (1 µM)
The negative controls had the same %DMSO as the corresponding positive controls
Normalisation of the bioluminescent signal was carried out against the negative control output given for each independent pre-incubation condition
Statistical two-tailed Student’s t-test (95% confidence) was carried out between a given sample and the corresponding negative control both at the same pre-incubation conditions
a pre-incubation of Ag with enzyme leads to almost complete quenching (no signal) for even the lowest Ag concentration
whereas ATP or substrate pre-incubation with Ag salt leads to higher signal than that without pre-incubation (so weakens its quenching effect)
A somewhat similar but weaker effect has been observed for Fe(III) at IC50 concentration of 4.7 µM and also above (20 µM) and for high Zn concentrations (1 mM and 1.25 mM)
in case of Fe(II)-1 salt at lower concentrations (25 μM and 75 µM)
pre-incubation with substrate first leads to maintaining of the level of quenching observed when no pre-incubation is applied (same as for IC50 value in pure SC buffer)
whereas enzyme or ATP-pre-incubation causes higher signal (weaker quenching) than that without pre-incubation
The effect is reversed for 0.5-mM concentration
at which near-complete quenching is observed for enzyme pre-incubation
with least quenching (most residual signal) at ATP pre-incubation
For both NLuc and RLuc exposed to Ag salt or Zn salt
pre-incubation of metal ion salt with enzyme leads to lower bioluminescent signal (stronger signal quenching) than pre-incubation with the substrate
The same is true for NLuc with Fe(II)-1 and Fe(III)
the order of pre-incubation seems to have little to no effect on Fe(II)-1-induced quenching
with less residual signal (stronger quenching) for Fe(III) at higher concentrations
Cu(II) presence quenches the signal more strongly (lower residual signal) in case of pre-incubation with a substrate
with much lower (or even none) quenching observed when pre-incubated with an enzyme first
Normalised percentage of bioluminescent signal for (A) NLuc and (B) RLuc assays with the original EDTA concentration in screening conditions (black; 0 mM for NLuc
and 0.25 mM for RLuc) and the same EDTA concentration as in FLuc assay screening conditions (grey; 0.5 mM)
Normalisation of the bioluminescent signal was carried out against the negative control output given by the original screening conditions (lowest EDTA concentration) of the corresponding bioluminescent assay plate
Statistical two-tailed Student’s t-test (95% confidence) was carried out between a given sample (at a given EDTA concentration) and the negative control at lowest EDTA concentration
The samples were deemed statistically different from blank for p < 0.05 and were marked with black “*.”
0.5-mM EDTA slightly increases the bioluminescent signal for all samples
with Ag at 68 μM and 200 µM concentrations and Cu(II) at 132 µM concentrations (all near the IC50 values of the original SC assay) being the most affected (the largest increase in bioluminescent signal in the presence of EDTA)
and (C) RLuc assays with 0 mM (black columns)
and 2 mM (black columns) concentration of glutathione (GSH)
Normalisation of the bioluminescent signal was carried out against the negative control output given by the original screening conditions (lowest GSH concentration) of the corresponding bioluminescent assay plate
Statistical two-tailed Student’s t-test (95% confidence) was carried out between a given sample (at a given GSH concentration) and the negative control at lowest GSH concentration
presents a statistically significant increase in activity with 0.2-mM GSH and even more with 2-mM GSH
leads to the weakening of inhibition of RLuc by all of the tested metals
with the effect being most pronounced for Ag and Cu(II)
Screening and subsequent investigations have been performed for a broad panel of environmentally and biologically relevant metal ions and their concentration ranges (Sigel and Sigel, 2000; Maret, 2016; Shamsollahi and Partovinia, 2019; Adams et al., 2020)
increasing the significance of any observed metal ion-induced quenching of bioluminescent signals
These effects could arise from various processes
including direct interaction with a luciferase enzyme
interference with substrate binding or turnover
substrate and reactants’ sequestration
or alterations in the chemical environment required for the bioluminescent reaction
As all of those processes are broadly dependent on the same type of affinities of metal ions to various ligands such as amino acid residues on enzymes
discerning these mechanisms remains challenging
Diverse experimental designs discussed above allowed quantifying the effects and providing insights into the role of reactants and buffer components in metal-mediated interference with luciferase-driven bioluminescent signal generation
Nearly all tested metal ions’ salts led to statistically significant reduction of bioluminescent signal of the FLuc system even at 1-mM concentration
Alkali metal ions salts showed the weakest inhibition that is relatively independent of the FLuc assay buffers (similar for both SC and H)
which might suggest a non-specific quenching mechanism (e.g.
their ubiquitous presence in buffers and biological as well as environmental samples
often at well into millimolar concentrations
can have a significant effect on bioluminescent readout and requires careful consideration
supporting direct metal ion–enzyme interaction as a cause of inhibition observed in our experiments
leading to a change in the stability of the light-emitting oxyluciferin product
still exhibits the strongest inhibition when pre-incubated with FLuc
substrate-metal ion pre-incubation leads to stronger quenching than ATP-metal ion pre-incubation
This supports previously reported hypothesis that apart from direct enzyme–metal ion interaction
FeSO4 can also reduce bioluminescent signal through the precipitation of D-luciferin substrate
NLuc and RLuc that do not require ATP and both use similar substrate types that are
displayed relatively higher tolerance to metal ion interference
with fewer metal ion salts exhibiting significant inhibitory effects at low concentrations and most IC50 values oscillating in the higher micromolar to millimolar range
Higher FLuc sensitivity is particularly visible when comparing low metal-affinity HEPES-based assay variants that were designed to minimise buffer–metal binding and the buffer variability between different luciferases (HEPES buffer has been shown to have negligible binding to metal ions; however
each of those assays still differ by the additives that came out essential to ensure sufficient assay quality)
all active metal ion salts tested exhibited from two (Fe(II) salts in RLuc H vs
FLuc H assay) to over thousand times (Cu(I) for NLuc H vs
FLuc H assay) higher IC50 values (lower inhibition) for NLuc H and RLuc H assays than in the FLuc H one
The variability in inhibition efficiency is much smaller between NLuc and RLuc (only up to 10 times the difference)
with NLuc being generally more resistant than RLuc
This is in line with the fact that NLuc is a bioengineered version of natural RLuc
The exceptionally stronger inhibition of NLuc than RLuc in the H assay by Zn
and Pt(II) might stem from the differential presence of ascorbate in the RLuc H assay
which is a known and highly potent Zn ionophore (i.e.
binds strongly to Zn2+ and potentially also to chemically similar Cd2+ and Pt2+ ions
depleting the effective concentration of “free” metal ions in the RLuc H assay)
With limited insights into the mechanism of NLuc and RLuc activity and regulation (Liu and Escher, 1999; Loening et al. 2006; Schenkmayerova et al., 2023; Nemergut et al. 2023)
the mechanism of metal ion interference with RLuc- and NLuc-mediated bioluminescence is very poorly studied
Pre-incubation experiments described in this manuscript for NLuc SC and RLuc SC assay conditions shed some light on this process
pre-incubation of NLuc and RLuc with Ag and Zn
and RLuc with Fe(II) led to more pronounced quenching
indicating direct metal–enzyme interaction
Reversed effects observed for pre-incubation of furimazine (NLuc substrate) with Cu(II) and coelenterazine (RLuc substrate) with Cu(II) and Fe(III) suggest substrate interaction or sequestration by those metals
The nature and composition of the buffer system played a crucial role in modulating the extent of metal ion interference observed in the bioluminescent assays by binding/precipitation and
lowering of effective availability of metal ions for interaction with luciferase/substrate/reactants
was used to unify the buffering system across all luciferases; however
some additives (like BSA for NLuc and RLuc
and MgSO4 for FLuc) could not be eliminated as they turned out to be essential to ensure sufficient luciferase activity and quality
and robustness of the assays (Z′ factor and signal-to-background ratios above recommended thresholds)
Certain metal ions, especially Cu(II) and Fe(III) at millimolar concentration, led to substantial pH decrease in SC buffers and especially in FLuc assay, potentially disrupting the optimal pH range for FLuc activity (Schenkmayerova et al., 2023) and demonstrating superior buffering capacity of HEPES buffering systems
together with simpler buffer composition and higher sensitivity of all enzymes to metal ion effects in the HEPES buffer
such as direct enzyme inhibition or substrate interference
may be more prominent drivers of metal ion-induced quenching in this system
together with the fact that Zn and Cu(II) IC50 values for FLuc and RLuc (bot not NLuc) drastically increase when moving from simpler H to TRIS and EDTA-containing SC conditions
points at those chelators as key protective additives of analysed luciferase buffer systems
These findings collectively highlight the importance of considering buffer composition and potential interactions between buffer components and metal ions when evaluating their impact on bioluminescent assays
we screened comprehensive and diverse panel of metal ion interferents
highlighting their significant influence on bioluminescence-based high-throughput screening (HTS) assays involving firefly luciferase (FLuc)
It is noteworthy that the observed quenching effects occurred within biologically and environmentally relevant concentration ranges of metal ions
underscoring a significant impact on HTS campaigns and the subsequent interpretation of screening data
The susceptibility to metal ion-induced quenching varied among the three luciferase systems
with FLuc exhibiting the highest sensitivity
This variability and differential effects of buffer compositions
and GSH highlight the complexity of the underlying mechanisms
The findings have important implications for the design
and interpretation of bioluminescence-based HTS assays
emphasising the need for rigorous assay validation protocols and potentially incorporating mitigating strategies
such as the use of chelators or alternative luciferase systems
depending on the anticipated metal ion composition of the samples or compound libraries
we reported a panel of three pairs of high-quality HTS assays for the activity of each of the three luciferases
and often complementary sensitivities to different metal ions
This allows future users to select an assay that is best suited to the type of anticipated metal contamination
the FLuc HEPES assay serves as a wide-spectrum metal ion detection assay with sub-micromolar sensitivity for quality control of samples/compound libraries and for hit validation to flag possible metal-induced false-positives
By elucidating the influence of metal ions on these widely used bioluminescent reporter systems and providing a set of new high-quality HTS assays for the activity of the three most commonly used luciferases
we contribute to the ongoing efforts to enhance the robustness
and interpretability of HTS campaigns in drug discovery and biological research
The original contributions presented in the study are included in the article/Supplementary Material; further enquiries can be directed to the corresponding author
The author(s) declare that financial support was received for the research
The authors acknowledge the financial support of the Foundation for Polish Science (FNP) for the Homing (grant no POIR.04.04.00-00-441F/17-00 (FC
the National Science Centre of Poland (NCN) for the Sonata (grant no 2017/26/D/NZ1/01234 (MK
the European Union for the grant iCare—Integrated assessment and advanced characterisation neuro-nanotoxicity (no HORIZON-CL4-2022-DIGITAL-EMERGING-01 (DK
and the Polish Ministry of Science and Higher Education (previously MNiSW (decision no DIR/WK/2018/06)) for involvement in the joint international project entitled European Infrastructure of Open Screening Platforms of Chemical Biology European Research Infrastructure Consortium (POL-OPENSCREEN
The authors are also pleased to recognise the expert support and access to the Centre for Chemical Biology (ERIC) infrastructure at IBCH PAS supported by the Ministry of Science and Higher Education (decision no DIR/WK/2018/06)
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2024.1436389/full#supplementary-material
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Received: 22 May 2024; Accepted: 15 October 2024;Published: 26 November 2024
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This oceanfront apartment with a wraparound balcony and sweeping view of the Atlantic Ocean was purchased for $3.4 million in 2006 by Daniyar Kesikbaev
the brother of then-Kazakh President Nursultan Nazarbaev
became part of a rancorous lawsuit in the United States between Bolat and his ex-wife
That dispute also involved millions of dollars in Manhattan real estate as well as what Bolat’s lawyer described as “over $50 million” worth of jewelry purchased with his client’s money
The Real Deal, a real estate industry site, reported shortly after the sale that the apartment had been on the market since the previous fall
around a year after Kesikbaev transferred ownership to Bolat
The fight over the Florida apartment was not the only property dispute for Bolat Nazarbaev in the Miami area
he sued a developer for alleged breach of contract over a balcony he claimed was insufficiently private
His complaint stated that Bolat paid a $1.2 million deposit for an apartment that the developer indicated would feature “private” balconies. In reality, the balcony for the apartment he planned to purchase “was not private at all,” the complaint said
“The privacy of the balcony is particularly important for the buyer in this case. Bolat Nazarbayev is the brother of the President of Kazakhstan. A private balcony is necessary for security reasons,” the Courthouse News Service cited the complaint as saying
Bolat requested that the developer return his deposit
and court records indicate the two sides reached a confidential settlement
When it was last sold, the Margo Way mansion, located in a cul-de-sac in one of the wealthiest postal codes in the United States, was reportedly among the most expensive homes sold in state history
The purchaser was hidden behind an anonymous company
Initial press speculation suggested a Russian oligarch was behind the purchase
federal court suggest that the home purchase was funded by Bolat Nazarbaev
the brother of former Kazakh President Nursultan Nazarbaev
just a few months after Bolat became embroiled in a bitter legal fight with his ex-wife
over ownership of several Manhattan properties
A proposed settlement submitted by Bolat’s lawyer in New York State Supreme Court in March 2014 stated that his client would relinquish any claim against Kurmangalieva relating to the property “located at 2 Margo Way
New Jersey.” Court papers filed as part of the lawsuit identified 2 Margo Way as the residence where Kesikbaev and Kurmangalieva lived
New Jersey property records show that the property has not been sold since the anonymous New Jersey-incorporated company bought it for $20 million in 2012
Precisely where the money for the home purchase came from remains unclear
But in a February 2014 e-mail to Kurmangalieva’s U.S
Bolat Nazarbaev’s attorney wrote that “we believe” the New Jersey property “was purchased with Mr
Nazarbayev’s money” and estimated its value as more than $20 million
attorney John Snyder also indicated that Bolat Nazarbaev had acquired more than $50 million in jewelry that Kurmangalieva was holding onto
Nazarbayev has agreed to release claims against your clients relating to jewelry that Ms
Kurmangaliyeva has appropriated for herself
valued in excess of $50 million,” Snyder wrote
Kurmangalieva has been listed as an executive or shareholder in several Kazakh companies
Kesikbaev married the daughter of Malaysia’s prime minister
a seller of expensive Hermes handbags in New York sued Kurmangalieva after she refused to pay a bill of more than $400,000 for the bags she had purchased
Kurmangalieva at one point threatened the seller
telling her in a phone call: “You know who my son is
He is the son-in-law of the prime minister of Malaysia
Do you know what we can do to you and your family
Kurmangalieva and Bolat Nazarbaev have a son together: Khanbolat Nazarbaev
Nazarbaev’s New York lawyer from the lawsuit told RFE/RL that he no longer represented Nazarbaev and did not know how to contact him
E-mails sent to Nazarbaev-linked investment companies in Almaty and to a charity he heads went unanswered as of publication
A message sent to a WhatsApp number for Kurmangalieva was opened and read but was not responded to
Kesikbaev did not respond to a Facebook message seeking comment
Property records show that more than a decade ago, the owner of the 215-237 block on Baker Street was a company called Farmont Baker Street Limited
That record is part of a larger backstory of the property, which was the focus of an exposé by the anti-corruption watchdog Global Witness published in July 2015
then-Kazakh President Nursultan Nazarbaev’s former son-in-law
The properties were acquired between 2008 and 2010 by four British-registered companies that either share a common parent — a shell company in the British Virgin Islands — or a direct corporate lineage
according to Global Witness and property and corporate registry records reviewed by RFE/RL
Among the strongest evidence tying the property to Rakhat Aliev
is the extensive overlap among directors between these companies and others directly owned by Aliev prior to his death
But documents obtained by RFE/RL and first published by the London-based organization SourceMaterial show that as of 2015
the owner was a British Virgin Islands shell company called Farmont Investors Corporation
Corporate documents showed Farmont Investors was ultimately controlled by Nurali Aliev’s mother
Darigha Nazarbaeva -- the former wife of Rakhat Aliev and the elder daughter of Nazarbaev
Not long after the publication of the Global Witness report
ownership of the Baker Street property was switched to another company
According to SourceMaterial
this appeared to be an effort to avoid new regulations aimed at cutting down on corrupt money flowing into British real estate
E-mails sent to the London law firm that has represented Nazarbaeva were not responded to prior to publication
An e-mail seeking comment from a public relations agency that Nazarbaeva and Aliev have used in the past went unanswered as of publication
SourceMaterial said that Nazarbaeva’s lawyers declined to answer detailed questions about the Baker Street property’s ownership
Documents reviewed by RFE/RL suggest that the new ownership structure under Landmark Network continues to have multiple ties to Darigha
including overlapping officers who worked for her deceased ex-husband
The Global Witness report helped lead to an investigation by the National Crime Agency and so-called “unexplained wealth orders” targeting properties owned by Nurali and Darigha
but rather others in the London area that shared several past corporate owners and directors — and linked back to Nurali Aliev and Darigha
a British court rejected the crime agency’s arguments that the properties were acquired by Rakhat Aliev prior to his death as a way to launder money
is perched on a south-facing hillside plot overlooking the Mediterranean Sea in one of Cannes’ most exclusive neighborhoods
Many of the houses in the neighborhood are hidden behind high walls and appear to have bodyguards and security cameras
the last time the property changed owners was in January 2008
when a company called Khanika purchased it for 22.9 million euros ($34 million)
Khanika’s ownership structure or beneficial owners could not be determined
though a company with a similar name appears in court records in nearby Monaco and a Monaco-based bank appeared to provide financing for the transaction
A real estate brochure published in August 2020 described the property as fulfilling “all the criteria corresponding to the mythology of the Riviera: a historical past
illustrious owners and an inordinate splendor
The property was one of several that Bolat Nazarbaev and his ex-wife Maira battled over in a bitter U.S
lawsuit that grew out of their divorce in the early 2010s
court records indicate a settlement was reached sometime in 2014
though it is unclear what kind of agreement was reached concerning the Cannes property
The court records also suggest the property was at one point owned by a trust registered in the name of the couple’s son
the nephew of former Kazakh President Nursultan Nazarbaev
Khanbolat was 9 years old when his parents divorced
it was not clear if the property was still for sale
When RFE/RL journalists photographed the house in September
Laundry was visible hanging to dry in the courtyard and several open windows could be seen on the house
41 Reeves Mews were purchased together in 2007 for 25.8 million pounds ($50.9 million) by a shell company called Merix International Ventures
41 Upper Grosvenor had been vacant for about seven years
which triggered both annoyance and curiosity in the neighborhood
was bought in August 2007 for 11.9 million pounds ($24.4 million) by a company in Luxembourg owned by Kulibaev
was bought later that year by a Luxembourg-registered company called Lynn Properties for 8.35 million pounds ($16.9 million)
the four properties are effectively part of a single unit
According to a 2015 lawsuit filed in British court by a real estate investor who claimed he had been jilted out of millions of dollars
Merix International Ventures was a wholly-owned subsidiary of a British Virgin Islands company called Kipros
whose ultimate beneficial owner was Kulibaev
That’s backed up by corporate registry documents
all four properties -- the two Upper Grosvenor and the two Reeves Mews properties -- belonged to Kulibaev through Merix
and the Luxembourg-incorporated Vitala Investment Holding
located in a historic building in Manhattan’s famed Wall Street district
were also caught up in the legal fight between Bolat Nazarbaev and his ex-wife
had used his stepfather’s money to buy the two apartments some six years earlier as an investment for Bolat
Daniyar took possession of them for himself and his mother
apartment 702 was sold by Kesikbaev in 2011 for $940,000
was sold by Bolat for an undisclosed price in 2016
Perched on a southern-facing hillside overlooking Spain’s Mediterranean coast
the sprawling property known locally as Can Juncadella was first developed by a Spanish industrialist around the end of World War II
It was later bought by a Japanese company that operated a school on the grounds
\nThe property was sold in 2007 and since then it has become a regular source of legal controversy
the estate is widely believed to belong to former Kazakh President Nursultan Nazarbaev himself
a theory referenced in a November 2012 court ruling in the nearby town of Blanes
The mansion is “said to be the property of the President of the Republic of Kazakhstan,” the ruling stated
Unconfirmed news reports said Nazarbaev himself attended a party at the property
Nazarbaev’s press secretary did not answer repeated phone calls made over several days by RFE/RL
An e-mail sent to the main office for the Nur Otan political party
The assertion of Kazakh ownership is backed up in corporate filings
a company registered in Barcelona called Flinder Data acquired the property for an unknown price
Flinder Data was owned by a Dutch-incorporated company called Faltain
which was in turn owned by another entity called Steppe Unity Cooperative
Steppe Unity is owned in part by Timur Kulibaev
one of Kazakhstan’s wealthiest men and husband of the former Kazakh president’s daughter
Flinder Data’s registered agent is Olga Aristova
who served in the same role for several companies linked to Kulibaev
Aristova was preceded in her role at Flinder by a Kazakh named Assylbek Karibaev
who worked for a subsidiary of KazMunayGas
who also headed the country’s sovereign-wealth fund and
controls the country’s largest bank by assets
A London law firm that has represented Kulibaev for many years
did not respond to e-mails seeking comment
E-mails sent to the charitable foundations that Dinara Kulibaeva heads in Switzerland and Kazakhstan also went unanswered
Much of the paper trail regarding the Spanish property’s ownership stems from court battles over environmental permits
A local environmental group called SOS Lloret tried to block the expansion of the buildings on the property in the early 2010s
saying the property was located in a sensitive
The Catalonian regional legislature also joined the fray
passing an ultimately unsuccessful measure to halt the expansion
the property’s owners sought to block access to a section of public road that ran close to the estate
but environmental groups won a series of rulings in local courts
the region’s highest court dismissed an appeal by Flinder
while the fight over the road played out in courts
the property’s owners aggressively tried to block people from walking on the road
erecting menacing signs and loudspeakers that blared warnings
They told RFE/RL that some of the loudspeakers also broadcast sounds of gunfire -- an apparent attempt to frighten locals
the Chateau de Bellerive was for many years owned by Sadruddin Aga Khan
the former UN diplomat and jet-setting global celebrity known for his philanthropic work
the lakeside property was purchased by Dinara Kulibaeva
the daughter of former Kazakh President Nursultan Nazarbaev,for 62 million Swiss francs ($63.8 million)
Kulibaeva expanded her lakeside holdings in January 2020
when she purchased an adjoining tract of land totalling about 1.5 hectares for 44 million Swiss francs ($49.3 million)
which includes a smaller house -- a historical building reportedly once owned by a 19th-century French military commander -- was acquired from an extended member of the Sadruddin family
The Swiss magazine Bilan last year ranked Kulibaeva as Switzerland’s 59th wealthiest person
the Hotel Corso is owned by a Czech-registered company called Kalma II
whose daughter is married to the son of Timur and Dinara Kulibaev
the company Kalma II is headquartered at the same address as the hotel
The company’s stated activities include “hospitality activities
residential and nonresidential premises and cosmetic services.”
Katranova is a general director of a company called Vostochnoye Rudoupravlenie
the largest producer and supplier of specialized minerals used by several oil- and gas-producing companies in Kazakhstan and the former Soviet Union
She has held positions in several other major companies in Kazakhstan
which is a subsidiary of the state-owned railroad Kazakhstan Temir Zholy
The hotel appeared to not be open when visited by RFE/RL reporters in September
Waiters working in cafes and restaurants nearby said the hotel had not been operational in recent memory
E-mails sent by RFE/RL to three Kazakh companies on whose boards Katranova sits
No one answered the main office phone at Hotel Corso on two separate days
The hotel was acquired by a Czech-registered company owned by Boranbaev in April 2000
four months after the purchase of Villa Ahlan
has reregistered several times since its founding in 1999
Initially described as “rental of flats and nonresidential premises without providing other than basic services associated with the lease of flats and nonresidential premises,” the company’s declared activities were changed in 2014 to “operation of nongovernmental medical facilities in the scope of physiatry and balneology” -- the medical study of therapeutic baths
The company also added as its declared activities “washing
and personal goods.” Another listed executive with the company is Zelina Katranova
whose daughter is married to another of Nazarbaev grandsons
Czech corporate records show Mingon-II is linked to several other non-Czech companies
including KazMunayGas-Service and Razvedka Dobycha KazMunayGas
both of which are subsidiaries of Kazakh state-owned oil and gas giant KazMunayGas
is a shareholder and former top executive in KazMunayGas
the building known as Myslivna used to be a popular cafe known as the Jagerhaus
according to historical postcards from the time
employees of the Czech Interior Ministry came to vacation and take medicinal baths
The entire property was sold by the Interior Ministry in 2017 to a company registered in the Czech Republic
located in the municipality of Karlovy Vary
The property was initially listed for auction at 41.4 million Czech crowns ($1.8 million)
but ended up being sold below the advertised price in November 2016
holds executive positions in several Czech-registered companies that belong to Boranbaev
who is related to Nazarbaev by marriage: his daughter married Nazarbaev’s grandson
was equally owned by two Dutch-registered firms
whose parent companies link directly back to Timur Kulibaev
ADNTS Holdings’ only owner is ADNTS Investments
registered in Singapore as a private company
The director of this company is a Singaporean man who is also listed as a secretary in Steppe Capital
a Singaporean company whose only shareholder is Kulibaev
linked to the Spanish hillside estate that has been identified as belonging to Kulibaev
Dostyk Holdings’ only shareholder is Steppe Unity Cooperatief
which is also registered in Amsterdam and is a subsidiary of Kulibaev’s Steppe Capital
Dostyk Holdings exited its ownership of Margotrade in November
Margotrade’s new 50 percent owner is a Kazakh company
which was incorporated in October and is owned by two Kulibaev companies
when RFE/RL reporters visited the Karlovy Vary property
with peeling paint and overgrown shrubbery
Though a car was parked next to the property’s outbuilding
no one answered when RFE/RL reporters tried to enter the grounds
ringing a gate bell and calling out to see if anyone was present
The London law firm that has represented Kulibaev for many years
RFE/RL also left a voice mail with Kulibaev’s lawyer at the same firm
Located in one of the most expensive districts on Geneva’s outskirts
the lakeshore property drew the attention of Swiss authorities when Kulibaeva sought to purchase it
Swiss regulations restrict some investments by foreigners; a local magistrate ultimately approved the purchase in 2009
According to a 2013 investigation by Swiss public broadcaster RTS, Kulibaeva had to seek permission from the Swiss government to acquire the property because its area exceeded the ceiling of 3,000 square meters set for citizens of non-European countries
The magistrate who allowed the purchase to go forward cited likely visits by her husband
"The frequent stays of her husband...as well as those of her father
which published an excerpt of the decision
"The importance of the receptions that Madame Kulibayeva would have to organize
as well as the special security measures that would be required as a result
also justify the acquisition of a building characterized by the size of its surface.”
In a 2016 interview with a Swiss business magazine
who previously had lived in eastern Switzerland near the lakeside town of Lugano
which Forbes magazine at the time estimated at $2.4 billion
This is neither a goal nor a source of pride,” Kulibaeva said
Boranbaev is the father of a Kazakh woman who
was the wife of former Kazakh President Nursultan Nazarbaev’s grandson
The property is located within Wentworth Estate
a 700-hectare location southwest of London that was once part of a grand country estate in the family of the royal duke of Wellington
The estate itself is a collection of gentle hills and leafy roads that is now best known as home to the Wentworth Club
U.K. property records show Boranbaev bought his property there in November 2014 through a New Zealand-incorporated company he controlled
the location has gained notoriety for the luxuriously priced homes
but also for the British and foreign notables who have lived there at one time or another
The list includes former Chilean dictator Augusto Pinochet
the late Russian oligarch and Kremlin confidant Boris Berezovsky
The husband of Boranbaev’s daughter, Alima, was Aisultan Nazarbaev, one of Nazarbaev’s grandsons and the son of Darigha Nazarbaeva. He was found dead in London in August 2020. According to preliminary conclusions by British authorities, the cause of death was cardiac arrest. The Financial Times reported earlier this month that an autopsy listed the cause as toxicity from cocaine
Aisultan had said publicly that he battled drug addiction
When Boranbaev bought the property in 2014
The owner of an interior-design studio listed on the property’s planning records gave an interview in 2016 discussing her firm’s work on what she called the “Pinehurst Project.” But she did not reveal the identity or nationality of the client
“On a project like this, the sky is the limit, and what I like about our clients is that they are incredibly mature about what they want,” the designer, Charu Gandhi, was quoted as saying
“These are not people who stumbled into having this lifestyle
Gandhi’s studio did not respond to an e-mail seeking comment on its work for Boranbaev
E-mails sent by RFE/RL to the Almaty soccer club that Boranbaev owns
An e-mail sent to a Gmail account that Boranbaev is known to use also went unanswered
Bolat Nazarbaev — the younger brother of former Kazakh President Nursultan Nazarbaev — and his then-wife Maira Kurmangalieva purchased the unit in a building overlooking Manhattan’s famed Central Park
The couple divorced three years later and the apartment became central to a bitter and public legal fight that played out in New York courts and the city’s tabloids
as Bolat accused Kurmangalieva and his stepson (her son)
of fraudulently taking ownership of the unit
Bolat is listed as a shareholder or head of numerous Kazakh companies operating in a range of sectors
The couple ultimately settled their lawsuit and Bolat regained sole ownership of the unit in 2014
He sold it two years later for a reported $17.9 million
The sports center near Karlovy Vary sits on various plots of land that were bought from various owners between 2010 and 2012
The entire property is owned by a company whose only shareholder is Boranbaev
Boranbaev also owns several sport and fitness centers
The mansion at Sunninghill Park was long known as the family home for Prince Andrew
who was given the property in 1986 as a wedding gift by his mother
The royal couple divorced in 1996 and the house was sold in 2007 for 3 million pounds above the asking price to a company in Luxembourg called Unity Assets Corporation
The property ended up sitting unoccupied for several years
much to the consternation of neighbors and historic preservationists
In 2016, Kulibaev tore down the property and moved forward with plans for a newer residence
RFE/RL also left a voice mail with Kulibaev’s lawyer that went unanswered as of publication
Two of the three properties -- on Denewood Road and Bishops Avenue
respectively -- were purchased by shell companies in 2008; the third
Britain’s National Crime Agency (NCA) argued in court filings that “the properties were acquired as a means of laundering the proceeds of unlawful conduct by Mr
Aliev was a prominent and powerful Kazakh who built a fortune in banking, oil, media, and other industries, and held several prominent government positions, including deputy chairman of the state security service known as the KNB
former Kazakh President Nursultan Nazarbaev’s elder daughter
Five months after his death, in a report about Kazakh investments in British real estate, the London corruption watchdog Global Witness described Aliev as allegedly having “used his position as the former deputy head of the country’s secret police to amass a vast business empire.”
A British judge overturned all three orders in April 2020
ruling that the agency\'s “assumption” that the money to buy the three properties came from Rakhat Aliev “as a means of laundering his money” was unreliable
"It is frustrating and disappointing that she has had to take this action to fight these draconian proceedings and clear her name,” a spokesman for Nazarbaeva said in a statement in April after the court issued its ruling
Representatives for Nazarbaeva and Nurali Aliev told the U.K
court that the funding for two of the homes was linked to Nurbank
one of Kazakhstan’s largest private lenders
Nurali Aliev bought the Hampstead Mansion on London’s “billionaire’s row” with money that came “principally” from a $65 million loan from Nurbank
while Nazarbaeva bought the Manresa Road property using proceeds from the sale of her shares in the Kazakh lender
An e-mail seeking comment from a public relations agency that Nazarbaeva and Aliev have used in the past also went unanswered as of publication
Rakhat exited the bank’s ownership in 2007
after which both Nurali Aliev and Nazarbaeva became Nurbank shareholders
At the time of Nurali’s purchase of the Hampstead Mansion
Nurbank was “ultimately controlled” by the mother-and-son duo
Nurali was elected chairman of the Nurbank board in October 2007
Nazarbaeva was elected to the board at the same time
court that she bought the Denewood Road property
located in a leafy suburb of northern London
using the proceeds of shares in the Kazakh sugar producer Kant that she sold
They said that Rakhat Aliev had transferred these shares to her in connection with their divorce in 2007 and that “unlike other assets then held by [Aliev] in Kazakhstan,” Kazakh authorities did not identify them as “being any part of his suspected proceeds of crime.”
Her representatives told the court that she was an independently successful businesswoman and “one of many entrepreneurial individuals who capitalized on the economic reforms in Kazakhstan at this time,” including in the sugar industry
They said that records related to her business trading “sugar
and cigarettes” in the years following the Soviet collapse could not be found despite “significant efforts” to do so
But Nazarbaeva estimates that this business potentially brought her as much as $45 million between 1992 and 1995
The Czech-registered company Mignon-II purchased the Villa Ahlan hotel in December 1999 -- the first of two hotel purchases by the company
Czech media reported that the hotel belonged to Boranbaev and noted that then-Kazakh President Nursultan Nazarbaev was a regular visitor to Karlovy Vary
even speculating that Nazarbaev was a part owner of the hotel
Records do not indicate any ownership role for Nazarbaev
Nazarbaev stayed at Villa Ahlan during a 2002 visit to Karlovy Vary, Russia’s state-run news agency RIA Novosti cited the municipality’s press service as saying at the time. The report noted that the hotel’s owners were “citizens of Kazakhstan,” but did not identify them
Former Ukrainian President Leonid Kuchma, who was known as a friend of Nazarbaev, stayed at the Villa Ahlan in 2005
the property on Lake Lugano housed a famed 19th-century building known as Villa Galli
popular among generations of Lugano locals for a luxury restaurant
Pacolli’s construction company, Mabatex
In 2008, Pacolli publicly revealed the ownership of the property where La Romantica was housed, saying that he owned three neighboring buildings
for whom he said he was the official representative
Pacolli denied that he had purchased La Romantica “on behalf of third parties” and threatened legal action against those who suggested this was the case
“no member of the family of President Nazarbaev is or ever has been the owner” of La Romantica or Stott Limited
Despite public outcry from historic preservationists
Pacolli demolished the aging La Romantica in 2013
the developer has been fighting with local authorities over zoning permits
The land is now home to a tennis club and three nondescript office buildings
An investigation by Swiss journalists published in 2010, meanwhile, found that Stott Limited was 50 percent owned by another company, Transasian Oil, headed by an Indian businessman and partner of Timur Kulibaev
whose name appears on several Kulibaev-owned companies
Transasian Oil loaned Stott more than 10 million Swiss francs ($12.4 million)
according to the investigation by Swiss public broadcaster RTS
and received the Villa Galli property as collateral
The debt pledge on the property totaled 8.5 million Swiss francs ($10.5 million)
making Transasian Oil the ultimate owner of Villa Galli
In a 2015 report on the threat facing Switzerland from money laundering and terrorism
Swiss Federal Police detailed an example of a scheme in which an unnamed Swiss property is purchased under circumstances that raise suspicions
While the scheme does not name the people or companies involved
it closely mirrors details in the RTS investigation and publicly known information about the Villa Galli property and the individuals it has been linked to
the person identified as “Suspect A” in the Swiss Federal Police scheme is described as someone “accused of having benefited from his position as a manager in the national oil company of an oil-producing country to enrich himself illegally.” This person’s money
“would have passed through different companies to finally be used by a front man to acquire property in Switzerland,” the document states
the company resembling Stott gains ownership of the property using a loan of 8.55 million Swiss francs from a company resembling Transasian Oil that is ultimately controlled by someone who has been “a confidant and front man” of “Suspect A” for “many years.”
The mortgage note for the historic building is then held by the company resembling Transasian Oil as collateral for the loan that the firm resembling Stott uses to buy the property
Asked whether the property cited in the scheme in the 2015 report was the La Romantica building
Swiss Federal Police spokeswoman Katrin Schmitter said: “We are subject to official secrecy and cannot comment on specific cases.”
that the examples “described in our reports are generally based on real-life cases.”
The federal police report also echoes the real-life case of Kulibaev
who was investigated by the Swiss Attorney-General’s Office for possible money laundering
The probe was dropped in 2013 after authorities said they found no evidence of wrongdoing
The report states that the allegations against “Suspect A” are determined to be “unfounded” and the proceedings dropped due to “lack of evidence as to the criminal origin of the assets.”
the property cited in the scheme is “demolished” amid media reports that “Suspect A” and “an acquaintance and presumed accomplice” intend “to replace it with a luxury hotel.”
RFE/RL also left a voice mail with Kulibaev’s lawyer
Pacolli’s longtime representative in Lugano said Stott Ltd
Behget Pacolli” and “Stott is the 100 percent owner” of the La Romantica property
The representative said the purchase of properties more than a decade ago
and the construction of a luxury hotel now planned for the former location of La Romantica
“There [was] no working relationship with Mr.Kulibaev at all!” she said in her e-mail
She did not respond to a follow-up e-mail seeking further details of when Pacolli gained control of Stott
and the discrepancy with Pacolli’s earlier public statements about the ownership of the Villa Galli properties
Swiss journalists also reported that Dinara Kulibaeva lived in a nearby villa between January 2007 and December 2009
which they said she rented from Pacolli for a reported 50,000 Swiss francs ($57,000) annually
Pacolli’s representative confirmed that his villa in Lugano was rented at one point for an unspecified 12-month period to the Kulibaev family “while their son and nephew were visiting the American school in Lugano
and for that period of time the two students were living in the villa.”
the tenancy has been terminated,” she told RFE/RL
The Nazarbaev Family’s Opulent Offshore Real Estate Empire
GENEVA – In the gentle dawn of Switzerland’s late summer
Lake Geneva’s ripples lap against the properties on the eastern shore in the suburb of Anières
Some of the buildings are understated in their wealth
with slate shingles or mansard roofs or Corinthian columns
Some have gazebos on manicured lawns looking west to the Jura Mountains
or docks where motorboats and kayaks are parked
Many have gates and surveillance cameras to protect from curious passersby
399 Route D’Hermance: a 3,200-square-meter three-level villa with a butterfly staircase
In an area known for having some of the most expensive housing in the region
the daughter of Kazakhstan’s longtime ruler
who are among Kazakhstan’s wealthiest people
purchased the villa in 2009 for a reported $75 million
And they are among several immediate and extended relatives of Nazarbaev who own lavish real estate in the West
relatives of Nazarbaev have purchased hundreds of millions of dollars in posh real estate in Europe and the United States
a string of high-end properties on luxurious lakesides
and overlooking the azure waters of Spain’s Costa Brava
A new RFE/RL investigation provides the most comprehensive overview to date of the properties in this sprawling real estate network linked to Nazarbaev’s relatives
The findings are not an exhaustive record of every foreign property owned by a relative of the former Kazakh president
who was officially granted the title “Leader of the Nation” in 2010 and currently serves as chairman of the country’s powerful Security Council and heads its ruling political party
But they offer an unprecedented window into the scale of the real estate investments by Nazarbaev’s relatives
and how many in close proximity to Kazakhstan’s ruling family ended up with luxury assets in exclusive locations
RFE/RL identified at least $785 million in European and U.S
real estate purchases made by Nazarbaev’s family members and their in-laws in six countries over a 20-year span
This figure includes a handful of properties that have since been sold
including multimillion-dollar apartments in the United States bought by Nazarbaev’s brother
It does not include a sprawling Spanish estate owned by Kulibaev
for which a purchase price could not be found
These acquisitions have been funded by the vast fortunes Nazarbaev’s relatives have amassed in the oil-rich nation’s energy
while at various times also serving in official government posts
Nazarbaev’s patronage is widely seen as crucial to the wealth built by his relatives
who have repeatedly and vehemently insisted they are successful businesspeople independent of their family and political connections
who has been dogged for years by accusations that his wealth
mainly from his work in the oil-and-gas industry
The Financial Times on December 2 said it had uncovered a secret scheme that allegedly channeled tens of millions of dollars from contracts related to a massive gas pipeline to China to Kulibaev
His lawyers denied specifics of the report to the Financial Times and did not respond to queries from RFE/RL
Several of these properties documented by RFE/RL have been the subject of legal challenges
and British freezing orders on three London residences that were later overturned by a court
The investments in pricey foreign properties also come against the backdrop of the country’s overall increase in national wealth since the Soviet collapse
This increased prosperity has lifted livelihoods for many average Kazakhs -- but it has also helped the politically connected elite transform into jet-setting tycoons and fodder for newspaper gossip pages
And with 80-year-old Nazarbaev in his twilight
will succeed him when he fully departs from Kazakh politics -- and what might happen to the fortunes of those closest to him
The political economy that Nazarbaev has built
it’s built on one man,” said Kate Mallinson
a London-based consultant and researcher of Central Asian politics
His relatives and closest allies have “hedged the bets on the future
not knowing what will happen -- and so they’ve had to put assets outside the country,” Mallinson told RFE/RL
the head of Kazakhstan’s oldest and largest human-rights organization
said “it is hard to separate the government from the [Nazarbaev] family” and “hard to say how it will be in Kazakhstan” after Nazarbaev dies
“You cannot rely on protection from the rule of law when you live in such political systems,” Zhovtis said
41-42 Upper Grosvenor Street; 41-42 Reeves Mews
RFE/RL journalists report the news in 23 countries where a free press is banned by the government or not fully established
We provide what many people cannot get locally: uncensored news
RFE/RL is registered with the IRS as a private
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RFE/RL's editorial independence is protected by U.S
Independent journalism at the University of Twente
The Institute of Electrical and Electronics Engineers (IEEE) has featured Pep Canyelles Pericas as their Engineer of the Week
especially because I’m still relatively junior in my career,’ says the University of Twente scientist
Canyelles Pericas: ‘It brings a lot of visibility and recognition for my work
but it helps if people know what I’m up to
It’s nice to be acknowledged for my research and it has helped me to reconnect with a lot of people from my network
We have to wait and see how much it will bring in the long run
but I think it’s important to engage with the general public and explain what you do in language that everyone understands
As scientists we are often paid from public funds and it’s important to show what we do with the funding.’
‘An engineer of the week should be an IEEE member with a variety of backgrounds and expertise
I focus a lot on tech transfer in between academia and industry
The Institute of Electrical and Electronics Engineers (IEEE) is the world's largest association of technical professionals with more than more than 396,000 members in over 160 countries around the world
Its objectives are the educational and technical advancement of electrical and electronic engineering
computer engineering and similar disciplines
‘To align the space between industry and academia. I enjoy working at the university and I’m thankful to MESA+ for their support in exploring my potential. I want to bridge the gap no matter on which side I end up. Ultimately, I want to work on technologies that can be applied fast and be of use to people.’
David Fernandez Rivas heeft woensdagavond een prijs in de wacht gesleept. Zowel de vakjury als het publiek viel voor de bezieling van de universitair docent van de UT en bekroonden hem met de Prins Friso Ingenieursprijs 2021.
David Fernandez Rivas won a prize on Wednesday night. Both the professional jury and the public fell for the inspiration of the UT associate professor and awarded him with the Prins Friso Ingenieursprijs 2021 (Prince Friso Engineering Prize).
UT-atletiekvereniging Kronos loopt als titelverdediger van het algemeen klassement warm voor de alternatieve Batavierenrace. De ambities zijn wat minder hooggespannen dan de vorige jaren, maar de hoop is er op een klassieke Bata-atmosfeer.
Ondanks de kritiek van universiteiten zet de Europese Commissie het plan door: Horizon Europe moet ook ingezet kunnen worden voor militaire toepassingen (dual use). Vooral bedrijven moeten daarvan profiteren.
Vanaf september 2026 gaan studenten in het hoger onderwijs 2.694 euro collegegeld per jaar betalen. Dat is een stijging van bijna honderd euro. Reden daarvoor is de relatief hoge inflatie van afgelopen jaar.
With the magazine ROOTS we want to connect students and companies. We do this by bringing stories of starters on the labor market. They talk about living and working in the region. We also publish advertorials. In this way, companies come into the spotlight of students and students get an idea of the life that awaits them and what opportunities there are in the region.
LEA BELIAEVA BANDER | Barcelona
First published: November 27, 2023 05:10 PM
Christmas has descended upon Catalonia, a perfect time to learn about Catalan holiday traditions such as eating turrons (nougats), placing the caganer (pooping figurine) in nativity scenes, or feeding the Tió de Nadal (Christmas log). And all of these things can be found in Christmas markets all over Catalonia.
This is the oldest Christmas market in Catalonia, open every Christmas since 1786. This year it will have 200 stalls dedicated to decorations, ornaments, nativity scenes, Christmas trees and more, making it the largest of its kind in the world, according to organizers.
Nadal al Port, Port Vell Christmas Market
Nadal al Port, located in the harbor area of Port Vell, is one of the newest Christmas markets in Barcelona, having only been inaugurated in 2018. Located right by the water on the Moll de la Fusta, visitors can enjoy a local gastronomy market, take a ride on the Ferris Wheel, see Christmas light shows and installations, or even try the 400m2 ice rink.
This market is another traditional Christmas Market in Barcelona, dating back to 1877 when street vendors would set up stalls to sell their goods. Today visitors can buy Christmas presents, decorations and food in over 100 stalls.
For Art Nouveau fans, there are two events that cannot be missed this year.
The first is the Sagrada Familia Christmas Market, located by the Nativity Facade of the famous Gaudí cathedral. Inaugurated in 1962, it has 150 stalls, that, according to the organizers, have "everything you need to celebrate Christmas at home.".
A short walk from that Christmas Market, there’s the Christmas Garden at the Sant Pau Art Nouveau Site (Recinte Modernista de Sant Pau). For the third year in a row, the old hospital is bathed in Christmas lights and other audiovisual installations. The Sant Pau Site also includes an ice rink.
For residents and visitors who are interested in alternative Christmas markets in Barcelona, there are various great options:
For the past 15 years, an old closed-down German toy factory, Antiga Fàbrica Lehmann, located in L'Esquerra d'Eixample, has been a creative hub for local designers and artists, and every year the cobblestone-clad patio of the factory is transformed into a Christmas Market, where visitors can buy handcrafted goods like jewellery, ceramics, clothing, artwork, as well as books and food.
Another alternative Christmas market is the Market for Sustainable Consumption and Social and Solidary Economy, held in the center of the city, ion Pl. Catalunya.
A total of fifty small businesses and organizations will sell craft products, clothing, books, food and other small gift items all produced with sustainability and solidarity in mind.
The new edition of the Christmas Market of Caldes de Montbui will be held from December 5th to 10th, with more stalls and family activities in the historic center of the town. The highlight of the event a thermal soup cooked in the biggest pot in all of Catalonia, which can hold 1,600 liters of soup. There will also be dozens of stalls selling food, Christmas products and antiques.
Not strictly Christmas themed, the market sets out to recreate medival life through workshops, shows, theatre and music for visitors of all ages. The fair will also include stalls with locally produced products such as food, jewelry, herbs and decorations.
This is one of the most popular events for children. Mura is located in the Bages region and, during the weeks before Christmas up until after the New Year, the Tió de Nadal logs invade the streets of the town.
Christmas and Torró Fair in Cardedeu
The fair dates back to 1272, according to organizers in the town located in northern Catalonia. The centerpiece of the fair is the torró, a traditional Christmas sweet, which will be sold in most of the stalls. Other items include gifts, handcrafted goods, nativity scenes and Christmas decorations.
The Fair of Santa Llúcia de Canyelles is held on December 2nd and 3rd. There are more than 350 stalls, exhibitions, children's programs and other activites between 9 am and 9 pm.
The characteristic wooden stalls of the Girona Christmas Market have once again filled the Independence Square in the heart of the city. In most of the stalls, visitors can buy handcrafted goods such as Christmas decorations, ceramics and other gifts, as well as a huge selection of locally produced foods.
The tiny town of Espinelves, located in north-central Catalonia just outside Vic, holds an annual Christmas tree market, the Fira d'Alvet. Vendors sell locally grown trees and Christmas decorations. Aditionally, the fair features around a hundred stalls selling hand-made crafts and food.
From December 8 to 10, the Olot Nativity Scene Fair will take place in the Esteve Ferrer square, with dozens of wooden stalls where artisans will exhibit and sell figurines and sculptures. The Plaça Major will host the Christmas Market Km. 0, where you will find gift products made by local producers.
The city of Lleida doesn't just have one, but two Christmas markets.
Located in the center of the commercial hub of the city, the Santa Llúcia Market, offers a variety of Christmas-related goods such as decorations, nativity scenes and trees.
The other market, the Lleida Christmas Market, is located next to the main Cathedral. Here, visitors can buy handcrafted products and foods from 25 different stalls.
Tarragona’s Rambla Nova will be packed with traditional wooden house stalls this season, selling Christmas decorations, trees, small presents, and typical Christmas sweets such as xurros, waffles and chocolate.
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A Publisher Correction to this article was published on 25 April 2018
This article has been updated
β-cell proliferation is a rare event in adult pancreatic islets
To study the replication-related β-cell biology we designed a replicating β-cells sorting system for gene expression experiments
Replicating β-cells were identified by EdU incorporation and purified by flow cytometry
For β-cell separation islet cells were sorted by size
granularity and Newport Green fluorescence emission that was combined with emitted fluorescence for EdU-labelled replicating cells sorting
The purity of the resulting sorted populations was evaluated by insulin staining and EdU for β-cell identification and for replicating cells
Total RNA was isolated from purified cell-sorted populations for gene expression analysis
Cell sorting of dispersed islet cells resulted in 96.2% purity for insulin positivity in the collected β-cell fraction and 100% efficiency of the EdU-based cell separation
RNA integrity was similar between FACS-sorted replicating and quiescent β-cells
Global transcriptome analysis of replicating vs quiescent β-cells showed the expected enrichment of categories related to cell division and DNA replication
key genes in the spindle check-point were the most upregulated genes in replicating β-cells
This work provides a method that allows for the isolation of replicating β-cells
a very scarce population in adult pancreatic islets
Pancreatic β-cells are the unique cell type in the body that produces and secretes insulin in response to small variations in blood glucose levels to tightly control systemic glucose homeostasis
Β-cell mass reduction is a central event in the development of type 1 and type 2 diabetes
and β-cell regeneration is a potential curative treatment of the disease
Despite the advantages provided by this system
its use is restricted to the study of β-cell replication in mouse islets
we sought to develop a method for replicating β-cells sorting based on EdU incorporation
suitable for global gene expression analysis and applicable to most of experimental designs and animal species
FELASA guidelines and recommendations for the use of laboratory animals were followed (European and local government guidelines) and animal procedures were reviewed and approved by the Animal Research Committee of the University of Barcelona (identification number: DAAM 7082)
Isolated islets were hand-picked under a stereomicroscope two or three times
until a population of pure islets was obtained
Islets were cultured in RPMI 1640 (Sigma Immunochemicals
USA) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin containing 10% heat-inactivated foetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2
USA) were added into the culture medium at final concentration of 10 µM
For the analysis of continuous labelling with EdU
islets were cultured in 5.5 mM or 22.2 mM glucose with or without EdU for 7 and 14 days
Culture medium was replaced on a daily basis and BrdU was added to the islets for the last 24 h of culture
islets were cultured in 5.5 mM glucose with EdU overnight starting on the day of isolation
Dispersed islet cells were washed once with PBS and incubated with 25 µM NG at 37 °C for 30 min
EdU labelling was performed by means of the Click-iT® EdU Alexa Fluor®647 Flow cytometry Assay Kit (Invitrogen
cells were fixed in 2% paraformaldehyde–PBS at room temperature (RT) for 15 min.
and washed once with 1% BSA-PBS before mild permeabilization with a saponin-based reagent for additional 15 min
The click reaction buffer was prepared following the manufacturer’s instructions and added to the washed cells for 30 min
labelled cells were washed twice with FACS buffer (PBS
0.5%BSA) and transferred into 35 µm nylon mesh cell strainer caped-tubes (BD biosciences
400 units of RNasin plus RNase inhibitor (Promega) were added to the buffer or solution used in each step from the cell fixation with PFA until cell sorting
Unstained cells and FMO (fluorescence minus one)
were used as negative control in each experiment and served as autofluorescence control
Sorting was done using a Beckman-Coulter MoFlo Astrios cell sorter equipped with a 100-μm flow tip and operated at a sheath pressure of 25 psi
The laser illumination power was set to 150 mW for NG excitation at 488 nm
and to 100 mW for Alexa 647 excitation at 640 nm
and 671/30 nm for EdU Alexa 647 fluorescence
cells were filtered through a 35 μm strainer to eliminate all remaining cell clumps
doublets are excluded by using pulse processing (FSC-H vs
FSC-A) followed by the exclusion of debris gating on a two physical parameter dot plot (FSC/SSC)
Mitotic and post-mitotic cell subpopulations were selected as EdU+ or EdU- respectively
An average sorting rate of 200 events per second was maintained
Sorted cells were collected in 150 μl of PKD buffer (Qiagen)
Cultured islets were fixed overnight in 4% paraformaldehyde-PBS at 4 °C
sectioned and immunostained after deparaffinization and rehydration
For EdU detection in cultured islets the click chemistry-based Click-iT EdU assay (Click-iT® EdU Alexa Fluor® 594 Imaging Kit; Molecular Probes
Life Technologies) was used according to the manufacturer’s instructions
sections were permeabilized with saponin and incubated with the Click-iT reaction cocktail for 1 h at room temperature
This labelling method was combined with insulin immunofluorescence
Β-cell purity in sorted populations was determined by insulin and c-peptide immunofluorescence as follows
Sorted populations were directly collected in 1% BSA-PBS
washed twice with PBS and incubated overnight with the rabbit anti-insulin antibody (1/100) or mouse-anti c-peptide antibody (1/50
Abcam) diluted in 1% BSA-PBS + 0.3% triton
Donkey anti-rabbit Alexafluor-488 labelled antibody (1/400) was used for β-cell visualization
Cells were loaded into the haemocytometer for counting
At least 200 cells were counted for each sorted population
The experiment was replicated 8 times with islets isolated from 8 different rats
Total RNA was extracted with RNeasy FFPE kit (Qiagen
UK) according to manufacturer’s instructions with the following variation
Sorted cells were directly collected in PKD buffer
immediately treated with 200 μg/ml proteinase K (Qiagen) at 56 °C for 3 h with continuous shaking
This modification significantly increased the RNA extraction yield
mRNA was linearly amplified with the Arcturus® RiboAmp® HS PLUS RNA Amplification Kit (Arcturus
USA) or Complete Whole Transcriptome Amplification Kit (Sigma WTA2) following the manufacturer’s protocol
RNA quality was assessed by using the Agilent RNA Nano kit in the Bioanalyzer 2100 (Agilent Technologies
Results are expressed as means ± SEM for 4–8 independent experiments. Statistical analysis were performed using GraphPad Prism 6 software, and differences among means were evaluated using the Student’s paired t test with p < 0.05 considered significant.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Continuous labelling of islets with EdU ex vivo
Rat pancreatic islets were cultured at 5.5 or 22.2 mM glucose in the presence or absence of 10 µM EdU
10 µM BrdU was added into the culture medium for the last 24 h
(A,B) β-cell replication (BrdU incorporation) and (C,D) β-cell apoptosis (TUNEL) of islets exposed to EdU for 7 days (A,C) or 14 days (B,D)
(E) Representative image of EdU and insulin staining on islets cultured for 7 (top) or 14 days (bottom) at 22.2 mM glucose with continuous EdU labelling
(F) Quantification of β-cells positive for EdU
Efficiency of replicating and quiescent β-cell sorting
(A) Replicating and quiescent β-cells were sorted after gating on SSC-Ahi/FSC-Ahi (R2) and FSC-Ahi/NG-Ahi (R9) for β-cell population
and EdU647+ (R5) and EdU647− (R4) for replicating and quiescent cells
(B) To assess the purity of each cell population
Mitotic β-cell population is illustrated as a composite of pictures from 4 different fields to show a representative number of cells
Insets framed in red show replicating (top) and quiescent (bottom) β-cells at higher magnification
The number of cells collected per rat pancreas ranged from 760 to 1200 cells in the proliferative β-cell fraction and from 4 × 104 to 6 × 104 cells in the quiescent β-cell fraction
RNA was isolated from replicating and quiescent β-cell fractions
RNA extraction from the quiescent β-cell fraction that consisted of approximately 2 × 104 cells
whereas for the replicating fraction with ≈1000 cells
the collected RNA yielded ~5 ng of RNA (~5 pg of RNA/cell)
Similar results were obtained in the RNA extraction from 1000 quiescent β-cells
excluding the possibility that replicating β-cells had lower content of RNAs
the efficiency of RNA extraction correlated with the number of cells in the fraction
Gene expression of replicating β-cells
(A) Ct values of housekeeping genes used as endogenous controls amplified in replicating (empty) and quiescent β-cells (hashed)
Values are represented as median [IQR] of n = 6
(B) Differential expression of cell-cycle related genes and insulin in replicating over quiescent β-cells
In order to confirm the purity of the collected fractions
a panel of pancreatic cell types and proliferation markers were analysed in both fractions
keratin 20 (krt20) and pecam1 were undetectable in our samples
indicating the absence of contaminating acinar
duct or endothelial cells (data not shown)
mRNAs encoding for glucagon (gcn) and somatostatin (sst) were detected in 2 out of 11 samples from both fractions
and pancreatic polypeptide (ppy) in 6 out of 11
vimentin the major cytoskeletal component of mesenchymal cells was detectable in both β-cell fractions and levels of expression in the replicating β-cell fraction were doubled compared to the quiescent β-cell fraction
a marker for the epithelial to mesenchymal transition (EMT) was undetectable
Genetic programme of replicating β-cells
Selection according to p-value < 0.001 and absolute linear fold change > 2
Q: quiescent β-cells; R: replicating β-cells
(B) EnrichR analysis of most changing genes
Relevant and significantly enriched gene sets from Biocarta
KEGG and Reactome data bases are represented in the bar-plot
(C) Gene Set Enrichment Analysis showing selected Rat Gene Ontology data sets enriched in the replicating β-cells compared to the quiescent β-cells
(D) Quantitative RT-PCR determination of mRNA levels of selected genes from the top regulated transcripts unveiled in the gene array
The genes most significantly upregulated in replicating β-cells are mainly involved in the control of G2-M phases of the cell-cycle (Table 2 and Fig. 4D)
the Slc16a1 gene that encodes for the Monocarboxilate transporter 1
which conveys pyruvate and lactate through the plasma membrane and its expression is negligible in mature β-cells
was significantly upregulated in replicating β-cells
This study reports a method for the purification of replicating and quiescent pancreatic β-cells for downstream gene expression analysis
This technique is based on the incorporation of EdU to the replicating DNA strands during the S phase of the cell cycle
and makes it compatible with diverse experimental designs and animal species
In the present study we have determined the effects of long-term EdU exposure on β-cell replication and survival
We show that β-cell replication and viability are unaffected in cultured islets exposed to 10 μM of EdU for 7 days; whereas longer exposure has a negative impact on β-cell survival
which compromises RNA isolation from fixed cells
the inclusion of a protein digestion step in the RNA extraction protocol
with the addition of Proteinase K in the cell lysis buffer
significantly improved the quality and yield of the RNA
granularity and NG labelling resulted in pure β-cell preparations
amylase and keratin 20 RNAs were undetectable in β-cell preparations
non β-cell pancreatic islet hormones (glucagon
somatostatin and pancreatic polypeptide) RNAs were detectable in some samples
indicating that contamination with endocrine non β-cells was not completely avoided
replicating β-cells could incur in inappropriate insulin release in response to glucose levels
Further work will reveal whether the genetic program of cycling β-cells is completely restored in daughter cells after mitosis
we present a method that allows for the isolation of replicating β-cells
even in situations where they are very scarce
We anticipate that it will be particularly useful for the study of β-cell replication of human islets
in which replicating β-cells are infrequent
we expect this method to have a broad applicability in the study of β-cell proliferation in the context of diabetes and β-cell regeneration
A correction to this article has been published and is linked from the HTML and PDF versions of this paper
Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans
Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating
Dynamics of beta-cell mass in the growing rat pancreas
Estimation with a simple mathematical model
Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy
reduced duct cell plasticity and impaired β-cell mass regeneration in middle-aged rats
A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation
A chemical method for fast and sensitive detection of DNA synthesis in vivo
Cell type specific applicability of 5-ethynyl-2′-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry
Adenoviral overproduction of interleukin-1 receptor antagonist increases beta cell replication and mass in syngeneically transplanted islets
Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe
Adenoviral overexpression of interleukin-1 receptor antagonist protein increases beta-cell replication in rat pancreatic islets
Gastrin induces ductal cell dedifferentiation and beta-cell neogenesis after 90% pancreatectomy
Controlling the false discovery rate: a practical and powerful approach to multiple testing
Journal of the Royal Statistical Society Series B 1
Orchestrating high-throughput genomic analysis with Bioconductor
limma powers differential expression analyses for RNA-sequencing and microarray studies
Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool
Mouse beta cell proliferation is inhibited by thymidine analogue labelling
Autofluorescence-activated cell sorting of pancreatic islet cells: purification of insulin-containing B-cells according to glucose-induced changes in cellular redox state
B and D cells and for the isolation of coupled cells from isolated rat islets
Preparation of rat islet B-cell-enriched fractions by light-scatter flow cytometry
Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas
Proliferation of sorted human and rat beta cells
Gene expression analysis of in vivo fluorescent cells
and MCM proteins: Markers of proliferation in the diagnosis of breast cancer
Combined modulation of polycomb and trithorax genes rejuvenates β cell replication
Bmi-1 regulates the Ink4a/Arf locus to control pancreatic beta-cell proliferation
Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin
The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal
The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth
Long live FOXO: unraveling the role of FOXO proteins in aging and longevity
Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation
Insulin demand regulates β cell number via the unfolded protein response
Insulin Signaling Regulates the FoxM1/PLK1/CENP-A Pathway to Promote Adaptive Pancreatic β Cell Proliferation
Activation of FoxM1 Revitalizes the Replicative Potential of Aged β-Cells in Male Mice and Enhances Insulin Secretion
The FoxM1 transcription factor is required to maintain pancreatic beta-cell mass
Overexpression of monocarboxylate transporter-1 (SLC16A1) in mouse pancreatic β-cells leads to relative hyperinsulinism during exercise
β-cell-specific gene repression: a mechanism to protect against inappropriate or maladjusted insulin secretion
Download references
This work was supported by grants from the Catalan Diabetes Association (NT)
Carlos III Health Institute (ISCIII) PI17/00108 co-funded by FEDER funds/European Regional Development Fund (ERDF)-“a Way to Build Europe” (EM) and by CIBERDEM which is a project of ISCIII
The authors thank the CRG Genomics and Bioinformatics facilities
Maria de Lluc Canyelles and Claudia Fernández contributed equally to this work
The Barcelona Institute of Science and Technology and UPF
obtained and analysed the data and contributed to discussion
conceived the method design and wrote the manuscript
All authors reviewed the final version of the manuscript
The authors declare that they have no competing interests
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DOI: https://doi.org/10.1038/s41598-017-17776-2
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Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients
The clinical and functional significance of CTCs is still under investigation
Protein profiling of CTCs would complement the recent advances in enumeration
transcriptomic and genomic characterization of these rare cells and help define their characteristics
Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients
The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs
a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines
a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs
Targeted single-CTC proteomics with the capacity for archivable
multiplexed protein analysis offers a unique
complementary taxonomy for understanding CTC biology and ascertaining clinical impact
Direct measurement of multiple proteins in single-CTCs comprises a critical complement to single-CTC transcriptomic and genomic studies
the current format of the single-cell western blot requires 1000s of cells to account for cell losses when settling into the microwells
single-cell resolution western blot (scWB) to measure a panel of proteins in single CTCs isolated from patients with primary estrogen receptor-positive (ER+) breast cancer
The rare-cell scWB quantifies multiple surface and intracellular signalling proteins—in each individual CTC—allowing estimates of biological protein expression variation among CTCs
as compared with a quantitative threshold for technical variation that we establish
We show that the rare-cell scWB is compatible with established CTC isolation tools
thus successfully analysing CTC populations with as few as two starting cells
In a pilot study of ER+ metastatic breast cancer patient-derived CTCs
we observe a lysis–hardy CTC phenotype and the unique capacity to normalize target protein expression by the number of CTCs analysed per assay
The rare-cell scWB offers a new approach to examining CTCs
with relevance spanning from understanding CTC biology to monitoring an individual’s response to therapy
Putative CTCs are visually identified using Hoescht 33342 nuclear stain
and each identified CTC is micropipetted (under microscopy) into a 50 μm diameter microwell (micrographs in inset)
Step iii: after the seating of one CTC into one microwell
single-cell western blotting proceeds as in-microwell chemical CTC lysis
covalent immobilization of proteins to the gel (photo-blotting) and in-gel immunoprobing
Step iv: single-CTC lysate is analysed by western blotting and rounds of immunoprobing support the multiplexing of 12 proteins
with expression is compared among patient-derived CTCs and to spiked cell line validation studies
25 μm (for the cell micrographs) and 250 μm (the separation micrographs)
After spiking the cancer cells into healthy blood
the samples were purified in the same manner as patient-derived CTCs
(a) Fluorescence micrographs and intensity plots from rare-cell scWB handling and analysis of healthy blood samples
each spiked with a cancer subtype: EGFR+(BT-20)
Negative controls include analysis of WBCs only and blank microwells (that is
Protein panel comprises the following: control and housekeeping proteins (GAPDH and β-tubulin)
(b) Comparative protein expression for each cancer cell (BT-20: n=27; SK-BR-3: n=27; MCF7: n=35)
Protein expression is graphed using a log-scale
Ranked oncoprotein expression for each cell line agrees with cancer subtype
Less than 1% of total protein signal is attributable to CD45
rare-cell scWB analyses of pure WBC populations reported only CD45 and GAPDH signal
with no measurable signal from any cancer-specific protein target
thus establishing co-expression of CD45 and GAPDH
and negative cancer marker response as selective for WBCs
When microwells were empty in the spiked cancer cell studies (‘blank’ wells)
the rare-cell scWB did not detect CD45 or GAPDH
In assessing potential ‘cross-talk’ between proximal microwells
we did not observe detectable protein in empty microwells proximal to cancer marker-positive cell-laden microwells
with the exception of low-level EpCAM background
the validation study suggests that (i) the cancer markers are selective for putative cancer cells (as expected from the literature) and (ii) the protein panel allows the rare-cell scWB to distinguish between cancer cells and WBCs
As is relevant to emerging clinical indicators of prognosis
the observation corroborates the utility of the rare-cell scWB to capture both single CTCs and perhaps also CTCs associated with other cells (although association mechanisms are not currently understood)
The primary molecular classification marker exhibited higher variability than other classification markers in two of the cancer cell lines (HER2: σSK-BR-32=2.08 × 1012
EpCAM expression in MCF7 and BT-20 cells had a wider distribution than that observed in the SK-BR-3 cell lines (σMCF72=3.00 × 1011; σSK-BR-32=1.80 × 1010; σBT-202=2.72 × 1011)
For surface proteins known to be minimally or not expressed in each cancer subtype
we observed minimal variability in expression among each population (for example
numerous and even unexpected relationships may be identified by deeper profiling
such as that provided by 8- to 12-plex scWB analysis of cancer cells
Micrographs of rare-cell scWB of patient-derived CTCs in representative cases where CD45 was not detected (CD45−)
We extended the lysis duration and elevated the lysis buffer temperature (that is
Na-DOC at 0.5% and lysis buffer duration of 20–25 s at 60–65 °C)
The increased temperature aids in reducing the CMC
we observed successful lysis and separations of CTCs from Patients 5
The number of CTCs counted after immunostaining did not match to the number of CTCs analysed by rare-cell scWB
as each measurement was performed on a unique blood fraction with no guarantee of a matched number of CTCs in each
The rare-cell scWB was suitable for protein analysis of CTCs from patients with both low (Patient 6) and high (Patient 10) CTC counts
the stringent lysis conditions were applied rare-cell scWB analyses of patient-derived CTCs from Patients 5–11
(a) Expression for each protein marker and each patient-derived CTC, with comparison with CD45 levels from scWB analyses of pure WBC controls. (b) CVs for protein expression (AUC) from the patient-derived CTCs. Dashed line indicates the threshold in protein expression variation established using GFP-expressing MCF7 cells (see Supplementary Fig. 3)
(c) Biaxial plots report protein expression for all markers for each patient-derived CTC from Patients 5 (n=6)
Protein target expression was normalized by microwell occupancy, which was one CTC per microwell with the rare-cell handling workflow described. We attribute the observed CTC-to-CTC variation in protein expression levels to biological differences, as all protein panel targets had variability that exceeded the technical variation cutoff (Fig. 4b)
we observed a wider distribution among the CTCs
as compared with the ER+ MCF7 cells (σCTC2=4.33 × 1012
This CTC EpCAM expression heterogeneity underscores the challenge for immunocapture-based enrichment
the patient-derived CTCs exhibited a larger CV than the MCF7 cells
suggesting that the patient-derived CTCs have higher variation in GAPDH (CVCTC=123%
in ER protein expression the patient-derived CTCs exhibited a narrower distribution than the MCF7 cells (σCTC2=8.20 × 109
these findings point to potential limitations of using cancer cell lines as models for patient-derived CTCs
To further contextualize CTC-to-CTC variation in protein expression, we compared each patient-derived CTC with all other patient-derived CTCs for Patients 5, 6 and 10 (Fig. 4c)
In contrast to the cell line spiking experiments
with patient-derived CTCs we observed low variability in GAPDH expression between Patients 5
we observed high CVs for panCK (CVP5=110% CVP10=71%
In accordance with our breast cancer cell line observations
the ER+ breast cancer patient-derived CTCs also exhibited correlation between ERK and GAPDH (rCTCs=0.579
P<0.01; n=20) and between the pairs ERK with β-tubulin (rCTCs=0.691
P<0.01; n=20) and EpCAM with β-tubulin (rCTCs=0.6
Among the patient-derived CTCs, we detected no statistically significant outlier CTCs (F-test)—with regards to exceptionally high or low protein expression—when target expression was normalized to the number of CTCs analysed in each scWB (that is, one CTC analysed per microwell). However, the GAPDH expression suggests two distinct CTC sub-populations (Fig. 4c)
which was confirmed with a F-test comparing a one-population model and a two-subpopulation model for the data resulting in an F-statistic of 3.89 and a P-value of 0.035 (degrees of freedom: (1,4))
thus suggesting two possible populations of GAPDH expression
The scWB leverages short distances and timescales to rapidly complete lysis
PAGE and protein blotting of proteins in single CTCs
The careful control of sample and analysis affords the unique option to forgo normalization by protein housekeeping proteins and directly assign protein levels on a per CTC basis
Important to longitudinal studies, the scWB is archivable30
which allows later-date profiling of new targets of interest with previously analysed single-CTC lysates
CTC lysates are covalently immobilized to the PA gel layer on the scWB device
a stable linkage compatible with long-term storage for retrospective CTC analysis
Although chemical fixation of CTCs also yields biospecimens suitable for long-term storage and retrospective studies
cell fixation is incompatible with stripping procedures and suffers from several pre-analytical variables including the following: epitope disfigurement during fixation especially changes in posttranslational modifications
loss of antigenicity owing to over- and under-fixation
analytical variables including target cross-reactivity with moderate specificity antibodies
lab-to-lab variation and qualitative but not quantitative analyses
Although the rare-cell scWB requires further long-term storage performance characterization and larger-scale patient studies
the targeted proteomics tool introduced here presents a promising approach to multiplexed
archival protein analysis of single CTCs with direct relevance to longitudinal studies
the microfluidic form factor of the assay may find utility in low-resource settings
especially with further engineering integration of the cell purification and handling fluidics
The cell line and CTC experiments used an 8%T PA gel
with arrays of 50 μm diameter and 60 μm-deep microwells
All PA gels were chemically polymerized with 0.08% APS and 0.08% TEMED
A separate SU-8 master was created to fabricate the PDMS mesofluidic insert used to localize large volumes (∼700 μl) of enriched cells over the microwell array in the PA gel layer
The insert dimensions were 37.5 mm × 50 mm with an opening comprising the volume reservoir of 20 mm × 30 mm
PDMS polymer base and curing agent were mixed (ratio 10:1)
poured over the insert master and cured for 2 h at 70 °C
we acquired healthy donor blood and spiked with cell lines representing three major breast cancer subtypes: triple-negative (BT-20)
MCF7 and SK-BR-3 cells were obtained from the American Type Culture Collection and authenticated using short tandem repeat analysis (Promega)
All cell lines tested negative for mycoplasma
BT-20 (ER−/PR−/HER2−) was maintained in Eagle’s minimal essential medium supplemented with 1% penicillin/streptomycin and 10% FBS
MCF7 (ER+/PR−/HER2−) was maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin
0.01 mg ml−1 insulin (Invitrogen) and 10% FBS
SK-BR-3 (ER−/PR−/HER2+) was maintained in McCoy’s 5A supplemented with 1% penicillin/streptomycin and 10% FBS
was obtained from the American Type Culture Collection and authenticated using short tandem repeat analysis (Promega)
The cell line was maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin
All cell lines were cultured in an incubator held at 37 °C under 5% CO2 and tested for mycoplasma contamination
Twelve patients with advanced breast cancer were recruited
according to a protocol approved by the Institutional Review Board (Stanford IRB 350–Panel 3–Protocol 5630) from the Department of Oncology at the Stanford School of Medicine
stored at room temperature and processed within 5 h after collection
The microfluidic device was first primed with PBS
the diluted blood sample was processed through the Vortex HT chip (8 ml min−1) followed by a wash step with PBS to remove contaminating red blood cells and WBCs (8 ml min−1)
Stopping the flow dissipates the vortices and releases the cancer cells from the microscale reservoirs for direct deposition on the top surface of the scWB platform
The enriched volume was ∼300 μl and was contained by a mesofluidic PDMS insert that sits atop the scWB
300–600 cells from 1 cell line were spiked into 1 ml healthy donor blood and processed using the Vortex chip
the cells isolated in the vortices were directly collected into the mesofluidic PDMS insert seated on top of the scWB PA gel for cell positioning into microwells
For both cell line spiking and patient-derived cell experiments
a volume of blood was reserved for subsequent red blood cell lysis to perform control experiments with WBCs
WBCs were prepared by lysing the red blood cells with Buffer EL (Qiagen)
0.5 ml of whole blood was combined with 2.5 ml of Buffer EL (Qiagen)
The tube was inverted several times and incubated for 10–15 min at room temperature
After centrifugation at 228 g for 5 min at room temperature
The pellet was re-suspended with 2.5 ml of Buffer EL and the process repeated
the WBCs were washed once with 1 ml of Buffer EL
The scWB assay can be completed within ∼20 h
Primary antibodies and fold dilutions against GAPDH (1:20
goat polyclonal antibody (pAb); SAB2500450
Sigma) were the immunoprobes in both breast cancer cell lines (BT-20
MCF7 and SK-BR-3) and patient-derived CTCs
Abcam) followed by anti-goat AlexaFluor 555-conjugated secondary antibody (A21432
Secondary antibodies to goat IgG pre-labelled with AlexaFluor 488 and 555 (A11055 and A21432)
mouse IgG pre-labelled with AlexaFluor 488
and rabbit IgG pre-labelled with AlexaFluor 488
A31572 and A31573) were used as prepared by the vendor (Invitrogen)
All secondary antibodies were applied as a 1:20 dilution
Flow cytometry analysis was performed on MCF7 cells to ascertain the effects of enzymatic detachment on the EpCAM antigen
MCF7 cells were detached from tissue culture plates either by trypsin-EDTA (0.25%
Gibco 25200072) or by EDTA alone (Ultrapure 0.5 M EDTA Gibco 15575020 diluted in PBS to 5 mM)
Half the cells were labelled with anti-EpCAM–AlexaFluor488 (mouse
eBioscience) and half were labelled with mouse IgG AlexaFluor488 (A21202) as an isotype control
2 × 106 cells were resuspended in 100 μl of 3% BSA (Sigma A2058) in PBS containing antibody at a concentration of 1 μg ml−1 and incubated over ice for 30 min
then resuspended in resuspension buffer (1% BSA
Cells were analysed on a Guava flow cytometer (Millipore)
A total of 10,000 events were collected per sample
four samples per experimental group (n=4) and data were compiled and analysed using FlowJo software
SK-BR-3 (n=34 and n=30) and MCF7 (n=42 and 40)
The null hypothesis that the GAPDH protein expression distributions are equivalent across the technical replicates was supported
Cells with similar (<5% variation) GFP AUC were binned and considered a homogeneous GFP-expressing sample
with a 1.27–3.37% difference in AUC from the lowest and highest GFP AUC of each bin observed
The technical variation cutoff was defined as 3 s.d
above the average CV of protein expression (for a 99.7% confidence interval)
Quantification of protein PAGE and probing used in-house MATLAB scripts as described in Kang et al.29 Band widths were characterized by Gaussian curve fitting in MATLAB (R2014b
Curve Fitting Toolbox) if the Gaussian had a R2-value>0.7
the integrated intensity for the region of interest was calculated
Multiple statistical analyses were performed to compare protein distribution
To determine significance between the different protein CVs observed
we performed a t-test statistic and used a permutation test to determine the P-values
The Levene’s test was used to determine non-equivalence of the variance between the markers for each cell line (BT-20
To classify a group of CTCs as a sub-population based on GAPDH expression in Patient 5
an F-test for model selection was performed
Model 1 assumed one population exists and model 2 assumed two sub-populations exist
The F-test compares the two models with the null hypothesis
considering the data follows model 1 instead of model 2
To detect correlation in protein expression between proteins, a Spearman’s rank correlation was performed, as the correlation of protein expression between two proteins was expected to be monotonic but not necessarily linear. Two proteins in the panel were sequentially paired (Supplementary Table 3) to determine possible correlations
Only correlations with a P-value ⩽0.01 were considered significant
The authors declare that all the data are available within the article file and its Supplementary Information or from the corresponding author upon reasonable request
Profiling protein expression in circulating tumour cells using microfluidic western blotting
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Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device
Isolation of rare circulating tumour cells in cancer patients by microchip technology
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Changes in circulating tumor cell detection in patients with localized breast cancer before and after surgery
mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients
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and mutation persistence in cultured disseminated tumor cells from bone marrow
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Global analysis of protein expression in yeast
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and genetic mutations from rare circulating tumor cells
EPISPOT assay: detection of viable DTCs/CTCs in solid tumor patients
Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits
Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents
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Truncated p110 ERBB2 induces mammary epithelial cell migration
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EpCAM: Structure and function in health and disease
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Microfluidic single-cell whole-transcriptome sequencing
Heterogeneity of estrogen receptor expression in circulating tumor cells from metastatic breast cancer patients
Rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells
KI-67 and AR by immunohistochemical analysis in breast cancer cell lines
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Choosing the right cell line for breast cancer research
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Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers
Tumor metastasis: mechanistic insights and clinical challenges
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We thank the breast cancer patients and healthy donors who generously consented to provide the blood samples necessary for this study
M.D.) who assisted with patient recruitment
The research reported in this publication was supported in part by the US National Institutes of Health and the National Cancer Institute (R21CA183679 to A.E.H.)
the National Institute of Biomedical Imaging and Bioengineering (R21EB019880 to A.E.H.)
and the Office of the Director's New Innovator program (DP2OD007294 to A.E.H.) with a Diversity Supplement (to E.S.)
a CIRM Predoctoral Fellowship and Obra Social `la Caixa' Fellowship (to E.R.C.) and a National Science Foundation Graduate Research Fellowship (to T.A.D.
UC Berkeley Biomolecular Nanotechnology Center (BNC) provided support for device fabrication
The UC Berkeley–UCSF Graduate Program in Bioengineering
performed single-CTC western blottings and analysis
performed fluorescence-activated cell sorting-based validation experiments
purified protein control experiments and experiments for scWB benchmarking against fluorescence imaging
coordinated patient recruitment and healthy blood samples collection
designed software for fluorescence quantitation
has financial interest in intellectual property related to the device and assay described here
and may benefit from royalties from licensing
has financial interest in commercialization efforts
have financial interests in Vortex Biosciences
All other authors declare no competing financial interest
Supplementary Figures and Supplementary Tables (PDF 925 kb)
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MusicInnovation& Creativity12.13.14June 2025BarcelonaProgrammeTicketsSónar+DPractical infoPlaylistsPartnersBack25/09/2023You can now watch the Piromusical at La Mercè 2023
plus explore the full playlist On the evening of Monday 25th of September
Sónar closed its 30th anniversary celebrations by providing the soundtrack for the closing ceremony of La Mercè in Barcelona
drawing the curtain on the city's biggest annual celebration
The creative team at Pirotècnia Igual (Canyelles) studied the soundtrack provided by Sónar for over a month in order to decide which effects to combine with each moment
depending on the rhythm and intensity of the music
The team then spent over 20 days developing the spectacle
which involved over 1,300kg of pyrotechnic material spread across 700 installations
over 15,000 discharges were fired during the show
accurate to within a hundredth of a second
the opening notes of Ryuichi Sakamoto's ‘Chasm’ rang out
a piece chosen especially by Sónar to pay tribute to the legendary Japanese composer
The show lasted 30 minutes and featured five segments of music mixed and mastered by Phran
a DJ and producer who’s a regular performer at Sónar
The soundtrack took the audience on a global journey through sounds
and rhythms from all five continents: a cross-cultural journey and a giant celebration
featuring classic tracks from some of the most legendary names in modern music
From Rosalía and Bad Gyal to Daft Punk and Pet Shop Boys
And from Björk and Aphex Twin to Miss Kittin and Jeff Mills
Just some of the artists from the history of the festival that were shared with over 100,000 people who attended the show
plus the 1 million spectators who followed it live on TV3
Listen to all 52 tracks in their entirety in the playlist for the Piromusical de La Mercè 2023
ya Now available via the Sónar Spotify account
Stray beyond Barcelona and you’ll find that Catalonia has some delightful spots for the adventurous boater hoping to make the most out of the Med
Tucked beneath a wooded hill and an old castle
Blanes (pictured below) is a pleasant fishing and yacht harbour
a welcome change after cruising up the mostly flat coastline from Barcelona
The town has good beaches and an intriguing ruined palace
The cliffs start rising immediately beyond Blanes and a procession of headlands stretches away to the distant corner of Catalonia
which make the Costa Brava so enticing from the sea
Many smaller calas can only be reached by boat and you anchor in such piercingly clear water that plunging overboard is irresistible
Three miles from Blanes you skirt the packed resort of Lloret de Mar before the coast turns wild again past a string of inlets
Cala Gran is delightful and then you reach the tiny club marina at Canyelles
Tucked under a wooded cliff near a dazzling beach
Cala Canyelles has a narrow gap into a shallow pool where locals moor stern-to the jetties
visiting boats up to about 35ft can squeeze into the first section behind the breakwater
but in settled weather you can anchor off the beach
Beyond Point Garbí is an impressive sweeping bay where a long mole protects the relaxed
pleasure-seeking marina at Sant Feliu de Guíxols
the winding village-style marina of Port d’Aro is set between a wooded park and a halcyon beach
Port d’Aro is a safe place to winter and has good facilities for getting work done
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General view of the exit of Barcelona on the Avinguda Meridiana
with the neighbourhood of Vallbona to the right of the bridge
and Ciutat Meridiana and Torre Baró to the left. © Myriam Meloni i Arnau Bach
a photographic project by Myriam Meloni and Arnau Bach
we see the Barcelona behind the ring roads
The Barcelona of the Rec Comtal irrigation canal and the old Ribes road
The Barcelona that every train commuter relates to the image
More than drawing closer to the farthest Barcelona
the photographs welcome us to the city’s first neighbourhoods
Myriam Meloni and Arnau Bach offer a profound contemporary photographic portrait – verging on the ethnographic – of the four border neighbourhoods in the far north of the city: “The primary interest is to photographically portray the limits of Barcelona
and Nou Barris is the district furthest from the centre”
the four neighbourhoods that mark the city limits and lend them a name are Canyelles
if you get sidetracked while walking through the thick forests or up the really steep slopes
which entailed not only photographing the main characters in the great little stories told in Linde
Co-author Myriam Meloni explains the approach she adopted when exploring the neighbourhoods and their people: “First we went to the residents’ associations
because they have the most connection with the social fabric of each neighbourhood
but from there we try not to be bound to be spokespeople for their demands
which would undoubtedly provide us with a more complete and
Esperanza went from living in a shack in the neighbourhood of El Carmel to living in a flat in Canyelles
The longstanding president of the Ciutat Meridiana Residents’ Association
these neighbourhoods have been home to people from very different backgrounds and with fairly low economic means
are the three neighbourhoods with the lowest average family income in the city
Vallbona and Torre Baró do not amass even 5,000 residents
The latter neighbourhood also has a population density of 28,000 people per square kilometre
although far from the population density of the neighbourhood La Florida
which ranks the highest in Europe with more than 74,000 inhabitants per square kilometre
The houses are also different depending on the neighbourhood
In Torre Baró and Vallbona many self-built
In Canyelles and Ciutat Meridiana there is a profusion of warren-type
many of which lack decent living conditions
Past (and present) of neighbourhood struggle
Filiberto Bravo is an institution in Ciutat Meridiana
Longstanding president of its residents’ association
his legacy of neighbourhood struggle is an encyclopaedia open to anyone who talks to him: “Filiberto’s vision of the neighbourhood is one of togetherness and social struggle
but the possible vision of Ciutat Meridiana held by a woman who has recently migrated from a small town in Morocco is a sense of freedom”
having a close-knit community that cares for one another but at the same time does not interfere in each other’s lives is critical for personal development in freedom”
Bellvitge on the northern border of Barcelona
as one of the testimonials presented in Linde notes
it is also class dignity: “When they proposed we come to live in Canyelles
[…] At night I could only think of the flat – Esperanza lived with her family in a shack in El Carmel – of how it was made
but for me it was a palace… And it still is.”
Although denouncing social injustice has helped keep the feeling of attachment alive among its residents after so many generations
lapsing into showing Ciutat Meridiana just as Evictionville
and the x of the equation was already resolved by Candel in Els altres Catalans [The Other Catalans]: “In reality
It is the sad fate of their status as a low proletariat.” In Linde
the exploration of reality of four of Barcelona’s poorest neighbourhoods seeks to express itself through beauty and eschewing drama
it moves away from the constant historicist examinations that are made of the now thirteen “nou barris” [new neighbourhoods]
the realities of the four neighbourhoods portrayed in the book are woven together in a narratively richer whole
“but in Linde it was not our intention to speak of the other objectively
but to afford a glimpse within a much more complex reality”
When a journalist has to cover a story about precariousness
they should always be attuned to the signal that warns of the danger of yielding to the romanticisation of poverty
Stories of precariousness are passed on orally by the person concerned; the journalist listens to them
internalises them and contextualises them (or
they should); then they portray that story
they publish it for the scrutiny and judgment of public opinion
neither with the publication nor with the payment received by the journalist
but continues to be the unique and current reality of that person and their environment
The great balancing act entails telling the story in a manner that is mindful of otherness
and that is only achieved over time and through an honest relationship with the sources
among other things: “The main idea was to decipher and understand the day-to-day life of these neighbourhoods
which end up being the fringes of the fringes when they are not and should not be considered as such
Beyond the economic reality and the historical institutional neglect of these neighbourhoods
there is another factor that explains how the distinctive characteristics of their residents are structured and how the feeling of attachment has been ignited here: all four are recent neighbourhoods
built in many cases by the actual residents who still reside in them
who inherited the homes that their parents built
four walls and a roof were raised at night so that
the wardens couldn’t demolish the construction
It is not the only example of neighbourhoods built home by home by their own residents: Huertas Clavería tells us in his Barrios de Barcelona [Barcelona’s Neighbourhoods] how the residents provided some rudimentary steps to spare the residents the slopes in Torre Baró
Another example: during the hijacking of bus line 47 in May 1978 (which served to demonstrate to the city council that the bus could climb the slopes of these neighbourhoods)
too steep for the Pegaso Monotral steered by the bus driver Manuel Vital
people continue to make small improvements in these neighbourhoods
without asking for permission or pardon: “Here
the feeling of attachment is very different from that of a neighbourhood that was built centuries ago
by people already forgotten and whose history has been lost”
Many were built by their owners in the mid-20th century
Until the Congost bridge was built in 2005
Only a goat path connected it with the adjacent neighbourhoods of Torre Baró and Ciutat Meridiana
someone prepares what is needed and in a couple of days children are already playing soccer
young people will adapt it to rehearse urban music choreographies: “The legislation is the same as in the rest of the city
Nor do they need to direct thousands of tourists each day.” Myriam Meloni rounds off the reflection: “There
every piece of public space has been allocated a use
It ends up being a matter of economic interest
each piece of city has a huge potential for economic revenue; but not here”
The centralist and productive conception of here and there is blurred in these neighbourhoods
Candel was referring to this type of peripheral neighbourhoods with his famous Donde la ciudad cambia su nombre [Where the City Changes Its Name]
and while it is true that the residents (including the photographer who has spent a bit more than a year in these neighbourhoods
as read in the previous paragraph) say “go to Barcelona” when they are already in it (something that also happens in the neighbourhoods that centuries ago were independent towns)
the historical aspiration of the residents of Canyelles
Vallbona and Ciutat Meridiana is to overcome this otherness and be treated just like the rest of Barcelona
Giving new meaning to sometimes monstrous spaces
The relationship with nature is much closer at these latitudes of the city
the last vestiges of the Rec Comtal irrigation canal are still used by young people to go for a dip
and the last vegetable gardens in the city also lie in this area
In Linde we become acquainted with the story of Rosita
a wild boar that roams the area: “When I call her”
“she always comes to me and we spend time together
Blas works the land and has raised animals
the landscape changes in a few hundred metres
From the mountain surroundings we enter the hub of roads (C-17
the city’s quintessential non-place that has been reappropriated by its residents
Ciutat Meridiana and Vallbona they have learned to live next to the mountains
they have still had more to learn to live hemmed-in between piles of concrete
The entry road links to Barcelona to the north have marked the life of these neighbourhoods
separating them from the rest of the city and from each other
motorway entries and exits and columns of implausible diameters to hold it all together ooze through the hovels
the little vegetable gardens and the waste grounds of Barcelona’s northernmost neighbourhoods
The photographs of these landscapes featured in Linde convey that overwhelming sense of insignificance of human measure in the presence of such architectural abominations
Two members of the dance troupe La Virgen del Quincho
named in honour of a very popular virgin in Ecuador
who rehearse every Sunday on wasteland in Ciutat Meridiana
The Ciutat Meridiana sloping lift connects to Torre Baró suburban train station and Ciutat Meridiana's line 11 metro station. © Myriam Meloni i Arnau Bach
Many of the residents who use these non-places to meet up come from realities in which many people had to live in miniscule places
such that all social life took place on the street
many of the flats in Canyelles and Ciutat Meridiana are tiny and are still inhabited by lots of people
so to a certain extent they are not only reproducing customs
but also they are still adapting to the circumstances
“This also occurs in the Raval neighbourhood
Occupation of the public space is a very cultural thing
It is a reappropriation of space without too much press
The residents of these neighbourhoods have given a new meaning to the non-place
meeting up with their families and friends
In short: “The space outside homes is given new meaning
turning it into something to be shared by the community”
Under tons of cement and countless cars that travel in and out and cross the city limits in a frenzy of movement
at ground level and availing of the shade under the bridges
And they are dressed in regional costumes that they have been sewing for weeks
from being there so long and having made the place their own
they have also internalised the constant and piercing noise of rolling tyres and rattling engines
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spicy sobrasada sausage spread generously on to sweet
toast and marmalade will always seem disappointing
It’s just one of Mallorca’s many delicacies and it’s served at a new five-star hotel that has opened in Santanyi in the southeast of the island."},"children":[]}]},{"name":"paragraph","children":[{"name":"text","attributes":{"value":"Thanks to its peaceful lifestyle and picturesque surroundings
Santanyi has been attracting increasing international interest
And the arrival of Can Ferrereta means that the town is now a serious player in the luxury holiday market."},"children":[]}]},{"name":"paragraph","children":[{"name":"text","attributes":{"value":"The hotel
which is full of intriguing historical touches
is on the site of a Mallorcan stately home thought to have been built in the 17th and 18th centuries
An ornate stone pathway leads you through the entrance; it was"},"children":[]},{"name":"paywall","children":[{"name":"text","attributes":{"value":" originally a passage for horses and other animals
past an iron sculpture by the Catalan artist Josep Riera i Aragó that resembles an aeroplane propeller."},"children":[]}]}]},{"name":"paywall","children":[{"name":"image","attributes":{"id":"6318cbe6-3233-4013-8e2d-310631760292","display":"primary","caption":"The hotel’s Ocre restaurant","title":"Can Ferrereta
Mallorca","credits":"ARTURO+LAUREN","url":"https://www.thetimes.com/imageserver/image/%2Fmethode%2Ftimes%2Fprod%2Fweb%2Fbin%2F112ea6dc-c39b-11eb-a26e-4c086490cfe1.jpg?crop=5804%2C3869%2C0%2C0","ratio":"1500:1000","relativeHorizontalOffset":0,"relativeVerticalOffset":0,"relativeWidth":0.9649210307564422,"relativeHeight":1},"children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Art is a theme: the hotel is bursting with works
including a quarter-tonne bronze sculpture by Jaume Plensa next to the hotel’s 25m outdoor swimming pool
and there’s even an original Miró in my bedroom."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"The grandeur of the building is complemented by friendly
welcoming touches; the idea was to create the feeling of staying with (rather extravagant) relatives
tells me."}}]},{"name":"ad","children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Can Ferrereta
forms part of an independent portfolio (which includes Sant Francesc
a hotel in a 19th-century neoclassical mansion in Palma) run by their eldest son
I speak to Soldevila Jr in the hotel’s cosy library
which has shelves stuffed with books on style
wine and travel."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Can Ferrereta
The plot had remained in the same family for centuries until the Soldevila Ferrers came across it in 2018
a big restoration job was needed and Bastidas Architecture
was given the task of preserving the building’s characterful features
such as the stone arches and wooden beams."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Even the new parts of the hotel
such as the annexe that houses 12 private suites
replicate the style of the original manor; the spa imitates the structure and layout of a Mallorcan barn typical of the agriculture of the region
while the pool’s showers are in a recreated"}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":" barraca de roter"}}]},{"name":"text","children":[],"attributes":{"value":"
a traditional stone hut for shepherds."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"According to Gerard Bastidas
shade is just as important as light in Mallorcan buildings
which are designed to protect you from the sun
Light and shadow influenced the interior design as well
“The property’s natural light has very romantic moments but it is also limited in some spaces,” explains Carla Navas from the Barcelona-based interior design studio Wit
“We wanted to keep that chiaroscuro feeling when you walk around.”"}}]},{"name":"inlineAd1","children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"They have succeeded
are known for the ochre colour of the buildings
The colour lends its name to Can Ferrereta’s restaurant
where the chef Alvar Albaladejo serves dishes incorporating traditional Balearic produce such as tap de corti paprika
mahones cheese and purple safarnaria carrots."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Wit has furnished the interior with items by international and home-grown brands
from Mogg and Carl Hansen to Blasco and Lo de Manuela
Woven rope contrasts with soft linen upholstery
while rustic wooden tables and seats bring a warm touch against the sandstone
sourced from the local quarry."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Leaving to explore the town
a remnant of the city walls built in the 16th century to defend against Ottoman and Moorish invaders who coveted this town and its gateway position to the island
Santanyi’s location is no less desirable today
although for rather different reasons — it’s 45 minutes by car from Palma airport and a short drive from beaches such as Cala Santanyi
Germans have been coming to Santanyi for years and not just on holiday
drawn by the laid-back Mallorcan way of life
arrived here in the mid-1990s and has been running his restaurant
serving seasonal Mediterranean dishes such as"}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":" llengua amb taperes "}}]},{"name":"text","children":[],"attributes":{"value":"(beef tongue with capers) and "}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":"frit mallorqui "}}]},{"name":"text","children":[],"attributes":{"value":"(the island’s traditional stew
made with pork or lamb)."}}]},{"name":"inlineAd2","children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Another reputed restaurateur in town
was born in Germany and raised in Mallorca from the age of two
Five years ago he opened his stylish establishment
Laudat — a charming spot for lunch or dinner with a romantic patio filled with lemon trees."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Laudat works with the local winery Armero i Adrover to produce a range of bespoke house wines — I try the white
made with a combination of prensal blanc and giro ros grapes
which nicely complements my sea bass fillet with caramelised fennel
Judit and her team serve tapas and decadent deserts."}}]},{"name":"image","attributes":{"id":"d2ab9afd-d86f-4843-b351-a376fe1fa608","display":"fullwidth","caption":"The market at Santanyi","title":"market in Santanyi
Spain","credits":"ALAMY","url":"https://www.thetimes.com/imageserver/image/%2Fmethode%2Ftimes%2Fprod%2Fweb%2Fbin%2F65e8a42a-c39b-11eb-a26e-4c086490cfe1.jpg?crop=6000%2C4000%2C0%2C0","ratio":"1500:1000","relativeHorizontalOffset":0,"relativeVerticalOffset":0,"relativeWidth":1,"relativeHeight":1},"children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"Santanyi particularly comes to life on Wednesdays and Saturdays
when its street market attracts crowds from neighbouring regions to browse summery dresses
There are fresh fruit and vegetables in the Plaça Major
next to the 18th-century Sant Andreu church
preserves and olive oils."}}]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"The bakery Panaderia Lozano tempts with savoury "}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":"panades"}}]},{"name":"text","children":[],"attributes":{"value":" stuffed with meat
as well as traditional pastries and cakes such as "}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":"lazos"}}]},{"name":"text","children":[],"attributes":{"value":" (twists of pastry with cream or chocolate)
"}},{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":"xuxos"}}]},{"name":"text","children":[],"attributes":{"value":" (fried
Ramón Canyelles produces contemporary clay homeware and sells more traditional earthenware
Across town I get an early peek at Terra Origens
a new concept store and café spread across three floors
scheduled to open this summer as a place to eat and shop; local artisans will also run workshops on pottery and weaving."}}]},{"name":"inlineAd3","children":[]},{"name":"paragraph","children":[{"name":"text","children":[],"attributes":{"value":"At dusk an enchanting luminosity envelops Santanyi as the sun begins to set and everything glows
No wonder so many have been drawn here and so many have stayed
And I have the bonus of that sausage breakfast at Can Ferrereta to look forward to."}}]},{"name":"paragraph","children":[{"name":"italic","children":[{"name":"text","children":[],"attributes":{"value":"Agnish Ray was a guest of Can Ferrereta
B&B doubles from £272 ("}},{"name":"link","children":[{"name":"text","children":[],"attributes":{"value":"hotelcanferrereta.com"}}],"attributes":{"href":"https://www.hotelcanferrereta.com/en/"}},{"name":"text","children":[],"attributes":{"value":")
Fly to Palma"}}]}]},{"name":"heading3","children":[{"name":"text","children":[],"attributes":{"value":"Three new buzzing hotels in the Balearics"}}]},{"name":"image","attributes":{"id":"e3a45cf2-0a32-4323-9e86-1d027a7806d6","display":"primary","caption":"Casa Pacha
Formentera","title":"CASA PACHA","credits":null,"url":"https://www.thetimes.com/imageserver/image/%2Fmethode%2Ftimes%2Fprod%2Fweb%2Fbin%2Fb1264294-c39b-11eb-a26e-4c086490cfe1.jpg?crop=3000%2C2000%2C0%2C0","ratio":"1500:1000","relativeHorizontalOffset":0,"relativeVerticalOffset":0,"relativeWidth":1,"relativeHeight":1},"children":[]},{"name":"paragraph","children":[{"name":"bold","children":[{"name":"text","children":[],"attributes":{"value":"Casa Pacha
Formentera"}}]},{"name":"break","children":[]},{"name":"text","children":[],"attributes":{"value":"Laid-back is the word for the style of luxury taking over the Balearics this summer
which means that the nightlife and leisure group Pacha is embracing all things barefoot with this new addition in Formentera
so expect ravishing sea views from the rooms
The interiors have been decked out by the Ibiza-based designer Patricia Galdón
as well as a few 1960s touches evoking the vintage spirit of Pacha lifestyle
"}},{"name":"bold","children":[{"name":"break","children":[]},{"name":"text","children":[],"attributes":{"value":"Details "}}]},{"name":"text","children":[],"attributes":{"value":"B&B doubles from £414 ("}},{"name":"link","children":[{"name":"text","children":[],"attributes":{"value":"casapacha.com"}}],"attributes":{"href":"https://www.casapacha.com/"}},{"name":"text","children":[],"attributes":{"value":")
Fly to Ibiza"}}]},{"name":"image","attributes":{"id":"19bcf222-df7c-43db-a482-a9aeeaba08df","display":"primary","caption":"Cristine Bedfor
Menorca","credits":"DANIEL SCÝFER","url":"https://www.thetimes.com/imageserver/image/%2Fmethode%2Ftimes%2Fprod%2Fweb%2Fbin%2Fd1b2dd88-c39b-11eb-a26e-4c086490cfe1.jpg?crop=5906%2C3937%2C0%2C0","ratio":"1500:1000","relativeHorizontalOffset":0,"relativeVerticalOffset":0,"relativeWidth":1,"relativeHeight":0.8889139760668322},"children":[]},{"name":"paragraph","children":[{"name":"bold","children":[{"name":"text","children":[],"attributes":{"value":"Cristine Bedfor
Menorca"}}]},{"name":"break","children":[]},{"name":"text","children":[],"attributes":{"value":"The novice hoteliers Cristina Lozano and Daniel Entrecanales have just opened their debut property
a colourful 21-bedroom guesthouse in the heart of Menorca’s capital
A fictitious persona invented by the owners gives the hotel its name
You’re encouraged to think of her as the perfect hostess
in which you can enjoy candlelit dinners and a sparkling pool
The interiors are designed by Lorenzo Castillo
where this new 20-acre resort opens in July
from a yoga platform with sea views to CrossFit
cryotherapy and even snorkelling meditation
much of it grown on the hotel’s organic farm
Tuck into seasonal island specialities such as tomato entrecôte
prepared by Israel’s celebrity chef Eyal Shani before heading down to the Live Cave at the waterfront for cocktails
and music from visiting artists."}},{"name":"bold","children":[{"name":"break","children":[]},{"name":"text","children":[],"attributes":{"value":"Details "}}]},{"name":"text","children":[],"attributes":{"value":"B&B doubles from £933 ("}},{"name":"link","children":[{"name":"text","children":[],"attributes":{"value":"sixsenses.com"}}],"attributes":{"href":"https://www.sixsenses.com/en/resorts/ibiza"}},{"name":"text","children":[],"attributes":{"value":")
Fly to Ibiza"}}]},{"name":"inlineAd4","children":[]},{"name":"paragraph","children":[{"name":"bold","children":[{"name":"text","children":[],"attributes":{"value":"International travel restrictions are in place
expert advice and inspiration for your next trip"}}]}]}]}]},"summary({\"maxCharCount\":200})":{"type":"json","json":[{"name":"paragraph","children":[{"name":"text","attributes":{"value":"Once you’ve tried soft
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It’s just one of Mallorca’s many delicacies and it’s served at a new five-star hotel that has opened in Santanyi in the southeast of the island
Thanks to its peaceful lifestyle and picturesque surroundings
And the arrival of Can Ferrereta means that the town is now a serious player in the luxury holiday market
An ornate stone pathway leads you through the entrance; it was originally a passage for horses and other animals
past an iron sculpture by the Catalan artist Josep Riera i Aragó that resembles an aeroplane propeller
The hotel’s Ocre restaurantARTURO+LAURENArt is a theme: the hotel is bursting with works
and there’s even an original Miró in my bedroom
The grandeur of the building is complemented by friendly
by which time its ownership was divided across several family members
The nearby Es Pontas sea archALAMYHaving fallen into disuse for several decades
while the pool’s showers are in a recreated barraca de roter
“We wanted to keep that chiaroscuro feeling when you walk around.”
One of Can Ferrereta’s junior suitesMallorca’s southern towns
mahones cheese and purple safarnaria carrots
Wit has furnished the interior with items by international and home-grown brands
where you can see wild olive forests and wetlands
Cala LlombardsGETTY IMAGESYou are likely to hear more German spoken here than Spanish or Mallorqui
serving seasonal Mediterranean dishes such as llengua amb taperes (beef tongue with capers) and frit mallorqui (the island’s traditional stew
Laudat — a charming spot for lunch or dinner with a romantic patio filled with lemon trees
Laudat works with the local winery Armero i Adrover to produce a range of bespoke house wines — I try the white
Judit and her team serve tapas and decadent deserts
The market at SantanyiALAMYSantanyi particularly comes to life on Wednesdays and Saturdays
The bakery Panaderia Lozano tempts with savoury panades stuffed with meat
as well as traditional pastries and cakes such as lazos (twists of pastry with cream or chocolate)
custard-filled eclairs) and ensaimadas (circular doughy treats)
You’ll do well for ceramic and handicraft gifts too
scheduled to open this summer as a place to eat and shop; local artisans will also run workshops on pottery and weaving
At dusk an enchanting luminosity envelops Santanyi as the sun begins to set and everything glows
And I have the bonus of that sausage breakfast at Can Ferrereta to look forward to
Agnish Ray was a guest of Can Ferrereta. B&B doubles from £272 (hotelcanferrereta.com)
International travel restrictions are in place. Check gov.uk for more information
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expert advice and inspiration for your next trip
The Local Europe ABVästmannagatan 43113 25 StockholmSweden
Barcelona recently announced that it will ban smoking on all of its 10 city beaches from July this year
This means there will be a total of five kilometres of smoke-free sand in Spain's second-largest city
Barcelona decided to ban smoking on four of its main beaches between May 29th and September 12th as a trial run
and this year has extended the ban all year round to all its beaches
while many others have taken to social media to express their anger at the rule
As well as being horrible to find between your toes when sunbathing or when your kids are building a sandcastle
cigarette butts are harmful to the environment and marine life
A discarded cigarette butt is made of over 97 percent cellulose acetate and contains 4,000+ chemical toxins
discarded cigarette butts are just half the issue
which on Spain's crowded beaches can affect the health and well-being of many others around
Barcelona is of course not the only Spanish city to have banned smoking on its public beaches
We take a look at which other beaches across the country have put a stop to it
many of Catalonia's beaches have banned smoking
Catalonia became the first region to designate a 'smoke-free' beach when the town of L’Escala introduced the rule back in 2006
it has been extended to a total of 19 beaches in the region
The region of Galicia has the most smoke-free beaches in Spain
with a whopping 187 ocean and river beaches imposing the ban
These include 70 beaches in the province of A Coruña
Some of the most popular and well-known beaches that are free from tobacco include Santa Cristina in Oleiros
Asturias designated smoke-free beaches for the first time in 2019 and now has 14 smoke-free beaches
The list currently includes Playa de Misiego
El Puntal y Miami in Villaviciosa and Playa de Los Quebrantos in Soto del Barco
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Marina Fabic and Faye Mallia are organising a holiday with a difference
where participants can learn the secrets of Spanish gastronomy while trying their hand at preparing traditional recipes
What better place to indulge in a culinary vacation than Catalonia
are organising a unique foodie visit to Spain – including cheese tastings and vineyard visits
as well as fun cooking classes with talented chefs and
while enjoying the architecture of Barcelona and Costa Brava seaside
Participants will be able to discover the exquisite and typical dishes of the area
the famous miniature canapés known as pintxos
some of the best wines in the world and an avant-garde cuisine
the group will prepare regional cuisine recipes such as the paella catalan and visit traditional markets and places to enjoy genuine Spanish gastronomy
The group will be hosted at a seaside villa perched in the coastal town of Roses
many restaurants and the fishing port that sells its fresh catches of fish daily
The villa offers the perfect location for a pleasant coastal walk
while a two-minute stroll from the house leads to Almadrava sandy cove and onto the rugged coast on a path to Canyelles Petites beach and further on to the larger town and beaches of Roses
Food at the villa will be in the hand of a local chef
who will focus on Mediterranean cuisine – foodies will also be able to enjoy several Michelin star restaurants
lying in the middle of the gastronomic belt of Spain
and touted by foodies as one of the best restaurant in the world
bars and terraces all offer splendid panoramic views and the cuisine of Girona
cold roast vegetables and the xató (escarola lettuce with anchovies and cod)
the group will prepare regional cuisine recipes such as the paella catalan
and visit traditional markets and places to enjoy genuine Spanish gastronomy
The combination of mountain and sea also reaches the kitchen in the form of chicken with spiny lobster or rabbit with snails
stews and rice dishes are other tasty recipes
the crema catalana (a kind of custard with caramelised sugar) and fresh cheese with honey (mel i mató) are outstanding
The best wines for this menu belong to the Ampurdán-Costa Brava Denomination of Origin
Wine lovers will enjoy many cooperatives and vineyards in the area
The Mediterranean character of Roses is reflected in the products of the land
In Roses begins the Wine Route DO Empordà
This constitutes the main focus of winemaking in the Empordà
providing an essential yardstick with which to discover and understand DO Empordà wineries
while exploring the zone with the highest density of vineyards and wineries that may be visited
Roses’ fishermen cooked suquet de peix
a simple dish of freshly-caught fish boiled with potatoes
this recipe has experienced an evolution and has been enriched with the incorporation of typically Mediterranean products
The holiday will also help the group become acquainted with some of the different regions in Spain through its most popular appetisers
desserts and main courses – the world-famous tapas
paella from Valencia and Andalusian gazpacho
cakes and pastries from Galicia and others
a good part of the province of Girona as well as Cadaqués or Port de la Selva can be toured
Empuries preserves important Greek remains while
at Figueres the Dalí Theatre-Museum offers tribute to the master of surrealism
declared a historic-artistic site and Girona
with its walled town known as La Força Vella
are some of the many other possibilities offered by this Catalan region
There will also be visits to traditional Catalan markets
attractive cities and natural environments
And of course you’ll enjoy the dishes you yourself have cooked
along with other typical delicacies from Spain’s gastronomic culture
For more information send an e-mail to Creative Holidays Malta on info@creativeholidaysmalta.com
please register for free or log in to your account.