To discriminate between self and non-self surfaces and facilitate immune surveillance, the complement system relies on the interplay between surface-directed activators and regulators. The dimeric modulator FHR-1 is hypothesized to competitively remove the complement regulator FH from surfaces that strongly fix opsonic C3b molecules—a process known as “deregulation.” The C-terminal regions of FH and FHR-1 provide the basis of this competition. They contain binding sites for C3b and host surface markers and are identical except for two substitutions: S1191L and V1197A . Intriguingly, an FHR-1 variant featuring the “SV” combination of FH predisposes to atypical hemolytic uremic syndrome (aHUS). The functional impact of these mutations on complement (de)regulation, and their pathophysiological consequences, have largely remained elusive. We have addressed these questions using recombinantly expressed wildtype, mutated, and truncated versions of FHR-1 and FH. The “SV” to “LA” substitutions did not affect glycosaminoglycan recognition and had only a small effect on C3b binding. In contrast, the two amino acids substantially affected the binding of FH and FHR-1 to α2,3-linked sialic acids as host surfaces markers, with the S-to-L substitution causing an almost complete loss of recognition. Even with sialic acid-binding constructs, notable deregulation was only detected on host and not foreign cells. The aHUS-associated “SV” mutation converts FHR-1 into a sialic acid binder which, supported by its dimeric nature, enables excessive FH deregulation and, thus, complement activation on host surfaces. While we also observed inhibitory activities of FHR-1 on C3 and C5 convertases, the high concentrations required render the physiological impact uncertain. In conclusion, the SV-to-LA substitution in the C-terminal regions of FH and FHR-1 diminishes its sialic acid-binding ability and results in an FHR-1 molecule that only moderately deregulates FH. Such FH deregulation by FHR-1 only occurs on host/host-like surfaces that recruit FH. Conversion of FHR-1 into a sialic acid binder potentiates the deregulatory capacity of FHR-1 and thus explains the pathophysiology of the aHUS-associated FHR-1 “SV” variant. The complement system is an essential component of innate immunity and consists of a cascade of soluble plasma proteins and several cellular receptors . Its actvia decay accelerating (DAA) or cofactor activity (CA) (Among the most important regulators protecting host surfaces are the soluble regulators factor H (FH) and its splice variant factor H-like protein 1 (FHL-1) , which aity (CA) . Moreoveity (CA) , 10. Howity (CA) , the C-tity (CA) , 13.via “deregulation”  . Additivia its two N-terminal domains, which increases its avidity for C3b and C3d  from paroxysmal nocturnal hemoglobinuria (PNH) patients under Eculizumab treatment or from healthy donors were used with approval from the Ethical Committee of Ulm University. Sera were collected in VACUETTE/S-Monovette serum collection tubes, aliquoted, frozen in liquid nitrogen and stored at −80˚C. Standardized normal human serum (NHS) and FI-depleted serum were obtained from CompTech.Pichia pastoris as previously described  CCP1-3, FHR-1, the complement receptor of the immunoglobulin family (CRIg), the mutant FHR-1(SV), the C-terminal FH fragments FH19-20 and FH18-20 and their mutated versions FH18-20(LV), FH18-20(SA), and FH18-20(LA) were recombinantly expressed in escribed –29, witheviously . All proHeparin affinity for all produced proteins was tested on a HiTrap™ Heparin HP column . Samples containing 0.5 mg of protein were dissolved in 5 ml PBS (pH 7.4) to a final concentration of 0.1 mg/ml and loaded on the column equilibrated in PBS. Each protein was eluted by a gradient, over 10 column volumes, from PBS to PBS supplemented with 0.5 M NaCl.Human microvascular endothelial cells  were cultured and the assay was performed as previously described  with incThe yeast opsonization assay was performed as previously described , except Fresh human blood from healthy donors was collected in EDTA pre-dosed blood collection tubes. After centrifugation of a 200 µl cell suspension, the plasma was discarded and the cells were washed four times with 10 ml PBS and suspended to the initial volume. Next, 100 µl RBCs were mixed with 100 µl 20 mM Mg-EGTA, inhibitors , 160 µl factor I-depleted serum  and PBS to a final volume of 400 µl and incubated for 1 h at 37°C. The cells were washed once with 1 ml PBS after incubation and the opsonization process was repeated. After opsonization, cells were washed two times with 200 µl 20 µM FH19-20 to displace any remaining FH and three times with 1 ml PBS. Then, the cell suspension volume was adjusted to 200 µl, distributed to fresh tubes, and incubated with 50 U neuraminidase or PBS for 1 h at 37°C. Subsequently, a 10 µl aliquot of the untreated or desialylated cells was incubated with 50 µl of a 1:100 diluted FITC-labeled mouse anti-human C3 antibody  for 30 min at room temperature and analyzed with flow cytometry after two wash steps with 1 ml PBS to control for proper C3b opsonization. If the measured C3b opsonization signal  on RBCs was not at least sixfold higher than non-opsonized cells, an additional opsonization process was applied. Having confirmed high opsonization levels, 10 µl cell suspensions were incubated with 10 µl of either analyte solution  or PBS for 30 min at room temperature and then washed twice with 1 ml PBS. In order to detect FH, the cells were incubated with a mouse anti-human FH antibody . After a 10 min incubation the cells were washed twice with 1 ml PBS, incubated with an APC-labeled goat anti-mouse antibody (Jackson ImmunoResearch) for 10 min, and then again washed twice with 1 ml PBS. Finally, the cells were analyzed using a BD FACSVerse flow cytometer with MFIs calculated from the resulting histograms. The MFI values were normalized to the signal obtained from C3b opsonized cells treated with FH.The hemolysis assay on PNH erythrocytes was performed as previously described , with mi9 cells/ml) were mixed with 20 µl NHS, 10 µl of analyte solution (protein concentration from 0.3 to 9.6 µM), and 10 µl Mg-EGTA dissolved in PBS to prevent classical pathway activation . The reactions were incubated for 30 min at 37°C and stopped by adding 150 µl ice-cold PBS supplemented with 5 mM EDTA. Afterward, the samples were spun down and the absorbance of the supernatants was measured at 405 nm.Sheep erythrocytes were supplied in Alsever’s solution (Fiebig-Nährstofftechnik) and washed with PBS. Then 10 µl of cell suspension .9 cells/ml), 10 µl of analyte solution (protein concentration from 0.6 to 9.6 µM), and 20 µl NHS diluted in PBS and supplemented with Mg-EGTA  was incubated for 30 min at 37°C. The reaction was then stopped by adding 120 µl ice-cold PBS supplemented with 5 mM EDTA. Afterward the samples were spun down and the absorbance of the supernatants was measured at 405 nm.Rabbit erythrocytes were supplied in Alsever’s solution (Fiebig-Nährstofftechnik) and washed with PBS. A mixture of 10 µl of cell suspension .The affinity of FHR-1, FHR-1(SV), FH18-20, FH18-20(LV), FH18-20(SA), and FH18-20(LA) to C3b was measured with surface plasmon resonance (SPR) spectroscopy according to previously described methods , 32 on a2-Biotin (Thermo Fisher Scientific), according to the manufacturer´s instructions. Approximately 1,500 response units (RUs) of biotinylated C3b were then immobilized onto a streptavidin chip , which had been previously conditioned and washed according to the manufacturer’s recommendations.After incubation with 1mM DTT at room temperature and buffer exchange, C3b was biotinylated with EZ-Link Maleimide-PEGFor the amine coupling, a carboxymethyldextran hydrogel biosensor chip  was conditioned and washed according to the manufacturer’s recommendations and then approximately 7,250 RUs of C3b were covalently attached to one of the flow cell surfaces.versus concentration were used for calculation of equilibrium dissociation constants KD .Protein series (1 in 2 dilution) were prepared in running buffer (PBS containing 0.005% Tween20) in the following ranges: FH18-20 and mutants thereof were injected at 0.16–40 µM on the SAP chip and 0.63–40 µM on the CMD500M chip; FHR-1 and FHR-1(SV) were injected at 0.04–10 µM on both chips. After injection of analytes for 2.5 min, buffer was flowed over the chips and dissociation was observed for 5 min. For regeneration, 1 M NaCl was injected for 0.5 min. Only reference-subtracted sensorgrams are shown. Plots of response at steady state N-acetyllactosaminide  and 6´-sialyl-N-acetyllactosaminide  (Carbosynth), were coupled to a streptavidin chip , previously conditioned and washed according to the manufacturer’s recommendations. For the binding experiments, a 1 in 2 dilution series of each analyte was flowed over the chip at the same concentrations as for the C3b binding assay on the SAP chip, FH19-20 was injected at 160–0.16 µM. Analytes were injected for 2.5 min and afterward dissociation was observed under buffer flow for 5 min. For regeneration, 1 M NaCl was injected for 0.5 min. All experiments were performed at 25°C and a flow rate of 25 µl/min.The binding of FHR-1, FHR-1(SV), FH18-20, FH18-20(LV), FH18-20(SA), FH18-20(LA), and FH19-20 to different sialic acid moieties was investigated by SPR. Approximately 150 RUs of two different biotinylated sialic acids, 3´-sialyl-2 at a flow rate of 25 µl/min. Finally, the TraceDrawer software was used to subtract the C3b binding signal of the analyte alone from the signal obtained for the analyte/convertase mixture.The inhibition of AP C3 convertase formation was tested by SPR. Approximately 6,000 RUs of C3b were immobilized by standard amine coupling on a CMD500M chip , which had been previously conditioned and washed according to the manufacturer’s recommendations. The AP C3 convertase signal alone was determined by injecting a mixture of FB (600 nM) and FD (100 nM) for 3 min and observing the natural C3 convertase decay for 5 min. In each experiment either the analyte alone , FHR-1, or FHR-1(SV), all at 9.6 µM) or a mixture of the analyte (9.6 µM), FB (600 nM), and FD (100 nM) was injected for 3 min and the natural C3 convertase decay was observed for 5 min. After every injection, 1 µM recombinant CR1 CCP1-3 was flowed over the chip for 0.5 min to remove the remaining convertases by accelerating the decay. For regeneration, 1 M NaCl was injected for 0.5 min. All experiments were performed at 25°C in PBS containing 0.005% Tween20 and 1 mM MgClD  for 2 min, followed by 5 min dissociation monitoring. Finally, C3 (1 µM) and each construct (at a concentration 10-fold higher than the KD) were co-injected for 2 min and dissociation was observed for 5 min. For regeneration, 1 M NaCl and buffer were flowed for 0.5 and 2 min, respectively, after each protein injection. All experiments were performed in duplicate, at 25°C in PBS containing 0.005% Tween20 and at a flow rate of 25 µl/min.SPR was used to evaluate if FH18-20, FH18-20(LA), FHR-1, or FHR-1(SV) inhibit C3 binding to C3b. Approximately 2,300 RUs of biotinylated C3b (prepared as previously described) were immobilized on a streptavidin chip , previously conditioned and washed according to the manufacturer’s recommendations. First, C3 binding to C3b was measured by injecting 1 µM C3 for 2 min and dissociation was observed for 5 min. Then, the binding signal to C3b was determined for each construct by injecting each protein at a concentration 10-fold higher than its respective measured KD values [100 µM FH18-20(SA) and 60 µM FH18-20(LV)]. As a control, the same measurements were performed with 100 µM FH19-20.Inhibition of C5 binding to C3b was performed by SPR in a similar manner as described for the inhibition of C3 binding to C3b, with only a few changes. For this experiment, 1,500 RUs of biotinylated C3b were immobilized on the streptavidin chip and a concentration of 0.15 µM C5 was used instead of C3. Additionally, the single mutants of FH18-20 were also tested using concentrations 10-fold higher than their K2O. Protein samples were prepared by buffer exchange into deuterated NMR buffer  using 3 kDa MWCO regenerated cellulose centrifugal filters . STD NMR spectra were collected using a train of Gaussian-shaped pulses with a total saturation time of 3 s, off- and on-resonance irradiation frequencies of −30 ppm and 7.3 ppm, respectively, and a total relaxation delay of 4 s. A 30 ms continuous-wave spin-lock pulse was used for the suppression of residual protein signals, and the water signal was suppressed using a 2.4 ms 180° shaped pulse . 4k scans were collected for each experiment, and spectra were referenced to the NHAc methyl signal at 298 K as a standard . Absolute STD values were determined and are reported as a % of the off-resonance signal intensity (specific resonances used have been indicated). Samples containing glycan but lacking protein were used to collect 1D reference spectra and perform control experiments to confirm that no direct irradiation of ligand occurs under the conditions used for protein-containing STD experiments. Glycan structures were solved in D2O and assignments performed using standard 2D experiments  with spectra referenced to a CH3OH internal standard .NMR spectra were recorded at 283 K on a Bruker Avance III 500 MHz NMR spectrometer equipped with a TXI room temperature probe head. Spectra were acquired and processed with Topspin 3.5 software (Bruker). Samples were prepared in 3 mm tubes and contained 5 mM glycan  and 50 µM recombinant FH protein in deuterated buffer. Glycans were added from a 40 mM stock solution in DIntrinsic tryptophan fluorescence was measured using a nano-differential scanning fluorimetry instrument  by exciting samples at 285 nm and measuring fluorescence emission at 330 and 350 nm. Measurements were taken at 293 and 310 K, using 10 µl sample volume per measurement in standard grade capillaries. Each concentration was measured in triplicate, and the full experiment run in duplicate to ensure reproducibility. Each sample contained 25 µM FH18-20 with the respective glycan concentration  in 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20, and 3 mM EDTA.P. pastoris and prepared to a high level of purity (To gain insights into the function of wildtype FHR-1 and the aHUS-associated FHR-1 variant (L290S and A296V), we expressed FHR-1*B (wild-type and mutated sequences) in addition to constructs comprising only the last three CCP domains of FHR-1*B and FH Figure 1KD when compared to the wildtype construct (10.0-12.1 µM). The affinity of the double mutant FH18-20(LA) for C3b was in the range of 5.8–8.3 µM and therefore similar to that of the single mutant FH18-20(LV). The KD of FHR-1 for C3b was in the range of 0.6–1.1 µM, while its double mutant FHR-1(SV) showed a slightly decreased affinity (1.4–1.5 µM)  . As previously reported, the affinity of FH18-20 for C3b was in the range of 9.9–12.2 µM. Consistent with the results published for corresponding mutations in the truncated FH fragment FH19-20 in Morgan et al. , the S11i.e., CCP20), affect the binding of FH18-20 for a model GAG, we performed affinity chromatography on a heparin column for all FH18-20 and FHR-1 constructs , which are also located in an FH GAG-binding site  and FH18-20(LA), showed strongly reduced binding to α2,3-linked sialic acid  of C3b . To veriTherefore, we experimentally confirmed the hypothesis that a serine-to-leucine substitution at position 1,191 in FH disrupts sialic acid binding, as predicted in Blaum et al. .Next we investigated how the differences in sialic acid binding affect FH deregulation on self  and self-like surfaces (sheep and guinea pig RBCs), which contain a layer of sialic acid that has been shown to be important for binding human FH –36.i.e., FHR-1, FH18-20(LV) and FH18-20(LA)] competed very mildly with serum FH and that this effect was more visible at higher protein concentrations. Conversely, FH18-20, FH18-20(SA), and FHR-1(SV) competed more efficiently with serum FH, although the single mutant FH18-20(SA) was about half as active as the wild type FH18-20  cells, thus reducing physiological protection from AP-mediated attack. We found that all constructs carrying the leucine in position 290/1,191  were able to compete with serum FH on both self and self-like surfaces when applied at high concentration, the sialic acid-binding constructs were by far the most efficient deregulators.To assess if deregulation occurs on different types of surfaces, we extended the analysis to foreign (rabbit RBCs and yeast cells) and foreign-like cells .By removing terminal sialic acid residues, neuraminidase turns self and self-like surfaces such as PNH or guinea pig RBCs into foreign-like surfaces. A hemolysis assay performed on neuraminidase-treated PNH RBCs revealed that all monomeric constructs showed a very similar level of hemolysis as the PBS and glycine buffer controls, independent of their ability to bind sialic acid, thus excluding the possibility that any deregulation was occurring. Surprisingly, the dimeric constructs FHR-1 and FHR-1(SV), when given at very high concentration, were able to partially protect these cells from lysis  dimers. However, when the dimeric constructs were given at very high concentrations (> 1 µM), rabbit RBCs were protected from lysis , FH18-20(SA) and FH18-20(LA)] were co-injected with C5. However, the FHR-1 and FHR-1(SV) dimers did both cause inhibition of C5 binding to C3b, as can be inferred from the fact that the binding signals of the constructs alone almost overlap with those of the analyte/C5 mixtures  equally inhibit C5 binding to C3b at very high concentrations, thus suggesting a further surface protection mechanism, whereby activation of the complement terminal pathway, and therefore cell lysis, may be reduced at non-physiological high concentrations of FHR-1.After addressing the question of why FHR-1 may protect foreign cell surfaces from complement activation, at least at high concentrations, insights into the mechanism of FH deregulation on host surfaces are still needed. We therefore employed an SPR experiment in which FH and either FHR-1 or FH18-20 are, first separately and then simultaneously, applied to immobilized C3b  7B. Thisin vitro opsonized human RBCs outnumbers a majority of FH and FHR-1 molecules in the assay and thus, competition cannot effectively take place. Utilizing C3b-coated SPR sensor chips, which are particularly well suited to detect weak interactions in a dynamic setting, a direct competition of FH and FHR-1 (or FHR-1(SV)) for deposited C3b was evident , fewer convertases are assembled when FB and FD are applied to deposited C3b  were applied. Previously, a plate-based assay using much lower FHR-1 concentrations (50 µg/ml) failed to detect any interference with formation of the AP C3 convertase  were able to substantially reduce the C3 density on these cells and thus considerably inhibit the formation of C3 convertases Figure 8In conclusion, our studies provide critical insights into the molecular and functional determinants of complement modulation by FHR-1. While we confirm that FHR-1 may inhibit the C5 convertase by restricting access of C5 to the convertase, and uncover that FHR-1 can impair the assembly of the AP C3 convertase, these effects were only observed at high concentrations and render the physiological relevance uncertain. Nevertheless, these insights will be valuable in light of the contradictory views in the literature. More importantly, however, we uncover that FHR-1 deregulates FH on host but not foreign cells, and provide a molecular rationale for the disease mechanisms of the aHUS-associated FHR-1(SV) variant. The amino acid substitutions L290S and, to a minor degree, A296V in this variant convert FHR-1 into a sialic acid-binding protein with substantially pronounced deregulation capacity on host cell surfaces under complement distress, as observed in aHUS and other disorders.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of Ulm University (Ethikkommission der Universität Ulm). The patients/participants provided their written informed consent to participate in this study.All authors contributed to the article and approved the submitted version.This work was supported by a grant from Deutsche Forschungsgemeinschaft (SCHM3018/2-2 to CS) and the Swiss National Science Foundation (31003A_176104 to DR).CS, BH, and HS are inventors of a patent application that describes the use of engineered complement regulatory proteins for therapeutic applications. CS and BH have received honoraria for speaking at symposia organized by Alexion Pharmaceuticals. BH acted as a consultant for various pharmaceutical companies. HS received research grants and honoraria for speaking in symposia or service on advisory boards from Alexion Pharmaceuticals, Roche, Apellis, and Ra Pharmaceuticals . DR is an inventor of patents and patent applications describing complement inhibitors and their clinical use. He received honoraria for speaker and consulting engagements from various pharmaceutical companies.The remaining 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.


Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and β2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-β2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-β2 inhibition. This study also highlights the first association between ADRβ2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRβ2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers. Untreated cells were added to wells containing 50 μL of pre-incubated media containing DMSO. Treated cells were added to wells containing 50 μL of pre-incubated media containing 10 µM norepinephrine. The plate was then incubated for 3 h at 37 °C in a humidified atmosphere containing 5% CO6 cells/mL in serum-free media  or 0.5% FCS containing media  were added to a 1.5 mL microtube along with 10 μM ICI-118,551. 50 μL/well of cell suspension  with or without antagonists was added to the top chamber of the plate followed by the addition of 150 μL of DMEM containing 10% FCS to the bottom chamber of each well. The cells were incubated for 30 min prior to the addition of norepinephrine (10 μM). The plate was incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. After incubation, the top chamber was inverted and carefully shaken to remove the culture medium and transferred to the black receiver plate. Each well of the top chamber was washed with 100 μL of warm 1× wash buffer and the top chamber inverted and carefully shaken to remove excess wash buffer and placed back into black receiver plate. To the bottom chamber of each well is added 100 μL of 1000× cell dissociation solution/ Calcein AM solution and the plate incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 60 min. The plate was read on a fluorescent plate reader at 485 nm excitation, 520 nm emission . Cells were serum-starved for 24 h prior to performing the assay. 1 × 106 cells/mL in serum-free media  or 0.5% FCS containing media  were added to a 1.5 mL microtube along with 10 μM ICI-118,551. 25 μL/well of cell suspension  were added to the top chamber of the plate followed by the addition of 150 μL of DMEM containing 10% FCS to the bottom chamber of each well. The cells were incubated for 30 min prior to the addition of 10 μM norepinephrine. The plate was incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. After incubation the top chamber was washed with 100 μL of warm 1× wash buffer and placed into a black receiver plate. To the bottom chamber of each well 100 μL of 1000× cell dissociation solution/Calcein AM solution is added and the plate incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 60 min to fluorescently label and dissociate cells from the membrane. After 60 min, the top chamber was removed and fluorescence was measured using a plate reader . Cells were serum-starved for 24 h prior to performing the assay. 1 × 106) and MDA-MB-468 (1.2 × 106) cells were seeded and grown to 90% confluency. On the day of treatment 1 mM stocks of norepinephrine, isoproterenol and ICI-118,551 were made up in serum free DMEM and passed through a 0.22 µm filter prior to serial dilution. Media was removed from each flask, and cells were washed three times with PBS. Untreated cells received 25 mL of serum free DMEM, and treated cells received 25 mL of DMEM containing 10 μM of the appropriate treatment condition. After 30 min incubation 10 µM norepinephrine or 10 µM isoproterenol was added into the relevant flasks. Flasks were then incubated at 37 °C, 5% CO2 for 24 h. Twenty four hours after treatment secretome samples were obtained by removing the media and centrifuging , filtered (0.22 µm) and concentrated using Amicon Ultra-15 Centrifugal Filter Units . Cell lysates were prepared using 9.5 M urea /2% v/v dithiothreitol /1% v/v n-octyl-beta-glycopyranoside  containing protease inhibitor . The lysates were harvested, chilled on ice for 5 min, sonicated for 5 min and this process was repeated three times before centrifugation for 10 min at 12,000× g and storage at −80 °C. MDA-MB-231 (1.3 × 10w/w) in a thermomixer (650 rpm) [18 cartridges  for solid phase extraction. A vacuum concentrator was then used to concentrate the samples before resuspension in 5% acetonitrile + 0.1% formic acid and subsequent analysis of the peptides on an AB Sciex TripleTOF 5600+ MS/MS instrument in both SWATH  and IDA (information dependent acquisition) acquisition modes . Cell lysate (100 µg) and secretome (100 µg) were diluted in 50 mM tri-ethyl ammonium bicarbonate (TEAB) before reduction (5 mM DTT at 56 °C for 20 min) and alkylation (15 mM iodoacetamide at room temperature for 15 min) and then digested for 16 h using Trypsin/Lys-C  at 37 °C at a 20:1 protein:protease ratio (650 rpm) . Next, s18 pre-column  at a flow rate of 10 µL min for 2 min. The sample was then eluted off the pre-column and onto a YMC Triart-C18 analytical column  that was in line with the Sciex TripleTOF 5600+ Duospray Source using a 50 µm electrode in positive mode, +5500V. The following linear gradients were used: for SWATH, mobile phase B increasing from 3% to 30% over 38 min, 30% to 40% over 5 min, 40% to 80% over 2 min for wash and re-equilibration . For IDA, mobile phase B increasing from 3% to 30% over 68 min, 30% to 40% over 5 min, 40% to 80% for column wash and re-equilibration over 2 min . Data independent acquisition was performed using 100 variable SWATH windows (optimised on sample type) (TOFMS m/z 400-1250) 25 ms accumulation time; 2.6 s cycle and IDA with a top 30 ion fragmentation (TOFMS m/z 400-1250) followed by 15 s exclusion using rolling collision energy, 50 ms accumulation time; 1.8 s cycle. Each sample was analysed on a Sciex TripleTOF 5600+ mass spectrometer coupled in line with an Eksigent ekspert nano LC 425 system running in micro flow (5 µL/min) mobile phase B (100% acetonitrile + 0.1% formic acid) over mobile phase A (0.1% formic acid). In brief, 8 µg of sample was injected and trapped onto a YMC Triart-CSpectral library generation, alignment and fold change analysis were performed as described previously . In brieMass spectrometry and curated protein profiling of the two basal-type cell lines (MDA-MB-231 and MDA-MB-468) identified increased LYPD3 expression, a pro-metastasis protein. LYPD3 protein expression in breast cancer tissue was confirmed by immunostaining three wax-embedded TMA slides  comprising 260 cases of invasive ductal adenocarcinoma, 50 cases of metastatic adenocarcinoma and 10 cases of normal breast tissue . Sections were immunostained using a monoclonal rabbit anti-human LYPD3 antibody  on a BenThe intensity of LYPD3 immunostaining was microscopically assessed for cytoplasmic staining using a five-point scoring technique, where a score of 0 represented nil staining; a score of 1: weak; 2: moderate; 3: strong; 4: strong cytoplasmic staining with additional cell membrane staining. p = 0.05 or less. The association between LYPD3 expression and clinical variables in malignant adenocarcinoma, metastasis and adjacent normal breast tissue was statistically tested using SPSS . Immunohistochemistry scores were dichotomously categorised  for Chi-square tests. Significance levels were Total cell lysates were used from the proteomic analysis. In brief, 50 µg of total protein from each sample was prepared with sample reducing buffer (60 mM Tris-HCl (pH 6.8), 2% SDS (sodium dodecyl sulphate), 10% glycerol and 0.01% bromophenol blue) at a ratio of 1:3 sample vs. reducing buffer. The sample was resolved on an SDS gel  with Tris/glycine/SDS gel running buffer (Geneflow) at a constant voltage of 150 V. After separation, samples were transferred onto nitrocellulose membranes using Tris/glycine/methanol transfer buffer at a constant current of 180 mA for 75 min at 4 °C. Membranes were blocked in 10% Marvel™ dried skimmed milk powder for 1 h before being probed with rabbit anti-LYPD3 antibody , rabbit anti-beta 2 adrenergic receptor antibody  and rabbit anti-beta actin antibody  overnight at 4 °C. The membranes were then washed and goat anti-rabbit IgG HRP-linked antibody was added . Membranes were washed and exposed to the Clarity Western ECL Substrate (1:1) and imaged using a Syngene G:Box and Genesys v1.5.4.0 software .5) were transfected with shRNAs and hexadimethrine bromide  at a final concentration of 8 µg/mL for 18 h. Resistant colonies were selected using media containing 2 µg/mL puromycin.Lentiviral shRNA plasmids and packaging mix (SHP001) were purchased from Sigma : shLYPD3  and shControl . MCF-7 cells (2.5 × 10LYPD3 gene expression profiling was performed using patient gene expression profiles generated through The Cancer Genome Atlas (TCGA)  and the For the first time, this study postulates that ADRβ2 signalling can regulate LYPD3. Furthermore, by knocking down LYPD3 expression in the MCF-7 breast cancer cell line we demonstrated that LYPD3 supports both basal and norepinephrine-induced cell migration. Thus, its level of expression could be an important indicator of breast tumour progression and this was supported by our findings in breast cancer tissue cohorts. This highlights LYPD3 as a promising therapeutic target but also supports the development of novel beta-blocker compounds that could be used prophylactically to try and reduce metastatic spread.


Herein, high-conductive LLZO thin films with cubic phase and amorphous domains between crystalline grains are prepared, via annealing the repetitive LLZO/Li2CO3/Ga2O3 multi-nanolayers at 600 °C for 2 h. The amorphous domains may provide additional vacant sites for Li+, and thus relax the accumulation of Li+ at grain boundaries. The significantly improved ionic conductivity across grain boundaries demonstrates that the high energy barrier for Li+ migration caused by space charge layer is effectively reduced. Benefiting from the Li+ transport paths with low energy barriers, the presented LLZO thin film exhibits a cutting-edge value of ionic conductivity as high as 6.36 × 10−4 S/cm, which is promising for applications in thin film lithium batteries.The high-resistive grain boundaries are the bottleneck for Li Thin l anodes –10. Howel anodes , 12.+ transport are one of the keys to achieving high ionic conductivity in solids [+ conducting performance. One is related to Li+ transport within a grain. The lattice sites possibly occupied by Li+ are energetically nonequivalent, and thus Li+ must get over an energy barrier (EBg) when it hops between these sites [+ transport across the grain boundaries (GBs) [+. A space charge layer would form because the unoccupied possible sites for Li+ around GBs are depleted  is much higher than EBg (~ 0.3 eV) for the case of LLZO [It is well-known that the energetically favorable paths for Lin solids , 14. Forse sites –18. The es (GBs) , 20. The+ occupation in the LLZO with cubic phase, which are tetrahedral 24d site (Li1) and distorted octahedral 96 h site (Li2), are close to energetically equivalent [g in the cubic LLZO is moderate (~ 0.3 eV). Although the cubic phase of LLZO is metastable at room temperature (RT), the strategies to stabilize it through the doping of high valence cations, such as Al3+, Ga3+, and Ta5+, have been well developed [−4 S/cm and activation energy of 0.47 eV [+ in the crystal lattice may further help to lower EBg [2O during thin film deposition [2O effectively compensated the lithium loss during sputtering-deposition. On the other hand, the strategy to address the conduction issues derived from high EBgb is few, although it is well-known the high-resistive GBs is the bottleneck for Li+ transport in LLZO [It has been reported that the possible sites for Liuivalent , 22, 23.eveloped . Lobe et 0.47 eV . It is gower EBg , 13. LLZposition , 35. Li2 in LLZO , 21.+ vacancies [gb (< 0.7 eV) [2CO3, and Ga2O3, and the following annealing  , 38. The2CO3, and Ga2O3 were sequentially deposited by radio frequency magnetron sputtering on polished MgO (100) substrates in pure Ar atmosphere. A multilayered thin film with the thickness of ~ 1500 nm (± 10%) was obtained by repeatedly deposited the triple-layered unit for 80 cycles , Li2CO3(99%), and Ga2O3(99.9%) mounted on 190 mm × 55 mm Cu backing plates are provided by Zhongnuo New Materials Manufacturing Co., China. The LLZO target used here is with desired cubic phase  for 2 h at 600 °C, 700 °C, and 800 °C, respectively.The ultrathin layers of LLZO, Lished MgO 00 substr2CO3, and Ga2O3 was determined by a step profiler  after annealing at 800 °C for 2 h .Meanwhile, there are no diffraction peaks of Li6Li+, La3+, Zr4+, and Ga3+ throughout the thin film #600-1  in the present LLZO thin film solid electrolyte #600-1.As mentioned above, the LiGBs Fig. . The micO or CO2 , can be ved Fig. a, b. It ved Fig. d. EDX maved Fig. c–f. Therectively . The Li+ntinuity . More imnuity [+ , 36–38. depleted , 20. In σtotal) can be calculated according to the equation:The EIS measurements of the presented LLZO thin films are conducted with the in-plane test configuration shown in Fig. L is the distance between the two contacting electrodes, S is the electrode area, and Rtotal is the total resistance of LLZO thin film determined through EIS measurements. The Nyquist plots of the measured impedance spectra  of LLZO thin films are also normalized to the distance of two parallel contacting electrodes, and can be calculated according to the following equation [where tra Fig. a and S2bequation :2\documCbulk and Cgb are the bulk capacitance and the grain boundary capacitance, which can be calculated using the equation (R (Rbulk and Rgb) and CPE (CPEbulk and CPEgb) [where equation  based ond CPEgb) , 40.3\dL and S) and the fitted values of the elements in the equivalent circuit  are summarized in Table Sσbulk, σgb, and σtotal at room temperature of the presented LLZO thin films. σtotal of #800-1 is lower than 10−8 S/cm since it is dominated by the Li-poor phase of LZO. The samples with Ga dopant and extra Li, #800-2, #700-1, and #600-1, possess the σtotal of 5.63 × 10−7, 3.89 × 10−5, and 6.36 × 10−4 S/cm, respectively. This trend may be caused by two reasons. First, the proportion of high-resistive LZO in the prepared thin films is trimmed down as the annealing temperature is reduced, which is demonstrated by their XRD patterns  of electrodes and the fitted values of the elements in the equivalent circuit  of the different thin films for calculating σtotal, σbulk, and σgb at room temperature. Figure S1. The thickness of #600-1 (1.516 μm) determined in its cross-sectional SEM image. Figure S2. XRD patterns of the LLZO target used in this study. Figure S3. The grain size of #600-1(~50 nm) determined by SEM image. Figure S4. The Nyquist plots of impedance spectra of LLZO thin-films #700-1 (a), #800-1 (green in b), and #800-2 (brown in b) measured at room temperature, inserts show the equivalent circuits for EIS analysis.


Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H The renin–angiotensin-aldosterone system (RAAS) plays a major role in cardiovascular physiology and physiopathology and is implicated in endothelial function, vascular remodeling and inflammation ,2. It isIschemia/reperfusion is a major pathophysiological phenomenon in cardiovascular diseases. Whether it is a result of athero-thrombosis, cardiovascular surgery or heart failure, first line consequences are observed in the vasculature of various organs, for example, myocardium, kidney or brain. Ischemia/reperfusion (I/R) induces vascular injuries and imbalanced vascular responses, leading to endothelial dysfunction. I/R conditioning studies for cardioprotection generally focused on beneficial effects of cardiomyocytes, thus forsaking coronary vasculature. However, these conditioning therapies may also affect blood vessels, and therefore participate in the whole organ protection . Among tInjured cells release extracellular adenosine triphosphate (eATP), which is recognized as a damage associated molecular pattern. It can affect vascular cells, through the modulation of EC apoptosis and activation but also smooth muscle cells (SMC) proliferation. eATP activates specific purinergic receptors, such as ionotropic P2X receptors and some members of the metabotropic P2Y receptor family. Liu and collaborators showed that eATP induced vasodilation in rat small mesenteric arteries by acting on both P2X7 and P2Y11 in ECs . In a raThis study aimed at assessing the role of P2Y11 modulation on vascular dysfunction in two models of intrigued stress conditions: high concentrations of AngII and (H/R) injury. We hypothesized that P2Y11 activation could reduce vascular dysfunction and mediate SMC and EC cross-talk after injuries, limiting intimal hyperplasia and endothelial dysfunction.p = 0.05) and significantly impaired relaxation in response to acetylcholine as compared to the control (CTL) group  group a. While L) group b. In theL) group c. HoweveL) group d. IncubaL) group d. These To assess whether the effect of P2Y11 ligands targeted EC, SMC or both cell types, we performed similar experiments on aortic rings denuded from endothelium. After EC removal, AngII still tended to increase the constrictive response to phenylephrine compared to CTL a, as obsTo assess the effect of P2Y11 ligands on nitric oxide bioavailability, we measured the vascular contractility in response to the eNOS inhibitor L-NAME. P2Y11 ligands demonstrated no significant effect in the CTL group c. AngII We then tried to evaluate how 30 min H/R or AngII incubation could modulate eNOS expression and its activation though the phosphorylation of ser1177 (p-eNOS) in HUVECs. First, we showed that HUVECs expressed P2Y11 receptor a. In theEndothelin-1 (Et-1) is one of the major signaling molecules that induce contraction . After 12O2 production in aorta segments. AngII exposure increased H2O2 production by the aortic rings compared to the CTL (from 1.05 ± 0.09 to 1.72 ± 0.2). In the latter, NF546 and NF340 had no effect on basal H2O2 production (2O2 release by vessels (from 1.72 ± 0.2 to 1.06 ± 0.15), while the P2Y11 antagonist had no effect. The combination of both NF546 and NF340 treatment had no effect on H2O2 production  release by HUVECs (from 1 to 0.45 ± 0.18). After reoxygenation, we measured significantly higher concentrations of eATP compared to the CTL, with a maximum reached around 30 min (from 1 to 5.9 ± 1.3), followed by a progressive return to basal secretion levels after prolonged reoxygenation b. The saVascular dysfunction can be characterized by a phenotypical switch of SMC from quiescent contractile to pro-proliferative synthetic phenotype, associated with intimal hyperplasia . We firsThe HCASMC phenotype was evaluated according to their expression level of α-SMA and TIMP-1 as markers of contractile phenotype, and vimentin and MMP-2 as markers of synthetic phenotype. P2Y11 agonist NF546 significantly increased α-SMA and TIMP-1 expression after AngII exposure (from 0.81 ± 0.15 to 1.61 ± 0.4 and from 1.15 ± 0.09 to 1.54 ± 0.13) and after H/R (from 1.03 ± 0.15 to 1.41 ± 0.17 and from 1.49 ± 0.17 to 1.73 ± 0.2) c,d. Despp = 0.06) and H/R group   or H/R (from 1.25 ± 0.13 to 1.35 ± 0.15). These effects were prevented by P2Y11 activation in CTL group (from 1.25 ± 0.13 to 1.15 ± 0.14), AngII group  a.To further improve our understanding of the crosstalk between ECs and SMCs, we assessed whether VEGF secretion by SMC was altered under stress conditions. AngII exposure did not increase VEGF secretion. However, VEGF secretion tended to be increased by H/R (from 395 ± 89.2 to 525.9 ± 121). VEGF secretion was decreased by the addition of the P2Y11R agonist NF546, in the CTL (from 378.9 ± 96.7 to 328.4 ± 91.1), AngII (from 430 ± 77 to 336.8 ± 64) and H/R (from 525.9 ± 121 to 382.2 ± 114) groups, and tended to be increased by the P2Y11R antagonist NF340 in the CTL, AngII and H/R groups b. 2O2 production. We reported that P2Y11R activation decreased SMC proliferation and phenotype switch in response to stress stimuli. In addition, P2Y11R activation decreased the ECs’ secretome pro-proliferative effect on SMC. We are the first to describe a role of P2Y11R activation in the prevention and treatment of vascular dysfunction, a key process in the pathophysiology of hypertension and myocardial I/R injury. In this study, we identified the purinergic G-protein coupled receptor P2Y11 as a potent target to modulate stress-induced vascular dysfunction. We showed that the activation of the P2Y11R-like improved vasomotor response in a rat AngII-induced vascular dysfunction model in an EC-dependent manner. This was modulated by NO and endothelin-1 release, but also by stress factors such as eATP and H2+] influx and vasoconstriction in a diabetic hyperglycemia model of vascular myocytes  [NO is a key molecule in the maintenance of vascular function and relaxation response . It is w2+] . Here, w2O2 production in our aortic rings model. This effect was suppressed by P2Y11R-like activation, further reinforcing its protective effect. Our group already showed that P2Y11R activation decreased mitochondrial oxidative stress in cardiomyocytes though the activation of PKCε -1-propane-sulfonate (CHAPS) solution  . 2, and 10 µM diclofenac (Sigma-Aldrich) to block the generation of prostaglandins, then the major vasodilator remaining active is the NO. Aortic rings were then stretched and equilibrated at a tension of 2 g (isometric tension). The maximal contraction level was evaluated in response to a 80 mM KCl solution. After washing steps and equilibration, aortic rings were exposed to angiotensin II (AngII)  or vehicle (control group) during 30 min. Both the control group and the AngII-treated group were incubated simultaneously with or without NF546 , NF340 , or both. These are respectively the specific agonist and antagonist of P2Y11R. Contraction was evaluated in response to phenylephrine (Sigma-Aldrich), and was adjusted to reach a preconstriction level of 80% of the contraction elicited by KCl. Relaxation was evaluated in response to cumulative doses of acetylcholine . Rat aortic rings were mounted on an isometric force transducer (Myograph DMT 620M) in an organ chamber. This chamber contained 5 mL of Krebs solution equilibrated 30 min at 37 °C with ONitric oxide bioavailability was assessed by evaluation of the vasoconstrictive response to the eNOS inhibitor N(ω)-nitro-L-arginine methyl ester . Aortic rings were preconditioned with phenylephrine to reach 10% of the maximal KCl-induced constriction before L-NAME treatment.Human umbilical vein endothelial cells (HUVECs) and human coronary artery smooth muscle cells (HCASMC) and their corresponding culture media  were purchased from Promocell and cultured according to the manufacturer’s instructions. Cells were used from passage 3 to 6 for experimental procedures. 2, 5% CO2, DPBS with CaMg) in a hypoxia chamber . DPBS was equilibrated overnight before the experiment. Several durations of reoxygenation  were used depending on the experiment and the type of cell. Hypoxia and reoxygenation durations were based on previous observations from our group [For H/R experiments, HUVEC and HCASMC were submitted to 5 h hypoxia . Modulation of P2Y11R was performed with its agonist NF546 (10 µM) and antagonist NF340 (10 µM); the maximal concentration effect was chosen according to Meis et al. . 2O2-released by aortic rings was evaluated by Ferrous iron xylenol orange Oxidation (FOX) assay using the PeroxiDetect Kit  according to the manufacturer’s instructions. A 30-min incubation was performed in the absence or presence of AngII. The two groups were also incubated at the same time with NF546, NF340 or both.H®-Multi Jr. luminometer . Endothelin-1 release was evaluated by ELISA assay with endotheline-1 (Et-1) ELISA kit . The supernatant samples were diluted by 10 in order to perform the assay. Vascular Endothelial Growth Factor (VEGF) secretion by SMC was analyzed in HCASMC supernatant by ELISA assay . The supernatant samples were diluted by 2 in order to perform the assay.Extracellular ATP secretion was evaluated in HUVEC supernatant with ATP Bioluminescence Assay Kit HS II , according to the manufacturer’s instructions. Luminescence was measured with a GloMaxThoracic aortas rings or whole cells pellet were lysed in RIPA buffer with protease inhibitor cocktail  and phosphatase inhibitors when needed . The protein concentration was determined by BCA assay . Proteins were separated by 10% SDS-PAGE and transferred on nitrocellulose membrane by semi-dry electroblotting. Non-specific sites were blocked by incubation in Tris-Buffered Saline (TBS)—tween 0.1% (TBST) -BSA 5% for 1 h at room temperature (RT), followed by primary antibody incubation overnight at 4 °C. Membranes were washed with TBST, then incubated with the appropriate secondary antibody coupled with horseradish peroxydase for 1 h at RT and revealed with Amersham™ECL™ Prime Western Blotting Detection Reagent  and PXi/PXi Touch gel imaging system . Proteins expression was quantified by densitometry with ImageJ software.The antibodies used were HSC70 , β-actin , hP2Y11 , hP2Y11-Peptide , eNOS , p-ser1177-eNOS  α-SMA , TIMP-1 , goat anti-mouse and anti-rabbit secondary antibodies . HCASMC, placed in 12-wells plates , were incubated with 10 µM EdU in different conditions. After 72 h, cells were harvested and EdU incorporation was evaluated by fluorescence staining  using flow cytometry (BD FACS Canto) according to the manufacturer’s instructions. For the assay of HUVEC secretome proliferation modulation, we subjected HUVEC to three conditions: basal condition (12 h—DMEM/F12), H/R condition  or AngII (12 h—DMEM/F12—AngII 100 µM). After the 12 h incubation, supernatant was harvested and centrifuged (10 min—1000 g), EdU (10 µM) was added, then supernatants were incubated 24 h with HCASMC in the same conditions as the proliferation assay. Analyses were performed as previously described. F test . p-values < 0.05 were considered statistically significant. Data given as mean ± SEM and box plot as median and mean + min to max. Data were analyzed using GraphPad Prism . Data were analyzed using Wilcoxon test. Data analysis of the dose–effect response curves were performed using the extra sum-of-squares


High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents.Functional enzymatic assays and patients’ plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels.High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub.Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17. Cancer growth varies widely with indolent and aggressive forms of CLL. In recent years, there has been substantial progress in the clinical management of CLL patients supported by a better risk stratification and the introduction of a number of novel therapeutic agents.TP53 mutations, which are associated with poor response to treatment and predict earlier relapse after achieving initial haematological remission.2 For treatment-naïve patients with these high-risk features, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib is indicated as an initial systemic therapy rather than chemoimmunotherapy.4 The latter is based on the purine analogue fludarabine or alkylating agent bendamustine or chlorambucil backbones combined to the monoclonal antibody rituximab directed against the CD20 B-cell marker.5 Other therapeutic small molecules include two distinct mechanisms of actions with venetoclax, a BCL-2 inhibitor,6 idelalisib, a phosphatidylinositol-3-kinase (PI3K) δ inhibitor, and duvelisib, a dual inhibitor of PI3K δ and γ isoforms.8 Phase 3 trials are ongoing for additional agents such as the second-generation oral BTK inhibitor acalabrutinib and the Syk/Jak inhibitor cerdulatinib.10 Whilst treatment has significantly advanced patients’ survival, it is also associated with incomplete clonal eradication, relapse, refractoriness or transformation to a more aggressive lymphoma (Richter’s syndrome).3Pre-treatment evaluation of informative prognostic markers helps the stratification of CLL patients into risk subgroups. These markers include unmutated immunoglobulin heavy chain variable region (U-IGHV), del(17p) or TP53, SF3B1, NOTCH1 and BIRC3 genes.12 Other genetic screens have recently identified further genes that could be implicated in fludarabine sensitivity such as ARID5B and BRAF.14 As targeted therapy agents have become more established in cancer therapy, recent reports attribute acquired hypermorphic mutations in genes of targeted pathways, including BTK and its immediate downstream effector phospholipase C, γ2 (PLCG2) critical for BCR signalling.15 Mechanisms of resistance to other targeted agents, so early in clinical use, have not yet been portrayed in CLL patients but work is ongoing in this area.Both innate and acquired resistance represent major challenges for long-term disease control and an intense area of research. Findings reveal that targeted agents have unique mechanisms of resistance compared with chemotherapy. Genetic anomalies associated with fludarabine refractoriness include mutations in 16 Accumulating evidence support an influence of the UGT pathway to drug resistance in many cancers including in leukaemias, namely in CLL patients treated with fludarabine and acute myeloid leukaemia (AML) patients receiving ribavirin or cytarabine-based treatments.19 However, for most drugs used in CLL, the glucuronidation pathway has not been comprehensively studied. Reports have established that high UGT2B17 expression is an adverse prognostic factor in CLL, associated with shorter treatment-free survival, overall survival and patients requiring more treatment.21 The link between UGT2B17, adverse CLL features and progression appears complex, and precise molecular mechanisms underlying these observations remain to be elucidated. In cohorts of CLL patients, UGT2B17 was associated with unmutated IgHV and predicted poor survival,17 and was further associated with progressive disease within the group of patients with a more favourable profile (mutated IgHV).21 A recent study supports that UGT2B17-mediated metabolism would participate in the regulation of signalling pathways critical for CLL progression.22 High UGT2B17 expression in leukaemic cells was further linked to a lack of response to fludarabine-containing regimens.17 UGT2B17 is one of the 19 functional human drug-conjugating UGT enzymes, also involved in regulating homoeostasis of a number of endogenous metabolites, however its role in conjugating anti-leukaemics remains largely unknown.16Additional contributing mechanisms of reduced drug sensitivity that may be shared by targeted agents and chemotherapy may include increased drug efflux by ATP-dependent transporters and drug inactivation by metabolising pathways such as the glucuronidation pathway by UDP-glucuronosyltransferase enzymes (UGTs). By conjugating lipophilic drugs to glucuronic acid (GlcA), UGTs impair biological activity and enhance water solubility of drugs, driving their elimination and a lack of drug efficacy.In the present study, we investigated the response to anti-leukaemic agents namely fludarabine, ibrutinib, idelalisib, bendamustine, chlorambucil, venetoclax, acalabrutinib, cerdulatinib and duvelisib in relation to UGT2B17 expression levels. We then sought to evaluate the UGT-mediated enzymatic inactivation of drugs relevant to CLL and the possible involvement of the UGT2B17 enzyme. Our data support that high UGT2B17 expression alters drug response by direct inactivation but likely also through other mechanisms. We further evidence that the UGT pathway is involved in the inactivation of the majority of anti-leukaemic agents used in CLL, suggesting that this pathway may be associated with drug resistance.UGT2B17 mRNA analysis was performed in CD19+-sorted cells of twenty CLL patients before and after the first cycle of treatment with fludarabine-containing regimens as described.23 Plasma samples for quantification of glucuronides in circulation were available for two patients in the 1st week after the first cycle of treatment with fludarabine-chlorambucil-rituximab and for 15 patients during treatment with ibrutinib. The B lymphoblastoid leukaemic cell lines included p53wild-type  and p53mutated (MEC-1) cell lines purchased from ATCC  or DSMZ . Cells overexpressing UGT2B17 were previously described.22 Cell culture components were all purchased from Wisent Bioproducts .Cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained from CLL patients who were diagnosed between 1987 and 2011 at Vienna General Hospital. Patients provided informed consent. The study was carried out in accordance with the Declaration of Helsinki and was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205). 2, 50 mM Tris-HCl (pH 7.5), 20 µg/mL alamethicin, 2 mM UDP-GlcA and 0.5 µg/mL leupeptin with the indicated substrate. Samples were then stopped by adding 100 µL cold methanol, centrifuged at 16,000×g for 10 min and supernatants analysed by mass spectrometry. The following negative controls were included for each analysis: reaction assays without substrate, without the microsomal fraction and without substrate and microsomal fraction.Glucuronidation assays were conducted using human liver, intestine and kidney microsomes  or supersomes expressing individual human UGT isoforms . In the absence of commercially available UGT2B11 supersomes, microsomal proteins derived from HEK293 expressing the recombinant UGT2B11 enzyme were used. Substrates included dihydrotestosterone (DHT) purchased from Steraloids , fludarabine and chlorambucil from Sigma-Aldrich  and bendamustine from Toronto Research Chemicals . All other compounds were purchased from Selleckchem . UGT assays were incubated at 37 °C for 6 h in a final volume of 100 µL of assay buffer containing 20 µg of proteins, 0.5 mM DTT, 10 mM MgClThe establishment of these analytical methods required the optimisation of chromatographic conditions, production of glucuronide standards using pooled liver microsomes and the use of deuterated molecules as internal standards . Drugs and their G metabolites were measured on an API 6500 mass spectrometer , operated in multiple reactions monitoring mode and equipped with a turbo ion-spray source. Electrospray ionisation was performed in the positive ion mode. The chromatographic system consisted of a Nexera . For fludarabine-G1 and G2, the chromatographic separation was achieved with an ACE Phenyl 3.0 µm packing material, 100 × 4.6 mm . The mobile phases were solvent A: water with 0.1% formic acid (v/v) and solvent B: methanol, at a flow rate of 0.9 ml/min. Fludarabine- G1 and G2 were eluted using the following programme: 0–2.0 min, isocratic 20% B; 2.0–2.1 min, linear gradient 20–65% B; 2.1–4.0 min, isocratic 65 % B; 4.0–4.1 min, linear gradient 65–90% B; 4.1–5.2 min, isocratic 90 % B; 5.2–5.3 min, linear gradient 90–20 % B; 5.3–8.5 min, isocratic 20% B. For cerdulatinib-G, chlorambucil dechlorinated metabolites G1 and G2, venetoclax-G, and the bendamustine derivative HP2-G, the chromatographic separation was achieved with a Gemini C18 3.0 µm packing material, 100 × 4.6 mm . The mobile phases were solvent A: 1 mM ammonium formate in water and solvent B: 1 mM ammonium formate in methanol at a flow rate of 0.9 ml/min. Cerdulatinib-G and chlorambucil dechlorinated metabolites G1 and G2 were eluted using the following programme: 0–5.0 min, linear gradient 10–90% B; 5.0–5.2 min, isocratic 90% B; 5.2–5.3 min, linear gradient 90–10% B; 5.3–8.3 min, isocratic 10 % B. Venetoclax-G was eluted using the following programme: 0–2.0 min, linear gradient 10–90% B; 2.0-6 min, isocratic 90% B; 6.0–6.1 min, linear gradient 90–10% B; 6.1–9.2 min, isocratic 10% B. The bendamustine metabolite HP2-G, was eluted using the following programme: 0–5.0 min, linear gradient 20–90% B; 5.0–5.2 min, isocratic 90% B; 5.2–5.3 min, linear gradient 90–20% B; 5.3–8.3 min, isocratic 20% B. For idelalisib-G1 and G2, the chromatographic separation was achieved with a Gemini C18 3.0 µm, 100 × 4.6 mm, using solvent A: 2 mM ammonium formate in water and solvent B: 2 mM ammonium formate in methanol at a flow rate of 0.9 ml/min. Idelalisib-G1 and G2 were eluted using the following programme: 0–3.0 min, isocratic 60 % B; 3.0–3.1 min, linear gradient 60–90% B; 3.1–4.0 min, isocratic 90% B; 4.0–4.1 min, linear gradient 90–60% B; 4.1–7.0 min, isocratic 60% B. For ibrutinib-G1 and G2, acalabrutinib-G and duvelisib-G, the chromatographic separation was achieved with an ACE Phenyl 3.0 µm, 100 × 4.6 mm, using a mobile phase of 75%, 75% and 65% methanol, respectively, eluted with 1 mM ammonium formate in water in isocratic mode at a flow rate of 0.9 ml/min. The systems were controlled through Analyst Software, version 1.6.1 from AB Sciex.4 cells/well (MEC1) or at 5 × 104 cells/well (JVM2) in 96-well U-bottom tissue culture plates . Drugs were added at time of plating at concentrations ranging from 1 nM to 100 µM (7–9 concentrations per drug), depending on the drug and based on cell viability. MTS cell viability assays  were conducted 72 h after treatment initiation according to the manufacturer’s instructions. Absorbance was read on a TECAN infinite M1000 plate reader  at 495 nm. Control cells were treated with corresponding vehicle concentration. To determine half maximal inhibitory concentrations (IC50), MTS readout of control samples treated with vehicle only was set to 100% cell viability, and relative cell viability of drug-treated cells was determined by dividing MTS values of the treated samples by the control. IC50 were calculated by fitting variable slope non-linear curves to normalised response data from drug treatments using GraphPad Prism v5 . Assays were replicated at least three times in triplicates.Cells were plated at 1 × 104 cells/well (MEC1) or 3 × 104 cells/well (JVM2) were exposed to varying drug concentrations as above for 72 h. Cells were washed, labelled for 12 min with Annexin V (1:200) and PI (2 µg/ml) in Annexin V binding buffer, then immediately analysed by flow cytometry on a FACSCelesta equipped with a high throughput sampler (BD Bioscience). Annexin V labelling assays were replicated at least twice in duplicates.Cytotoxicity of drug treatments was determined by labelling cells with AlexaFluor 647-conjugated Annexin V  and propidium iodide (Sigma). Cells plated in 96-well plates at 2 × 10UGT genes were measured by qPCR analysis of 10 ng of cDNA with Power SYBR green master mix (Thermo Fisher Scientific). For measures of other genes including housekeeping genes from normalisation, total RNA was DNase I-treated and purified using the RNeasy MinElute Cleanup kit (Qiagen) per manufacturer’s instructions and as described previously.22 RNA sequencing experiments were performed on MEC1, EHEB and JVM2 cells treated with fludarabine (10 µM and 50 µM), ibrutinib (1 µM and 5 µM) and idelalisib (5 µM and 10 µM) for 48 (MEC1), 72 (JVM2) or 96 (EHEB) hours. Sequencing data was quality trimmed using Trimmomatic v0.36 and aligned to GRCh38 using HISAT2 v2.1.24 Transcriptome assembly and generation of read-count matrices were performed using Ensembl GRCh38 transcriptome annotation with StringTie v1.3.4.25 Differential gene expression analysis was done with the edgeR package for R v3.5.1. Isoform quantification was done using kallisto v0.44.0 with a custom GTF formatted annotation containing alternative UGT transcript information.26 Sequencing data are available from the Gene Expression Omnibus (GEO) accession number GSE135030.Total RNA was extracted from MEC1, EHEB and JVM2 cells treated with fludarabine, ibrutinib or idelalisib using RNeasy plus mini spin kits . cDNA was generated using SuperScript IV reverse polymerase . 27 Public microarray data from PBMCs of untreated CLL patients and CD19+-sorted B-cells from patients treated with fludarabine-containing regimens were obtained from the Gene Expression Omnibus with the accession numbers GSE1315928 and GSE15490,23 respectively. In the latter study, responders included complete response or partial response, and non-responders comprised stable disease or progressive disease. Long-read sequencing data was downloaded from the Sequence Read Archive (SRA) using the accession number SRP036136 and aligned with HISAT before visualisation. Identification of possible upstream regulators was carried out using iRegulon29 for Cytoscape v3.2.1. ENCODE ChIP-seq data from samples GM12891, GM12878 and GM19099 was analysed for RELA binding within a 10 kb window centred on the TSS of genes co-expressed with UGT2B17 by bootstrapping. Differential expression analysis for RNA-seq data was done using the edgeR v3.22.5, while microarray data was analysed with limma v3.36.5 and affy v1.58. AnnotationHub v2.12.1, Iranges v2.14.12 and GenomicRanges v1.32.7 were used for analysis of ChIP-seq data. Clustering and functional enrichment analyses were performed using clusterProfiler v3.8.1, coseq v1.4.0, ComplexHeatmap v1.18.1 and DESeq2 v1.20.0 packages for R v3.5.1 (http://www.bioconductor.org).37Public expression data from 291 CLL patients was downloaded from the International Cancer Genome Consortium (ICGC) with the project code CLLE-ES.UGT2B17_n2 promoter (including exon 1c) was PCR amplified from LNCaP cell line genomic DNA using Phusion DNA Polymerase  as recommended, with the following primers : forward 5′-CTAGCAGACGCGTGAGATCCTAGTAGGAGGTTTTGGC-3′ and reverse 5′-CTAGCAGCTCGAGCAAGTTCCAGATGTCCAGACTC-3′. The PCR fragment was then digested with MluI and XhoI restriction enzymes and inserted in pGL3 basic vector (Promega) digested with the same enzymes, using the Rapid DNA Ligation Kit (Roche). Construct was verified by Sanger sequencing. MEC1 cells were co-transfected with 9.5 µg pGL3 constructs and 0.5 µg pRL-null basic renilla (Promega) using the Neon Transfection System (Thermo Fisher Scientific). Cells were then harvested, lysed and assessed for luciferase activity using the dual-luciferase reporter assay kit (Promega), as per manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly luciferase to renilla activity, relative to the pGL3 control. Assays were replicated four times in triplicates.The sequence corresponding to 2.5 kbp of P-values were calculated using Student’s T-test unless otherwise indicated. Differences in gene expression were considered significant if adjusted P-values were inferior to 0.05.Results from glucuronidation assays represent a minimum of two independent experiments. All other results represent at least three independent experiments. Statistics were calculated using GraphPad Prism v5  or R v3.5.1. 23 microarray data analysis of RNA from CD19+-sorted B-cells (GSE15490) showed that UGT2B17 was induced shortly after treatment initiation in 6 of 11 non-responders whereas it was induced in only 2 responders out of 9 (P = 0.0007) , with all cases showing low expression of all other UGT isoforms. This was sustained by the analysis of RNA sequencing data from 291 CLL cases indicating that UGT2B17 predominates in leukaemic cells  values higher by 1.5 to 4.3-fold (P < 0.013) for fludarabine and ibrutinib (MEC1 and JVM2) as well as idelalisib, chlorambucil and venetoclax (MEC1) established by MTS assays  Fig. . Patientlls Fig. . Cells oys Table .Fig. 1ExUGT2B17 expression in MEC1, JVM2, and EHEB cellular models  and was associated with elevated enzyme activity for DHT glucuronidation, a substrate for the UGT2B17 enzyme    Fig. . The low01) Fig. . Consist 2) Fig. . In the  2) Fig. . As obse 2) Fig.  comparat 2) Fig. . A more  2) Fig. .Fig. 2DrGiven the higher and induced expression of UGT2B17 in patients not responding to fludarabine-containing treatment regimen, we explored whether UGT2B17 may be involved in the glucuronidation of the drug itself. We also examined the conjugation of additional agents relevant to CLL. Our observations in CLL patients treated with fludarabine raised the possibility that UGT2B17 may be involved in the glucuronidation of the drug itself, and potentially also of B-cell receptor inhibitors ibrutinib and idelalisib, which contain functional hydroxyl and amino groups susceptible for conjugation into inactive glucuronides (G). Glucuronidation assays using pooled human liver microsomes enriched in UGT enzymes led to the formation of polar G derivatives. This was confirmed by their fragmentation patterns assessed by mass spectrometry (MS), namely a loss of the GlcA moiety corresponding to a m/z shift of 176 Da Fig. . Fludaran = 8) and UGT2B (n = 6) enzymes, using quantitative MS methods , 3 weeks to 2 months (T1), and between 4 and 9 months (T2) after treatment initiation  but with a considerable patient-to-patient variability (CV = 190%). Lastly, we explored whether glucuronidation may be involved in the conjugation of additional anti-leukaemics and the potential involvement of UGT2B17. MS analysis confirmed the formation of at least one G product by livers following incubations with bendamustine, chlorambucil and targeted agents venetoclax, acalibrutinib, cerdulatinib and duvelisib  involved, based on UGT expressed in metabolic liver, kidney and intestine tissues, and individual recombinant UGT1A  signalling pathway were significantly enriched in cluster 3  and exon 1b (P2) were evidenced in luciferase assays , P2 and P3 enhanced luciferase gene expression by 2.5- and 16.3-fold, respectively, in MEC1 cells -dependent mechanism involving the regulation of UGT1A protein stability in AML.UGT2B17, and high UGT2B17 expression was associated with reduced sensitivity to these drugs in B-cell models. While an influence of other drug-related mechanisms, including transporters and drug metabolising enzymes is possible, CYP3A4, which is another key metabolising enzyme for these drugs,39 was not perturbed in patients treated with fludarabine (not shown). It raises the possibility that therapeutic pressure induces UGT expression in B-cells. This rapid adaptation of neoplastic cells in the presence of a cytotoxic stressor and targeted therapies support that the UGT metabolic pathway is highly relevant in leukaemia, has the potential to affect drug response locally in malignant cells, and may be useful in predicting response to several CLL therapies. We also demonstrated that the glucuronidation pathway is involved in the conjugation of a number of additional anti-leukaemics  and that UGT2B17 plays a role in the inactivation of chlorambucil and cerdulatinib. These two anti-leukaemic agents target different cellular pathways than fludarabine and have distinct modes of action. High UGT2B17 expression may thus lead to lower response to these drugs in CLL patients but this remains to be demonstrated.Treatment with targeted agents ibrutinib and idelalisib also resulted in a marked transcriptional up-regulation of UGT2B17 transcription remain poorly understood and especially in lymphoid cells. We provide the first evidence that UGT2B17 expression in B-cells is driven by a non-canonical UGT2B17 promoter and the use of an alternative noncoding exon 1c coupled to the common protein-coding region leading to the canonical UGT2B17 enzyme. According to our analysis, the gene expression signature associated with high UGT2B17 expression in CLL patients and cell models comprises a number of genes targeted by NF-κB. This promoter may thus be targeted by NF-κB that plays a central role in CLL. The NF-κB pathway is associated with poor prognosis and response to drug treatment, and represents an emerging drug target in CLL.44 The mechanism underlying high UGT2B17 expression in B-cells remains to be fully explored.Crucial parts of the machinery governing 18Our data also point to other mechanisms of altered drug sensitivity associated with high UGT2B17 expression. The inactivation of ibrutinib and idelalisib is largely dependent on UGT1A4 as the conjugation of other anti-leukaemics tested herein . UGT1A4 is far less abundant than UGT2B17 in leukaemic B cells, and undetected at the mRNA level in most untreated CLL patients. Given that ibrutinib is mainly administered as an oral agent and subjected to first-pass hepatic metabolism, this drug is likely conjugated primarily in the liver expressing high levels of UGT1A4. In one of the lymphoid cell models, we showed drug-mediated induction of UGT1A4, but this finding remains to be demonstrated in CLL patients. Induction at the protein level also requires examination, given the recent report in drug-resistant AML cells suggesting reduced mRNA expression but an enhanced UGT1A protein expression mediated by increased protein stability upon drug treatment.22 which could favour progression in untreated CLL cases and subsequent drug resistance in treated patients. In our transcriptomic analysis, AMPK signalling was negatively associated with high UGT2B17 expression in conditions of both induced and high basal UGT2B17 expression in cell models and CLL patients, potentially linking B-cell metabolism to the glucuronidation pathway and subsequent adverse clinical outcomes. AMPK is a major regulator balancing energy supply and ultimately protects cells from harmful stresses by the coordination of multiple metabolic pathways.45 The activation of the AMPK pathway has been shown to affect growth and apoptosis in CLL.46 As a stress-response molecule mediating drug resistance through different mechanisms, AMPK is further involved in the metabolism reprogramming and induction of autophagy, also regulating the self-renewal ability of cancer stem cells.47 More recently, ibrutinib resistance was associated with a metabolic rewiring in CLL.48 Likewise, UGT proteins were shown to be part of complex protein networks. Their functional interaction with other metabolic proteins induced broad changes in cell metabolism and may contribute to tumorigenesis and drug response.50Consistent with its regulatory function of the levels of endogenous molecules, high UGT2B17 may deplete intracellular metabolites leading to aberrant cell signalling and dysregulated cell functions,50 values of tumour cells, namely for ibrutinib, were in the same range as those reported previously in B lymphoblastoid leukaemic cell lines and in primary cells from patients,53 supporting the relevance of our initial observations. The impact of UGT2B17 expression on response to drug in vivo and ex vivo, as well as the influence of the microenvironment on UGT2B17-related responses, remains to be examined in more details.The findings of this study have to be seen in light of some limitations, including the fact that drug cytotoxicity and treatment outcome in relation to UGT2B17 expression was investigated in cell models and a limited number of fludarabine-treated patients. However, IC19 Our observations warrant additional studies to appreciate the prevalence and the clinical implications of high UGT2B17 expression on outcomes of leukaemia patients.In summary, we unveiled a biochemical underpinning of reduced drug sensitivity related to the UGT2B17 metabolic pathway and drug inactivation. The evidence provided should prove useful for understanding and potentially overcoming drug refractoriness. The impact of glucuronidation in the inactivation of a number of anti-leukaemic drugs is underestimated since we established that most agents are subjected to this metabolic process, including ibrutinib, idelalisib, venetoclax and duvelisib as well as other small molecules under development such as acalibrutinib and cerdulatinib. This may well apply to a number of other cancer therapeutics given the recent report that GLI1-inducible glucuronidation imparts resistance to a broad spectrum of compounds including FDA-approved drugs such as methotrexate.Supplementary information


Alzheimer’s disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood–brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches. Fovessels . As a coovessels ,46. As s 0.0005) B.1–40Cy5 , suggesting that KBs are able to partly enhance basolateral-to-apical Aβ peptide transport. In fact, no significant difference in Aβ1–40Cy5 transport was observed in the other KB conditions. The values for [3H]inulin transport did not differ significantly in the presence of KBs , confirming that the BBB model was not compromised by KBs.Only the basolateral-to-apical transport of Aβ1–40Cy5 D was sige of KBs D, confire of KBs A,C afterand P-gp ,47,48,49These observations suggest that only the combination of the two KBs was able to increase basolateral-to-apical transport of Aβ peptide. The use of LRP1 and P-gp inhibitors demonstrated the functional involvement of LRP1 and P-gp activity in the efflux of Aβ peptide across the BBB.+ cells) and brain pericytes inulin (1.25 Ci. mmol−1) was purchased from Analytical Sciences . Fluorescent human amyloid beta-peptide (1–40)-Cy5 labeled (Aβ1-40Cy5) was purchased from Phoenix Pharmaceuticals . The powder was resuspended in 250 µL of DMSO  followed by an addition of 750 µL of RH-HSA 0.1% to obtain a solution at 1 µM, according to the manufacturer’s instructions, and stored at –20 °C to be used at 10 nM in RH-HSA 0.1% buffer. Receptor-associated protein  was purchased from Merck Millipore, dissolved in RH-HSA 0.1%, and freshly used. Elacridar was purchased from Sigma-Aldrich (United Kingdom), dissolved in DMSO at 0.5 mM, and stored at -20 °C to be used at 0.5 µM in RH-HSA 0.1% buffer.Endothelial cell medium  supplemented with 5% heat-inactivated fetal calf serum , 50 µg·mL+ cord blood hematopoietic stem cells with brain pericytes as previously described by Cecchelli et al. (2014)  assay . For each condition tested, cells were seeded in triplicate and incubated during 48 h in the absence or in the presence of different concentrations of KBs  once the BBB phenotype was established. Following treatments, 150 000 BLECs/insert were counted for each condition. MTT was added to the apical compartment for 2 h at 37 °C and 5% CO2. During this time, the glucose and βHB concentrations were measured in both the apical compartment and the basolateral compartment. The Precision-Xtra meter  with a glucose or β-ketone sticks was used. In these assays, the glucose and βHB concentrations measured by the sticks were representative of the glucose and βHB present in the culture medium. Data were expressed in mmol·L−1 for βHB and in g·L−1 for glucose. The βHB concentration within BLECs was measured at 48 h following the treatment. Briefly, after medium removal, BLECs were washed twice with RH buffer and lysed with RIPA lysis buffer . The βHB concentration in the BLECs was normalized by the protein amount using Bradford assay . The amount of βHB into the supernatant was assayed using β-ketone sticks, as described above. The βHB concentration was verified in the culture medium without cells at 37 °C and 5% CO2 to ensure the stability of βHB.After 5 days of co-culture, 20 mM AcAc or 20 mM βHB or 20 mM AcAc/20mM βHB (ratio treatment) was added to the apical compartment for 48 h at 37 °C and 5% COThe integrity of the BLEC monolayer after 48 h of treatment with KBs was evaluated using the method described by Dehouck et al. (1992) . Each co−1 KH2PO4 , 8.0 g·L−1 NaCl, 2.87 g·L−1 Na2HPO4-12H2O, and 0.2 g·L−1 KCl) with 10% (v/v) normal goat serum (NGS) was performed. Then, cells were incubated for 60 min with the primary antibody in PBS-CMF supplemented with 2% NGS at room temperature (RT), against the target protein as described in After 48 h of treatments with or without KBs, BLECs were fixed and permeabilized as described in After three washes in PBS-CMF supplemented with 2% NGS, the cells were incubated with secondary polyclonal antibody  diluted 1:500 in PBS-CMF supplemented with 2% NGS in the dark for 30 min at RT. Hoechst 33358 was used for nuclei staining. After mounting with Mowiol (Sigma-Aldrich) containing 1,4-diazabicyclo [2.2.2]octane (Sigma-Aldrich), images were taken using a Leica microscope  and processed using ImageJ32 software.After 48 h of treatment, BLECs were washed twice with cold RH buffer and scraped with 50 µL UT4 lysis buffer  or 50µL RIPA 1X lysis buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl)  supplemented with a cocktail of protease and phosphatase inhibitors purchased from Sigma-Aldrich . Lysates were then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was saved and sonicated three times at 40 W for 5 s. The lysate protein concentration was measured using the Bradford method. Then, 30 µg of total protein for LRP1 or 20 µg for the other proteins were mixed with Laemmli  supplemented with beta-mercaptoethanol  and boiled for 5 min at 95 °C. Details of the denaturation and reduction step of the protein extract are described in Then, the protein mixes were electrophoresed using Criterion™ TGX™ (Tris-Glycine eXtended) precast gels (Bio-rad) and subsequently electrotransferred to nitro cellulose membranes . Nonspecific binding sites were blocked by incubating the membrane with Tris-buffered saline 0.1% Tween 20  supplemented with 5% skimmed milk at RT for 1 h under agitation. Membranes were incubated in the appropriate primary antibodies  and incu1-40 across BLECs was investigated as previously described inulin was performed in the same way as the Aβ1-40Cy5 peptide transport, thus representing a paracellular marker. The BBB integrity was checked by measuring the PeLY as described in 1-40 transport was performed by adding 10 nM of Aβ1-40Cy5 peptide with or without 200 nM of RAP in the basolateral compartment. For P-gp inhibition studies, the basolateral-to-apical transport of Aβ1-40 transport was performed by adding 10 nM of Aβ1-40Cy5 in the basolateral compartment with or without 0.5 µM of elacridar in the apical compartment. All transport studies were performed in triplicate on a rocking platform at 37 °C for 30 min. At the end of the incubation period, aliquots of the donor and receiver solutions were collected, and the fluorescence compounds (Aβ1-40Cy5 and LY) were measured with a spectrofluorimeter (Synergy H1). The radioactivity of [3H]inulin was measured using a scintillation counter . The permeability  of Aβ1-40-Cy5 or [3H]-inulin was calculated according to the following equation: (1) Papp = (k.V(R))/(A.60) as previously described [−1) defined as the slope obtained by linear regression of the cumulative fraction absorbed (FAcum) as a function of time (min), V(R) was the volume in the receiver chamber (mL), and A was the area of the filter (cm2). Determination of the cumulative fraction absorbed (amount permeated), FAcum versus time, FAcum was calculated from: (2) FAcum = ∑ (C(Ri))/(C(Di)), where C(Ri) was the receiver concentration at the end of the interval i and C(Di) was the donor concentration at the beginning of interval i. A linear relationship should be obtained. The apical-to-basolateral and the basolateral-to-apical Papp were determined by Equation (1) for each molecule. For each filter, the calculated mass balance was determined as described in Forty-eight hours after KB treatments, the apical-to-basolateral and the basolateral-to-apical transport of Aβescribed ,44,46 . Data are presented as the mean ± standard error of the mean (SEM). The results of experiments were evaluated using the Mann Whitney t-test. The threshold for statistical significant was set to *