Uridine diphosphate-glucuronosyltransferases (UGTs) are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER) of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes). They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body’s chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon. They arUGTs are type-I transmembrane glycoproteins mainly located within the smooth endoplasmic reticulum (ER), although some isoforms can be found in the nuclear envelope ,4. UGTs 2) such that the compounds can be conjugated by phase 2 enzymes such as the UGTs. This necessitates the phase 1 metabolites crossing the ER membrane in order to access the UGT active site. Evidence suggests a functional interaction between the UGT and CYP proteins to facilitate the multistep detoxification process [Detoxification is divided into two phases . The bas process ,12,14,15Besides detoxification, glucuronidation can activate substrates such as certain procarcinogens, as well as morphine that can be converted to morphine 3- and 6-glucuronides, where the latter is a more potent analgesic than the parent drug . Glucuroα-toxin, or the pore-forming antibiotic alamethicin [The majority of the UGT enzyme protein, including its catalytic site, is believed to reside inside the ER lumen ; thus there are physical and physiological barriers to substrates accessing the active site of the enzymes within the ER lumen, as well as to the egress of reaction products from the ER and, subsequently, from the cell. This leads to the phenomenon of latency of UGT activity in preparations of ER . The phenomenon of latency derives from experimental observations that in isolated microsomal preparations, UGTs generally show increased activities after the membranes are physically or chemically disrupted ,26,27,28methicin ,37,38,39Three major hypotheses have been developed over the years to explain latency: compartmentation, conformation, and adenine-nucleotide inhibition. The first two hypotheses mainly address whether the ER membrane limits UGT activity by acting as a barrier blocking access of UDPGA  to the luminal space and thus the UGT active site, or by regulating the conformation of the UGT protein through lipid–protein  interactions. The last hypothesis was the most recently proposed, based on the inhibitory effects of adenine-containing nucleotides on the UGTs. Under this hypothesis, the membranes might limit UGT activity by sequestering these natural inhibitors in the lumen.The latency phenomenon makes accurate measurement of UGT activity in microsomal preparations challenging, however it is one of the most important factors to take into consideration in the design of UGT enzyme assays. Different methods of preparation of the microsomal fraction can have major impact on measurable latency, since the level of intactness of the membrane preparation clearly influences the enzyme activity. For example, it has been shown that preparation of microsomal fractions from freshly isolated liver is significantly preferable over frozen tissue, which tends to produce microsomal fractions with much lower levels of intactness . SimilarHere we review the major hypotheses that have been proposed to explain the phenomenon of latency as it applies to UGTs, and discuss future studies that might be needed to gain a greater understanding of its origins and implications.This hypothesis is based on the assumption that the highly hydrophilic co-substrate for the glucuronidation reaction, UDPGA, (and possibly certain aglycone substrates) is not able to diffuse across the ER membrane, and therefore that disruption of the membrane integrity is required to reveal the full extent of in vitro UGT activity in microsomal preparations. The discovery of transporters for nucleotide sugars, including UDPGA, has provided very strong evidence in support of this hypothesis.The barrier function of microsomal membranes was proposed by Winsnes in 1972  and was N-glycosylated in the ER lumen. Notably, other internal topological elements within the UGTs might be required to regulate the integration process, as demonstrated for UGT1A6 [The identification of complete cDNA sequences improved the understanding of many aspects of UGTs such as the ER targeting process and topology . Initialr UGT1A6 ,50. In tEscherichia coli, respectively [Although the intimate association between UGT protein and the ER membrane has so far prevented the generation of a complete crystal structure, the predicted topology of UGTs has been well developed. Vanstapel and Blanckaert predicted the catalytic site of bilirubin UGT faces ER lumen , which wectively ,61.N-acetylglucosamine (UDP-GlcNAc) has been key to advancing the compartmentation hypothesis.In addition, a transporter (or transporters) might be needed to facilitate the efflux of polar glucuronides out of the ER; this transporting activity has been biochemically demonstrated with a rate comparable to the rate of glucuronide synthesis . InteresIt is well known that UDP-GlcNAc can activate UGT activity in microsomal preparations in the presence of UDPGA, having been first demonstrated by Pogell and Leloir, who suggested the effect was partly due to inhibition of the breakdown of UDPGA . Later wN-ethylmaleimide (NEM) blocked the activation effect of UDP-GlcNAc on native UGT activity within intact microsomes, without a direct inhibition on the enzyme’s catalytic site [Further evidence came from the observations that addition of tic site . This pitic site ,69. HoweThe discovery of UDPGA transporters was preceded by studies recognizing a carrier-mediated, UDPGA transport activity at the rat liver ER membrane ,69,70,71N-acetylgalactosamine  into microsomal vesicles when expressed in Saccharomyces cerevisiae [S. cerevisiae) transported UDPGA in a manner that was much enhanced by UDP-GlcNAc and showed that UGTrel7 was a UDPGA/UDP-GlcNAc antiporter [A number of nucleotide sugar transporters (NSTs) have now been cloned, expressed and functionally characterized. UDP-galactose transporter-related protein 7 (UGTrel7), a human NST, was shown to transport both UDPGA and UDP-revisiae . Kobayasrevisiae . These ttiporter . Therefotiporter ,78, thesfrc) that encodes an NST transporting UDPGA, UDP-GlcNAc, and UDP-xylose from the cytoplasm to the lumen of ER/Golgi in Drosophila [hfrc 1 homologous to frc (Drosophila melanogaster), sqv-7 (Caenorhabditis elegans), and UGTrel7 (human) [UDPGA transport is a complex process, and probably involves other transport molecules, as described by Rowland et al. who demonstrated that UDPGA uptake into human liver microsomes involves more than one transporter . Other Nosophila ,81. Inte (human) . It encoThe concept that the conformation of the UGT enzyme protein is subject to influence by the hydrophobic lipid environment in the ER membrane, and it is this that controls the activity of enzymes, is the underlying basis of the conformation hypothesis. In contrast to the compartmentation hypothesis, the conformation hypothesis explains latency by assuming that the catalytic site of UGTs is accessible by substrates and UDPGA and the membrane disruption induces the formation of different kinetic forms of the enzyme with increased activity . Vmax towards p-nitrophenol, including the addition of phospholipase A or Triton X-100, and sonication [Vmax of UGTs could be due to a change in the protein–lipid interaction or greater access of substrates to the catalytic site after the membrane integrity had been lost, or a combination of both. Because Triton treatment only sped up the forward reaction which is the glucuronidation reaction catalyzed by UGTs [According to Vessey and Zakim, treatments altering the lipid portion of bovine liver microsomes increased UGT activity at nication . These t by UGTs , the act by UGTs ,83. Henc by UGTs . They cop-nitrophenol UGTs follow a random order kinetic mechanism after they carried out product inhibition and isotope exchange studies, and so the enzymes can firstly bind substrate or co-substrate and form rapid equilibrium [p-nitrophenol, UGTs showed increased values of Vmax and Km UDPGA, which suggested that occupation of the substrate binding site decreased the binding affinity for the co-substrate. The interaction between substrate and co-substrate binding sites was further investigated by Hochman and Zakim who used an enzyme reconstitution system where a delipidated form of purified UGT, called GT2p, was reconstituted into different species of phospholipids to study their influence on the enzyme properties [2p represented the second peak of eluted UGT after a hydroxylapatite column purification from pig liver microsomes, and showed a single band of protein following sodium dodecyl sulfate—polyacrylamide (7.5%) gel electrophoresis (SDS-PAGE) [2p activity affected the extent to which substrate binding decreased the binding affinity of the co-substrate. In parallel with this result, the α-glucuronidase activity of GT2p was differentially affected by the binding of structural variants of aromatic ethers that only occupy the substrate binding site without being conjugated. Therefore, the binding of a substrate seems to induce a conformational change in the enzyme that possesses an altered co-substrate binding affinity, which is dependent on the surrounding lipid environment.The interaction between substrate and co-substrate binding sites within a single UGT protein was another piece of evidence used to support the existence of multiple conformational forms ,85. Vessoperties . GT2p reDS-PAGE) . It was 2p was reconstituted into bilayers of 1,2-dimyristoylphosphatidylcholine or 1,2-dipalmitoylphosphatidylcholine lipids in the gel phase, the enzyme displayed non-Michaelis–Menten kinetics, however when the lipids were heated to liquid–crystal phase, the enzyme(s) displayed Michaelis–Menten kinetics [2p demonstrating non-Michaelis–Menten kinetics had two different binding sites for UDPGA per molecule of enzyme, and the appearance of Michaelis–Menten kinetics after phase transition occurred was in parallel with the disappearance of one binding site [1p, the first eluted fraction from a hydroxylapatite column as described by Hochman and Zakim [Vmax. This result further suggested that the interaction between UGTs and phospholipids does not depend on the specific chemical structures of phospholipids. Rather, as Rotenberg and Zakim suggested, the interaction occurs through a non-specific mechanism depending on the physical state of phospholipids [Further experiments suggested that the physical properties, rather than the chemical structures, of phospholipids surrounding UGTs seemed to play a role in the interaction with the enzymes and regulation of their kinetic patterns. For example, when GTkinetics ,88. The ing site . These ring site . For insnd Zakim , the lysholipids .2p reconstituted into different species of lysophosphatidylcholine [2p from oleoyl lysophosphatidylcholine to stearoyl lysophosphatidylcholine led to changes in the rate of glucuronidation depending on the specific aglycone substrates: a 20-fold decline for p-nitrophenol, no change for 1-naphthol and phenol, and a 3-fold decline for p-bromophenol. Hence, the specific lipid environment has a selective effect on the glucuronidation rate towards different aglycones. Two mechanisms have been proposed as to how the lipid–protein interaction affects the catalytic rate of UGT . Firstly, phospholipids may regulate enzyme–substrate interactions and determine how much inherent binding energy to UDPGA can be shared for catalysis to enhance the reaction rate [The experiments described above repeatedly emphasize the role of the lipid environment in the regulation of UGT conformation. The change in conformation can in turn alter the rate of glucuronidation presumably by changing the catalytic mechanism, as demonstrated for GTlcholine ,92. Specion rate .2p fraction of UGTs existed in three forms through conformational changes: the active form (E), inactive form (E’), and denatured form. The change between E and E’ is controlled by temperature and is reversible, whereas the denatured form is obtained by heating E’ to a certain extent. The specific temperatures required for the occurrence of these conformational changes are dependent on a specific lipid environment. In general, the reconstitution of pure, delipidated GT2p into different species of phosphatidylcholine, such as distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or 1-stearoyl-2-oleoylphosphatidylcholine, stabilized the E form with a greater change in the entropy of the whole system rather than the enthalpy, and slowed down denaturation rate of the E’ form. However, each of these phospholipids affected the thermal stability of GT2p to a different extent depending on the nature of the acyl chains, so the increase in temperature might alter enzyme conformation through modulating the physical state of phospholipids. Using the reconstitution method, Hochman and Zakim showed that only gel-phase phospholipids could reconstitute the sensitivity of the purified GT2p fraction to stimulation by UDP-GlcNAc, a physiological activator of UGTs [2p seems to have a favorable interaction with viscous lipids [Besides kinetic properties and avidity for substrate binding, the lipid environment also regulates the thermal stability of UGTs . Rotenbe of UGTs . Also, Gs lipids . These ds lipids ,90. The Vmax; diet supplementation that increased the membrane content of cholesterol in guinea pig liver ER showed similar results to that of the in vitro data. In both cases, the activity of UGTs was studied under the native membrane environment. Rotenberg and Zakim reconstituted delipidated GT2p into different phospholipids to study the influence of the lipid environment on the observed cholesterol effect [Alternatively, the gel-like regions might be formed by lipid rafts, membrane microdomains that are enriched in cholesterol, sphingolipids, and specific proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and Src-family kinases . These ml effect . They shBecause lipid rafts have been extensively studied with much evidence supporting their existence in living cells, technologies have been developed to monitor raft dynamics such as single molecule tracking together with advanced microscopic analyses ,97. Thes2p may be artificially affected by other components more than the phosphatidylcholine variants, and may not faithfully reveal UGT–phospholipid interactions. Also, the native structure and function of membrane proteins might be altered during purification before the enzymes are reconstituted into a new lipid environment. Lastly, if UGTs truly have a significant interaction with surrounding phospholipids in the ER membrane, then it is essential that the enzymes are reconstituted into a native lipid environment for functional studies [2p reconstituted in phosphatidylcholine variants, were mainly carried out on pig liver microsomes, since the activity of p-nitrophenol UGTs was greater than that from cow liver microsomes and so was easier to measure [p-chloromercuribenzoate as the membrane perturbants [p-nitrophenol which can be glucuronidated by more than one isoform such as UGT1A6 and 1A1 [The challenges of using purified membrane protein preparations on the outcome of these experiments has been mentioned several times by Zakim’s group ,87,90. I measure . It woulturbants . The ext and 1A1 . This meIt is true that the compartmentation hypothesis is more widely accepted in the field, however the studies by Zakim and colleagues, and others, provide a rich reservoir of data that do indicate that the regulation of UGT activity within the ER membrane environment is complex and may well involve multiple mechanisms.p-nitrophenol, estradiol, and estrone were suppressed by 4 mM ATP (adenosine triphosphate) in guinea pig liver microsomes [+ , and NADP+  inhibited the glucuronidation of 4-MU and estradiol by allosteric binding to UGTs at a site independent from the substrate and co-substrate binding sites, and proposed that membrane perturbations could lead to the release of these inhibitors and hence an increase in glucuronidation activities [+ were observed on the UGTs after Brij58 solubilization of the membranes, which suggests the allosteric binding site is luminal [+, or NADP+. Recently, the inhibitory effects of adenine and adenine-containing nucleotides on UGT activities have been proposed as another explanation of latency ,104,105.crosomes . Then, Ntivities . When Ni50 value measured from Brij58-treated human liver microsomes (33.8 μM), but much lower than that from rat liver microsomes (66.8 μM). Therefore, under physiological conditions, the level of ATP in the ER lumen might be sufficient to suppress UGT activity. These authors also showed that Brij58 and alamethicin treatments could lead to the release of ATP from rat liver microsomes in vitro. Two structural components have been thought to be important for inhibitory nucleotides to bind the allosteric effector site of UGTs [+, and NADP+, and so was thought to compete with the other adenine nucleotides for the common allosteric binding site. The second structural component is a di- or triphosphate moiety attached to the 5’-position of ribose, because neither AMP nor adenosine showed an inhibitory effect on the UGT activity. However, because adenine was able to inhibit UGT and lacks a phosphate group, the significance of the number of phosphates attached to ribose on the inhibitory effect of adenine nucleotides is called into question. Besides adenine-containing nucleotides, GTP and CTP are the only guanine- and cytidine nucleotides that inhibited UGTs, respectively, using 4-MU as the aglycone substrate [50 values, and so comparable inhibitory effects. GTP, however, was much less potent than ATP.In 2012, Ishii et al. compared alamethicin- and Brij58-treated microsomes from rat and human livers, and showed the inhibitory potency of adenine nucleotides was lower in the alamethicin-disrupted microsomes . The lum of UGTs ,105. Theubstrate ,105. CTP2+.UDP is a well-known inhibitor of UGTs. However, unlike adenine nucleotides, UDP is believed to compete with the co-substrate UDPGA for binding to UGTs, hence exerting the inhibitory effect on glucuronidation ,107,108.+, could play a role in the regulation of glucocorticoid levels [+ to produce cortisone from cortisol in hepatic ER lumen. As the level of NADP+ decreases, the inhibitory effect on the UGTs is also decreased, which enables UGTs to glucuronidate excess cortisol. Above all, information on the ER concentrations of adenine nucleotides relative to their cytosolic concentrations and IC50 values is needed to further investigate the physiological effects of the nucleotides on glucuronidation and metabolism.Despite being a possible explanation of latency, the adenine nucleotides, particularly NADPd levels ,109. SpeThe controversy over the underlying mechanisms that explain the latency of UGT activity that is observed in vitro in microsomal preparations has continued for more than half a century. It is clear that disruption of the microsomal membrane by physical and chemical treatments is necessary to reveal full UGT enzyme activity in vitro. Based on the available evidence, it is likely that much of the effect is due to the disruption of the membrane allowing increased access of UDPGA  to the active site of the enzymes. Critical evidence for this comes from the existence of transporter(s) for UDPGA that presumably regulate access of the co-substrate to the enzyme in vivo. However, it is equally clear that UGT enzyme activity can be regulated by membrane components including phospholipids, cholesterol and indeed other proteins, including other UGTs and CYPs. Some of the effects of membrane disrupting agents such as detergents may well be mediated through their impact on the membrane environment as well as on the UGT proteins themselves. Readers may ask “why does this matter?”, particularly in a time when we know much about the biology, genetics and regulation of UGTs and glucuronidation. One of the main reasons it remains important is that we are still unable to accurately extrapolate in vitro measurements of UGT activity to estimates of in vivo clearance of drugs that are metabolized by glucuronidation. This in vitro–in vivo extrapolation (IVIVE) consistently underpredicts clearance in vivo for such drugs despite significant advances in physiologically-based pharmacokinetic (PBBK) analysis and a far more sophisticated understanding of the physiological factors that influence UGT activity ,111. We


Rhodiola rosea (R. rosea) product has previously demonstrated CYP2C9 inhibition in humans. The purpose of this study was to provide in vitro inhibitory data for this particular interaction and to classify the mechanism of the interaction. Another aim was to examine the in vitro influence of ethanol on the CYP2C9 activity. Human CYP2C9 (wild type) isolated from a baculovirus‐infected cell system was incubated with 0.8 μmol/L losartan for 20 min. Sulfaphenazole was used as a positive control. The commercial R. rosea product “Arctic Root” was used as test inhibitor. Formation of the CYP2C9‐produced losartan metabolite EXP‐3174 was determined by validated LC‐MS/MS methodology. Possible mechanism‐based (irreversible) inhibition was evaluated using time‐ and NADPH‐dependent inhibition assays. Kinetic constants  were calculated from a Lineweaver‐Burk plot. Mode of inhibition was determined. CYP2C9 was inhibited by “Arctic Root” with an IC50 (extract concentration yielding 50% reduction in enzyme activity) of 19.2 ± 2.7 μg/mL. Inhibitor concentrations of 20 μg/mL and 40 μg/mL yielded Ki values of 16.37 μg/mL and 5.59 μg/mL, respectively. The Lineweaver‐Burk plot showed noncompetitive inhibition mode. No time‐ or NADPH‐dependent inhibition was observed. The presence of ethanol inhibited CYP2C9 activity in a concentration‐dependent manner. In conclusion, the commercial R. rosea product “Arctic Root” demonstrated noncompetitive inhibition of CYP2C9 in vitro. Further work identifying the constituents responsible for this inhibition is needed.A commercial  AUCarea under the curveISinternal standardRhodiola rosea L. (Crassulaceae) is a plant in the Crassulaceae family, and is mainly found in arctic and mountainous habitats  enzymes. A previously published study from our group found an in vitro inhibition of CYP3A4 enzyme activity by extracts of six R. rosea clones, with IC50 values (extract concentration yielding 50% reduction in enzyme activity) ranging from 1.7 to 3.1 μg/mL  or anticoagulant activity. This result was opposed by Spanakis et al. , salidroside, and tyrosol have been suggested as bioactive constituents , and Solution B  were purchased from BD Biosciences . All other chemicals were of HPLC grade.Losartan potassium , sulfaphenazole , and caffeine  were obtained from Sigma‐Aldrich . EXP‐3174 (lot no. 200013283) was kindly donated from Merck . Baculovirus expressed human wild‐type CYP2C9*1 , NADPH regenerating system Solution A  was obtained from a store for herbal drugs.The commercial One “Arctic Root” tablet was weighed, grounded in a mortar, and dissolved in 15 mL 50% ethanol. Herbal constituents were extracted at 37°C for 1 h. The extraction solution was transferred to a falcon tube, centrifuged at 1562 xg for 10 min, and decanted into a new container. The residue was added 5 mL of 50% ethanol and the extraction process was repeated. The first and second extracts were pooled and the extract solution was evaporated to dryness at 40°C under a gentle stream of air overnight and weighed. Dried extracts were kept at 4°C, avoiding light. Before experiments, the extract was dissolved in a small amount of 50% ethanol to make herbal stock solutions with known concentrations. Shelf life was set to 2 weeks.50 experiment (0–100 μg/mL) was expected to cover (and exceed) the herbal concentrations occurring in the small intestine, liver, and blood in vivo. Our estimates  and a NADPH regenerating system . R. rosea, the positive control inhibitor sulfaphenazole (1.0 μmol/L) or buffer/ethanol was added in volumes of 100 μL. As the herbal solution contained ethanol, all incubations were performed in 0.8% ethanol with adequate controls. The total incubation volume was 400 μL. After a 5 min acclimatizing in the water bath, the reaction was initiated by adding 20 μL of the regenerating NADPH system. The reaction was terminated on ice by the addition of 200 μL stop solution  caffeine). The formation of EXP‐3174 was linear from 5 to 20 min with CYP2C9 concentrations up to at least 30 nmol/L and a losartan concentration ranging from 0.05 to 40 μmol/L.CYP2C9 (12.5 nmol/L) was incubated in conical glass tubes in a shaking water bath for 15 min at 37°C in a 0.1 mmol/L potassium‐phosphate buffer (pH 7.4) containing losartan , whereafter the substrate was added to a concentration of 0.8 μmol/L and the incubation was continued for another 15 min. The reaction was terminated on ice by adding 200 μL of stop solution.For evaluation of mechanism‐based inhibition, time‐ and NADPH‐dependent inhibition assays were applied. In the time‐dependent assay, CYP2C9 was preincubated at 37°C with R. rosea as described above, but in the presence and absence of the NADPH regenerating system for 0 and 45 min. The reaction was initiated by adding substrate and continued as described above.The NADPH‐dependent assay was performed by preincubating CYP2C9 with EXP‐3174 was analyzed by previously published validated LC‐MSMS methodology, with minor adjustments .The ICKm and Vmax values for CYP2C9‐mediated metabolism of losartan were estimated by incubating CYP2C9 with increasing substrate concentrations (0.4–8.0 μmol/L), Km (app) and Vmax (app) were obtained in the presence of control inhibitor and R. rosea. Data were transformed, plotted in a Lineweaver‐Burk plot using Sigmaplot, and the mode of inhibition was determined by analyzing the plot and applying the equations presented in Table Ki was calculated by using the equations given in Table n = 3 replicates. A two‐sample t‐test was used to evaluate the effect of the herbal preparation and inhibitor control on CYP2C9 enzyme activity and linear regression analyses were performed on standard curves and inhibition plots. Dixon's Q‐test was used to identify and remove one potential outlier per dataset, if applicable  activity without inhibitor was 0.378 ± 0.061 pmol metabolite/(pmol enzyme min). The ICl/L Fig. .Figure R. rosea “Arctic Root” extract on CYP2C9‐mediated metabolism of losartan. The IC50 value was calculated to be 19.2 ± 2.7 μg/mL.Figure μg/mL), but without losartan for 0, 15, 30, and 45 min. Results were CYP2C9 activities of 49.5 ± 0.0, 53.8 ± 0.1, 54.5 ± 0.1, and 51.2 ± 0.1% compared to reference without inhibitor, respectively. NADPH dependency was evaluated by preincubating CYP2C9 with “Arctic Root” extract (20 μg/mL) with and without NADPH for 45 min. Results were CYP2C9 activities of 51.2 ± 0.1 and 52.2 ± 0.2%, respectively. No significant differences were found (two‐tailed t‐test) for time‐dependent or NADPH‐dependent inhibition.Time‐dependent inhibition was evaluated by preincubating CYP2C9 in the presence of NADPH and “Arctic Root” extract , sulfaphenazole (y = 32.3091x + 2.833), inhibitor 20 μg/mL (y = 3.1626x + 1.9445), and inhibitor 40 μg/mL (y = 19.3507x + 7.1215). Losartan concentrations for all incubations were 0.4, 0.6, 0.8, 1.2, and 8 μmol/L. Ki values were also calculated from the Lineweaver‐Burk dataset by using the equations given in Table The Lineweaver‐Burk plot is shown in Figure R. rosea product “Arctic Root” was investigated using cDNA baculovirus‐expressed human CYP2C9 Supersomes. In all experiments, specific positive inhibition control activities and analytical determination of metabolites fulfilled preset international acceptance criteria    and competitive at high concentration (40 μg/mL). This shift is uncommon, but similar findings have previously been described for gentiopicroside on CYP2A6 (Deng et al. N‐demethylation of aminopyrine by mixed function oxidase cytochrome P‐450 in rat (Roots and Hildebrandt Ki value close to the IC50 value for any inhibitor concentration. The calculated Ki for the 40 μg/mL inhibitor concentration was somewhat lower. One explanation for a shift like this, if there is any, could be linked to different constituents exerting effects toward the enzyme at different herbal concentrations. A competitive inhibitor has a Ki value of approximately half the corresponding IC50 value. In our study, sulfaphenazole, a known competitive inhibitor, had a Ki of 0.11 μmol/L and a IC50 of 0.25 μmol/L.The commercial R. rosea on CYP enzyme activity, the potential differences in biochemical activity of multiple unknown constituents must also be taken into consideration. The supposed bioactive constituents of R. rosea are salidroside, tyrosol, rosavin, rosarin, and rosin (Ming et al. R. rosea (Panossian et al. R. rosea constituents rhodiosin and rhodionin has recently been published with IC50 values of 0.47 and 0.19 μg/mL, respectively (Xu et al. R. rosea products, the specific constituents responsible for the inhibition must be identified.In the interpretation of both the in vitro and in vivo inhibitory potential and characteristics of R. rosea product “Arctic Root” and adds to the increasing evidence of both in vitro and in vivo CYP inhibitory potential of R. rosea. Further studies should evaluate possible CYP enzyme effects of other commercially available R. rosea products and in more detail attempt to identify which of the R. rosea constituents that are responsible for these effects.In conclusion, this study found a noncompetitive inhibition of CYP2C9 by the commercial The authors declare that they have no conflict of interest.Figure S1. In vitro CYP2C9 enzyme inhibition by the positive control inhibitor sulfaphenazole.Click here for additional data file. Click here for additional data file.


Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C  as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation.To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic  PIK3CA gene), and loss-of-function of the lipid phosphatase and tumour suppressor PTEN453) lipid, recruitment of PH domain-containing proteins to the plasma membrane and activation of effector proteins including AKT, mTOR and small GTPases such as Rac and RhoA789PI3K signalling is a major regulator of cell growth and metabolism and is one of the most frequently mutated pathways in cancer12Pik3ca and inactivation of PTEN are associated with overlapping but distinct tumour profiles and have different outcomes, both in the clinic and in mouse models of cancer6Pik3ca mutant tumours are often dependent on p110α121415Despite activating the same pathway, mutations in While there are a number of PI3K pathway inhibitors in clinical trials, including p110α, AKT, mTOR and pan-PI3K inhibitors, they have so far shown limited efficacy118Pik3ca mutation or Pten loss, we performed a phosphoproteomic screen using a label-free quantitative mass spectrometry method21To dissect the signalling mechanisms after Pi3kca is activated from the endogenous locus and in the heterozygous state, and in the case of Pten, often by homozygous loss. We also aimed to assess the impact of Pi3kca activation/Pten loss at short time points after induction, in the absence of any other oncogenic mutations as well as in the absence of acute stimulation. This contrasts with other studies that examine the effect of PI3K activation in transformed cell lines, at long time points after mutation (when presumably other mutations have occurred) and/or in the presence of acute stimuli. In order to achieve these aims, we took advantage of two inducible genetic models of PI3K activation: primary mouse embryonic fibroblasts (MEFs) with heterozygous expression of the Pik3caH1047R hot-spot mutation from the endogenous locus or homozygous loss of PtenPik3caH1047R/WT, Ptenfl/fl and appropriate control MEFs.We were particularly interested in identifying molecular changes that occur at early time points after mutation and in response to low-level activation as these may most closely mimic physiological signalling events. Indeed, in cancer, Pik3caH1047R and Pten loss as well as a number of cellular processes that were differentially-regulated by Pik3caH1047R expression or Pten loss. Validation of NT5C, one of the hits that was commonly-regulated by Pik3caH1047R and Pten loss, demonstrated that it is a novel AKT substrate as well identifying an unexpected role for this protein in regulating actin-dependent processes at the leading edge of the cell.Using this system we identified 7566 unique phosphopeptides from 3279 proteins. Analysis of regulated phosphopeptides identified events that were commonly regulated by both Pik3caH1047R hot-spot oncogenic mutation from the endogenous locus; further referred to as Pik3caH1047R/WT cells, with wild-type control cells referred to as Pik3caWT/WT and (2) homozygous loss of PtenPtenfl/fl, with wild-type control cells referred to as PtenWT/WT . Expression of either Pik3caH1047R/WT or loss of Pten resulted in a modest but sustained phosphorylation of downstream effectors including AKT, PRAS40, GSK3α/β and FKHR , and at short time points after induction  474 regulated phosphopeptides in the Ptenfl/fl analysis and iii) 189 regulated phosphopeptides in the Pik3caH1047R/WTversus Ptenfl/fl comparison (We defined a phosphopeptide to be regulated if changes in phosphorylation intensity (i.e. increases or decreases in phosphorylation) within a screen, or between the two screens, had a p-value of less than 0.05 (p < 0.05) . From thmparison . In all Pik3caH1047R/WT or Ptenfl/fl cells, which we defined here as either “commonly-regulated” or “differentially-regulated”, respectively  a p-value < 0.05 when comparing changes in phosphorylation intensity between the Pik3caH1047R/WT and the Ptenfl/fl screens  and these were included in the differentially-regulated list.We were particularly interested in identifying phosphosites that had either the same or different patterns of regulation in ectively . Commonlanalyses . Differe screens . There w2 = 0.92) between changes in phosphorylation intensity from the two screens, indicating that these peptides were in fact regulated in similar ways and are strong candidate PI3K effectors , Afadin (pS1795), and AAPK1 (pS496) . Furtherffectors .Pik3caH1047R/WT and Ptenfl/fl cells  between changes in phosphorylation between the two screens (Pik3caH1047R/WT and Ptenfl/fl screens. Four of these phosphorylation events (ARHGH (pS414), CETN2 (pS98), KIF1A (pT1528) and SRBS1 (pS1201)) were regulated in the same direction (i.e. upregulated in both screens or down-regulated in both screens), suggesting that they may in fact be commonly-regulated sites, differing only in the amplitude of their phosphorylation or the abundance of the total protein. However, there were two phosphosites, DNMT1 (pS958) and EMSY (pT171) that were regulated in different directions, with increased phosphorylation in Pik3caH1047R/WT cells and decreased phosphorylation in the Ptenfl/fl screen. These proteins may represent a novel group of effector proteins that are regulated independently by different modes of PI3K activation.We also identified 150 phosphopeptides that were differentially-regulated in fl cells . In supp screens . The majPik3ca- or Pten-regulated proteins , and interestingly, proteins associated with cytoplasmic vesicles. Further analysis of differentially-regulated hits, highlighted some specificity, whereby Pik3caH1047R expression was associated with regulation of proteins localized to nuclear specks and the nuclear envelope, while Pten loss was predominantly associated with proteins involved with DNA metabolism and repair.Analysis of differentially-regulated proteins  producedPik3caH1047R/WT or loss of Pten. Commonly-regulated phosphosites were enriched in two motifs, RXXS and SP  and pS1795 AFAD (peptide#0200). Both of these sites demonstrated enhanced phosphorylation phosphorylation in both blotting .Pik3caH1047R/WT and Ptenfl/fl cells  in order to further validate our mass spectrometry screen. NT5C, which demonstrated enhanced S184 phosphorylation in both fl cells  is knownPik3caH1047R/WT and Ptenfl/fl cells by western blot   and founern blot , consistnduction . S184 isnduction . To form α-pNT5C  or an an α-pNT5C  demonstrWe next examined how S184 phosphorylation on NT5C was regulated in cells, by testing its’ sensitivity to various inhibitors and stimuli. S184 phosphorylation was sensitive to inhibition with the PI3K pathway inhibitors A66 (p110α inhibitor) and MK-2206 (AKT inhibitor), but not to rapamycin, U0126 or GSK-650394, which inhibit mTORC1, MEK1/2 and SGK1, respectively . Interesin vitro nucleotidase assays, using Flag-tagged NT5C constructs ectopically expressed in HEK293 cells. In line with previous reportsin vitro catalytic activity or substrate specificity between wild-type NT5C and the non-phosphorylatable NT5C S184A mutant  and/or in the presence of acute stimuli. As far as we are aware, such analysis of low level PI3K pathway activation as observed in cancer has not been carried out to date.We used a label-free quantitative mass spectrometry method to obtain unbiased phosphoproteomic profiles from primary MEFs, which either heterozygously express the endogenous Pik3caH1047R and Ptenfl/fl screens, respectively. It is important to note that as samples were collected 48–96 h after mutation induction our screen is not restricted to acute effects of PI3K activation but may also reflect secondary effects which could rely on transcription or translation of regulating proteins or could be caused by changes in target protein abundance. The two categories of phosphorylation events that we were particularly interested in were those that were either commonly- or differentially-regulated between the Pik3caH1047R and Ptenfl/fl screens. In the category of commonly-regulated phosphopeptides, we identified 167 peptides. These included a number of known PI3K-regulated phosphorylation sites, such as on GRB10, GRB14, Afadin and AMPKα, as well as novel putative substrates of GSK3 and AKT, one of which, NT5C, we characterized in more detail.Using this system, we identified 7566 unique phosphopeptides from 3279 proteins, with 860 and 474 phosphopeptides regulated in the Pik3caH1047R cells and decreased phosphorylation in Ptenfl/fl cells. The identified site on DNMT1 (DNA (cytosine-5)-methyltransferase 1), pS958, is within a high stringency CDK5 consensus site. Intriguingly, DNMT1 is a known negative regulator of Pten transcription and is upregulated in some cancers42Pik3caH1047R compared to Pten loss.We also identified 150 peptides that had markedly different changes in phosphorylation intensity between the two screens. Two of these, DNMT1 (pS958) and EMSY (pT171), were regulated in both genetic contexts, with enhanced phosphorylation in Pik3ca mutation and cytoskeleton rearrangement and cell migrationFunctional annotation of the regulated proteins revealed some further trends. Firstly, we observed that all groups of regulated peptides were enriched for functions related to the cytoskeleton. This is in agreement with a previous phosphoproteomic screen which identified a strong link between oncogenic Pik3ca and Pten. Firstly, there was enrichment of proteins associated with the nuclear envelope  and nuclear specks  specifically in response to Pik3caH1047R activation, indicative of a specific role for p110α signalling in nuclear transport and mRNA splicing. In support of a putative p110α-specific effect in the nucleus, there is evidence that a nuclear pool of PIP3 exists, that is associated with nuclear specks and is insensitive to PTENPik3caH1047R and Pten loss. As phosphoinositide conversion plays an integral role in vesicular trafficking, it is tempting to speculate that gain-of-function of a phosphoinositide kinase (p110α) could have different effects on the composition of phosphoinositide lipid species compared to loss-of-function of the lipid phosphatase PTEN. Taken together, the data from our phosphoproteomic analysis supports further study of the role and importance of subcellular pools of phosphoinositides in diverse cellular processes.Our study also identified some processes that may be differentially-regulated downstream of Pik3caH1047R/WT and Ptenfl/fl cells, and is within an AKT substrate consensus site. Using a phospho-specific antibody we confirmed the mass spectrometric result and demonstrated that S184 in NT5C was phosphorylated by AKT in vitro and was sensitive to PI3K inhibitors in cells. Therefore we conclude that NT5C is a novel AKT substrate. However we do not discount the possibility that other PI3K-activated AGC kinases, such as SGK3, may also phosphorylate this S184 in a cell- or context-dependent manner. Despite being sensitive to PI3K inhibition, S184 phosphorylation displayed strikingly different kinetics than other known PI3K effectors such as AKT or PRAS40, in that phosphorylation and dephosphorylation did not appear to be acutely regulated, but instead occurred only after long-term stimulation or inhibition, respectively. Based on this, we speculate that S184 phosphorylation may not be subject to a constitutively active phosphatase and/or may require additional post-translational modifications to occur.In order to validate our mass spectrometric screen, we focused on NT5C . NT5C p184 was a commonly-regulated phosphorylation event, elevated in both Pik3caH1047R-expressing cells, they were not sensitive to NT5C depletion. A caveat with these results is that we were unable to achieve complete knockdown of NT5C by siRNA and the remaining protein may be sufficient to perform its nucleotidase function. However, although contrary to expectations, we propose that NT5C may not be the major 5′ 3′-nucleotidase activity in the cell. In support of this, data from publically available databases show a frequent amplification of NT5C in cancer, at both the transcript and protein level50Contrary to our original hypothesis, S184 phosphorylation did not have an impact on NT5C catalytic activity. Even more surprising, while we observed elevated pools of dNTPs in Data from an interaction databaseTaken together, we have produced an unbiased resource of two models of PI3K-regulated phosphorylation. Validation of one of the hits from the screen has identified NT5C as a novel AKT substrate, and point towards an underexplored role for PI3K in regulating actin dynamics at the cell edge. Future work will be important to more fully dissect the interaction between NT5C and the Arp2/3 complex and its role in PI3K-regulated cell motility. Our study adds to the growing realization that PI3K pathway activation in cells leads to an extensive remodelling of the transcriptome and proteome46525353Pik3caH1047R+neo/WT is a mouse line in which one of the two wild-type (WT) Pik3ca alleles is converted to Pik3caH1047R. Due to the presence of a neomycin (Neo) selection cassette in the targeted Pik3ca locus, the expression of the mutant p110αH1047R protein is dampened, resulting in minimal or no activation of the PI3K pathway. Pik3caH1047R+neo mice were crossed with CAG::Flpe-ERT2 micePik3caH1047R/WT; FlpER(T2)+/− mice are further referred to as Pik3caH1047R/WT mice. 4-OHT treatment resulted in the removal of Neo cassette, restoration of p110αH1047R expression levels to those of endogenous p110αWT and PI3K pathway activation. The presence of the Pik3caH1047R+neo mutant allele was monitored by PCR using neo cassette primers: 5′-GGTTCCAGCCTGAATAAAGCC-3′ and 5′-AGATCAGCAGCCTCTGTTCC-3′, giving rise to an expected PCR fragment of 307 bp. In each amplification cycle, denaturation was performed for 30 sec at 95 °C, annealing for 30 sec at 60 °C, and extension for 1 min at 72 °C; the number of cycles was 35 followed by a final extension of 10 min at 72 °C. Following Flp-mediated recombination the presence of the recombined allele was monitored using recombination primers: 5′-GGTTCCAGCCTGAATAAAGCC-3′ and 5′-CACAGCTGTCCTGGGTAAGG-3′ and the same PCR conditions described above where used. The size of the expected PCR fragments was 425 bp  or 340 bp (wild-type locus). The PTENfl mouse line in which exon 5 in the phosphatase domain of PTEN is flanked by loxP recombination sites, has been described+/− mice that express a germline 4-OHT-inducible CrePtenfl/flCreER(T2)+/− mice, further referred to as Ptenfl/fl mice. MEFs made from E13.5 embryos were treated with 1 μM 4-OHT for one or two consecutive days. Unless mentioned, all experiments with primary MEFs were performed 48–96 h post 4-OHT treatment; control cells were also treated with 4-OHT. Efficiency of recombination was assessed by PCR or western blot analysis.Mice were housed in individually-ventilated cages and cared for in accordance with UK Home Office guidelines and legislation, with procedures approved by the Ethics Committees of University College London and Queen Mary University of London. 2, 3% O2) which increases the proliferative lifespan of cultured cellsHT1080 cells were cultured in DMEM, high glucose, GlutaMAX™ Supplement (Invitrogen #61965–026) with 10% FBS and penicillin-streptomycin. All other cells were cultured in DMEM with 10% FBS and penicillin-streptomycin. For phosphoproteomic experiments primary MEFs were grown under normoxic conditions. For all other experiments, primary MEFs were grown under hypoxic conditions (5% COWhole-cell lysates or immunoprecipitates were resolved by SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes. Proteins were probed using primary antibodies obtained from the following sources : pAKT (S473) (#9271), pAKT (T308) (#4056), AKT (#9272), pERK1/2 (T202/Y204) (#9106), p110α (#4249), PTEN (#9552), pFoxO1/3a (T24/T32) (#9464), pGSK3 (S21/9) (#9331), pS6 (S240/4) (#2215), pp70S6K (T389) (#9205), pPRAS40 (T246) (#2640), pNDRG1 (T346) (#3217), pAFAD (S1718) (#5485), pAAPK1 (S485) (#2537), AMPK (#2532) and GST (#2624) from Cell Signaling Technology; ARPC1B (#HPA004832), Vinculin (#V9131) and Flag M2 (#F3165) from Sigma; NT5C (#NBP1-84563) from Novus Biologicals; p34 (#07-227) from Millipore and AFAD (ab90809) from Abcam. A rabbit affinity-purified polyclonal antibody against NT5C pS184  was developed in collaboration with GenScript, utilizing the RLLpSWSDNWREILDC peptide, covering amino acids 179–192 of human NT5C. Quantification of western blots was performed using ImageJ.Primary MEFs were immortalized by stable transfection of p53 siRNA2 enrichment of phosphopeptides, packed tips were sequentially washed with glycolic acid buffer 2, 99/1 H2O/ACN and eluted with four sequential washes of 5% NH4OH in 1% ACN. LC-MS/MS analysis and data processing was performed as describedSamples were processed as describedPeptide identification was performed by searching the MS/MS data against the SwissProt database restricted to mouse entries with the Mascot search enginehttps://david.ncifcrf.gov/summary.jsp) was used to perform functional annotation of regulated peptides, using the complete list of peptides identified within our mass spectrometric screen as background. Categories/Processes were enriched if they had a p-value < 0.05. The motif-x algorithm (http://motif-x.med.harvard.edu) was used to extend and extract motifs25http://scansite.mit.edu/) to identify predicted kinases and PhosphoSitePlus® (http://www.phosphosite.org/)Quantified phosphopeptides were analysed within the model-based statistical framework MSstats v3.3.1GAATTCAATGGCGGTGAAGCGGCCCGT-3′; Reverse (with added XbaI restriction site) 5′-ATCCTCTAGATCACAGGCTGGCTCGCTTGC-3′], cloned into the EcoRI and XbaI restriction sites within the multicloning site of 3xFlag-CMV-7.1 and fully sequenced. NT5C was then subcloned into pGEX4T3 and sequenced. Lentiviral plasmids were constructed using the Gateway system as follows: Flag-NT5C was subcloned into the entry vector pEntr1A, sequenced, following by cloning through recombination into the destination vector pLenti CMV Blast Dest (Addgene#17451). Point mutants of NT5C were generated by site-directed mutagenesis and sequenced. For transient knockdown of NT5C in human cells, ON-TARGET plus Human NT5C (30833) siRNA-SMART Pool was purchased from ThermoScientific (L-020511-01-0005). For stable knockdown of NT5C, shRNAmir were purchased from Open Biosystems as follows: non-silencing scrambled (ATCTCGCTTGGGCGAGAGTAAG), shRNAmir against mouse NT5C: NT5C#1 (V2LMM_33051), NT5C#2 (V3LMM_521869), shRNAmir against human NT5C: hNT5C#1 (V2LHS_85392), hNT5C#2 (V2LHS_85394).NT5C cDNA was amplified from a mouse cDNA library using the following primers [Forward  5′-TACGCCells were lysed in CHAPS lysis buffer ) and protease (Sigma) inhibitor cocktails). For analysis of protein interaction, anti-Flag M2-agarose immunoprecipitations were performed overnight at 4 °C. All other immunoprecipitations were performed with anti-Flag M2-agarose or glutathione-Sepharose 4 Fast Flow  for 2 h at 4 °C, washed four times with CHAPS lysis buffer and eluted by boiling in sample buffer.2, 2.5 mM EGTA, 12.5 mM β-glycerol phosphate, 0.5 mM sodium orthovanadate, 0.5 mM dithiothreitol (DTT)). Kinase assays were initiated by addition of 80 μM ATP and performed for 30 min at 30 °C. Reactions were stopped by addition SDS-PAGE sample buffer, separated by SDS-PAGE and phosphorylation detected by immunoblotting.Kinase assays were performed in a 30 μl reaction mixture containing active 250 ng AKT (purchased from Millipore) and bacterially produced GST-tagged NT5C proteins (1 μg) in kinase assay buffer . Phosphatase assays were performed in a 50 μl reaction containing 5 mM of nucleotide substrate in phosphatase reaction buffer. Reactions were carried out for 30 min at 37 °C. Reactions were stopped by addition of 200 μl dH2O, centrifuged and placed on ice. Supernatant containing released phosphate was measured using a malachite green-based colorimetric phosphate assay kit (Abcam #ab65622), according to the manufacturer’s instructions. Experiments were performed in duplicate and results averaged.Flag-NT5C immunoprecipitates were washed two times in CHAPS lysis buffer and two times with Phosphatase reaction buffer  and coat protein plasmid (pMDG) were used at a 1.5:1:1 ratio. Viral supernatants were collected after 48 and 72 h, cleared through a 0.45 μm filter and stored at −80 °C. cDNA lentivirus was used at a 1x concentration. For production of stable cell lines, lentiviral transduced cells were selected with puromycin and/or blasticidin.g, 4 °C for 10 min), 180 μl of supernatant was transferred to a new tube and stored at −80 °C. At the time of analysis, 50 μl of 1 M NH4Ac was added to the PCA extract, and the solution neutralised by addition of 20 μl of 10% NH3. Finally, 5 μl of internal standard 8-ChloroATP (Sigma) and 5 μl deionised water was added. The extract was transferred to LC-MS vials and 10 μl was injected in to the UPLC-MS/MS system. LC-MS grade water, methanol, acetonitrile and formic acid were from Fisher Scientific, UK. Calibration standards were prepared at 0, 1, 5 and 25 μM.Intracellular nucleotide pools were quantified according to the previously published method for determination of intracellular gemcitabine triphosphate in human PBMCs4Ac in ACN/H2O (30:70 v/v), pH 6.0 (buffer A), and 1 mM NH4Ac in ACN/H2O (30:70 v/v), pH 10.5 (buffer B). The mobile phase gradient was employed, comprising: buffer A = 95% at 0–0.5 min, from 95 to 0% over 1.25 min, held at 0% for 1.75 min, from 0 to 95% over 0.1 min, ending with 95% for 2.9 min, all at a flow rate of 500 μl/min. Eluting compounds of interest were detected using a triple stage quadrupole Vantage mass spectrometry system  equipped with an electrospray ion source. Samples were analyzed by Multiple Reaction Monitoring, negative ion modes at a spray voltage of 3000 V. Nitrogen was used as sheath and auxiliary gas at a flow rate of 50 and 20 arbitrary units, respectively. Argon was used as collision gas with pressure of 1.5 mTorr. The optimum transitional daughter ions mass and collision energy (CE) of each analyst were as follows: ATP 506.0 → 134.1.2 , CTP 482.0 → 159.0 , dATP 490.0 → 159.0 , dCTP 466.0 → 159.0 , dTTP 481.0 → 159.0 , GTP 522.0 → 159.0 , UTP 483.0 → 159.0  and ChloroATP 539.9 → 159.0 .Analytes were resolved using an ultra-performance liquid chromatography system  equipped with a Biobasic AX 5 μm, 100 × 2.1 mm column  and a mobile phase consisting of a mixture of 10 mM NH® 488 phalloidin (#A12379) and other Alexa Fluor- conjugated secondary antibodies were from Invitrogen.Cells were seeded on coverslips, fixed with 3.7% formaldehyde (15 min), permeabilized with 0.2% Triton-100 (5 min) and blocked in 3% BSA in PBS for 1 h at room temperature. Coverslips were incubated with primary antibody overnight at 4 °C, washed and incubated 1 h at room temperature with secondary antibodies and/or phalloidin conjugated to fluorophores. Coverslips were mounted with ProLong Diamond Antifade Mountant with DAPI (Molecular Probes) and imaged using a Zeiss 700 inverted microscope. Primary antibodies used were Flag M2 (#F3165) from Sigma; p34 (#07-227) from Millipore; Cortactin (#05-180) from Upstate. Alexa FluorCells were trypsinized, counted and seeded at 30,000 (HT1080) or 7,500 (MEFs) cells per well in 12-well plates pre-coated with fibronectin (5 μg/ml). Plates were incubated for 45 min before fixing with 3.7% formaldehyde (15 min), washing and staining with 0.1% Crystal Violet (30 min) at room temperature. Experiments were seeded in duplicate or triplicate and at least 5 fields of view were imaged per genotype. Cell area was manually quantified using ImageJ. For HT1080 cells, areas greater than 20,000 and less than 1000 were removed from the analysis. For primary MEF experiments, cell areas greater than 20,000 and less than 500 were removed from the analysis. Significance was analysed using one-way ANOVA plus Tukey’s multiple comparison’s test.Single Cell Motility Assay. HT1080 cells were seeded at 6000 cells per well in ViewPlate-96 Black plates (Perkin Elmer# 6005182) in triplicate or quadruplicate. The next day, cells were labeled with 0.4 μM CellTracker™ Orange CMRA Dye (Invitrogen) for 30 min to allow better cell visualization. Cells were imaged on ImageXpress Micro XL Widefield High Content Screening System (Molecular Devices). 4 sites per well were imaged every 10 min for 80 time points. Motility analysis was performed using multidimensional motion analysis within MetaXpress software. Significance was analysed using one-way ANOVA plus Tukey’s multiple comparison’s test.How to cite this article: Moniz, L. S. et al. Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate. Sci. Rep.7, 39985; doi: 10.1038/srep39985 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In silico inferences of cell-to-cell excitatory and inhibitory connectivity and GABA/Glutamate receptor time constant based on neural mass modeling within the Dynamic causal modeling framework, further suggested excitation/inhibition balance was under a strong circadian influence. These results indicate that circadian changes in EEG spectral properties, in measure of excitatory/inhibitory connectivity and in GABA/glutamate receptor function could support the maintenance of cognitive performance during a normal waking day, but also during overnight wakefulness. More generally, these findings demonstrate a slow daily regulation of cortical excitation/inhibition balance, which depends on circadian-timing and prior sleep-wake history.Several neuropsychiatric and neurological disorders have recently been characterized as dysfunctions arising from a ‘final common pathway’ of imbalanced excitation to inhibition within cortical networks. How the regulation of a cortical E/I ratio is affected by sleep and the circadian rhythm however, remains to be established. Here we addressed this issue through the analyses of TMS-evoked responses recorded over a 29 h sleep deprivation protocol conducted in young and healthy volunteers. Spectral analyses of TMS-evoked responses in frontal cortex revealed non-linear changes in gamma band evoked oscillations, compatible with an influence of circadian timing on inhibitory interneuron activity.  Neuronal function sustaining human cognition depends on the balanced and recurrent activity of excitatory and inhibitory circuits46It is commonly accepted that an increase in excitation is invariably accompanied by an increase in inhibition and vice-versa81112131416in vivo daily dynamics of neuronal ensemble is based on animal data8112124in vivo, even using invasive approaches, such as intracranial EEG recordings in patients2425Addressing directly this issue in humans remains a challenge, however. Most of what is currently known about the in silico model to the TMS-evoked EEG responses to infer a more detailed mesoscopic description of excitation/inhibition within a cortical column. We hypothesize that circadian phase would influence excitation and inhibition balance and that this influence would be most apparent through a non-linear, ~24 h sine-wave oscillatory patterns in excitation/inhibition parameters. We further postulated that this mesoscopic non-linear neuronal network dynamics would be related to changes in cortical excitability and behavioral measures acquired during the sleep deprivation protocol.Here, our aim is to assess the dynamics in human cortical excitation/inhibition balance over the 24 h day. We report results of an original analysis extension of previous data which consisted in neuropsychological and neurophysiological assessments during a 29 h sleep deprivation protocol in young and healthy individualsFollowing an 8-h nocturnal baseline sleep, 22 healthy young men  following a typical non-linear variation across the protocol  following a non-linear temporal profile (corrected = 0.01). This initial decrease was followed by a sharp increase during the biological night . This finding reflects that extended wakefulness is associated with changes in a fundamental mechanism shaping TMS-induced responses.The literature posits that different cortical areas oscillate in their intrinsic EEG frequency modes in response to TMS stimulations profile , with na7,131 = 4.62, p < 0.001), similar to the non-linear profile observed in the beta band (corrected < 0.001) followed by a steep increase during the biological night .We next assessed whether EEG power in the gamma band of the TMS-evoked response (30–50 Hz), which has been related to GABA activity of inhibitory interneuronseta band . A markein silico approach to infer excitation/inhibition balance using Dynamic Causal Modelling (DCM) for event related potentials (3536A receptors (GABAaR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and N-methyl-D-aspartate (NMDAR) receptorsWe then applied an tentials 33. DCM hminus AMPAR+NMDA]. Both GABAergic and glutamatergic receptors are indeed likely affected by sleep-wake history and time of day187,130 = 2.8, p = 0.01; corrected = 0.005). Conversely, GABAergic receptor time constant undergoes a large increase relative to glutamatergic receptor during the biological night . GABA/glutamate receptor time constant value then returns towards mean GABA vs. glutamatergic parameter levels.The first index comprised the relative time constants of inhibitory GABAaR and excitatory glutamatergic AMPAR and NMDAR [GABAaR minus sum of inhibitory connectivity from inhibitory interneurons to stellate and superficial pyramidal cells]. Similarly to the receptor time constant index, analysis show that neuronal cell-to-cell excitation/inhibition balance significantly varies with non-linear dynamics during the 29 h sleep deprivation protocol . During the biological night, however, excitatory connectivity increases relative to inhibitory connectivity up to the early morning sleep promoting zone .The recurrent coordination of excitation and inhibition within neural networks is subtended by the connectivity between excitatory pyramidal cells and stellate cells and inhibitory interneuronsp > 0.14). Thus, the dynamics of neuronal excitation/inhibition balance within a cortical region appears to be under a strong circadian influence.Therefore, intriguingly, it is as if both indices go in opposite directions, seemingly “counteracting” each other. Interestingly also, both neuronal indices seemed to “recover” in the morning following sleep deprivation, when the circadian system switches back to favoring wakefulness: values of both morning sessions  are indeed not statistically different from the mean  but not with excitatory/inhibitory cell-to-cell connectivity balances . No significant correlations are found with power in other frequency bands of the spontaneous waking EEG recordings .Further correlation analyses indicate that a relative increase in GABAa receptor over glutamatergic receptor time constant, as inferred from our r = 0.56, p < 0.001; slope: r = 0.52, p < 0.001; r = 0.31, p < 0.001; Likewise, GABA/glutamate receptor time constant balance strongly and significantly correlates with both the amplitude and slope of the TMS evoked responses , thus mirroring putative sleep and circadian mechanisms  and cell-to-cell excitation/inhibition connectivity  balances are significantly associated with performance to the task  . Ancovas also indicate significant associations between power in the theta frequency band of the spontaneous waking EEG and both DCM indices (p ≤ 0.05). Associations with other frequency bands of the spontaneous waking EEG remained non-significant (p > 0.15). Importantly, the relationship between our markers for neuronal excitation/inhibition balance and measures of system-level physiology and behavior are similar across all circadian phases for all parameters considered .Finally, we probed whether all these correlations would vary as a function of circadian phase by performing analyses of covariance on the same combination of variables as those used for correlations, but including circadian phase as a factor. Analyses of covariance confirm all previous significant correlations, as they lead to significant covariance with DCM parameters irrespective of circadian phase  were enrolled. Exclusion criteria included: (1) BMI ≤18 and ≥25; (2) psychiatric history, severe head trauma, sleep disorders; (3) addiction, chronic medication; (4) smoking, excessive alcohol consumption (>14 doses per week) or caffeine (>3 cups/day); (5) night shift workers during the last year; (6) transmeridian travel (>1 time zone) <2 months; (7) anxiety and/or depression. One participant was excluded from analyses due to melatonin phase-delay >6 h compared with the average of the sample, and one due to artifacts in EEG recordings throughout the protocol. Thus, data presented here include 22 participants  was set as stimulation target for right or left-handed, respectively. This brain area was identical toParticipants then underwent one laboratory polysomnography night to exclude any sleep disorders. Afterwards, they kept a regular sleep-wake schedule of 8 h sleep duration (+/−15 min) 1-week prior to the study. Compliance was verified using wrist actigraphy  and sleep diaries . Schedulper se, participants arrived at the laboratory 6 h prior to sleep time and were maintained in dim-light (<5 lux) thereafter. They were trained on the behavioral test battery before sleeping (in darkness) at their habitual bedtimes under EEG , and TMS were delivered by means of a Focal Bipulse 8-Coil (Eximia TMS) combined with a magnetic resonance–guided navigation system (Eximia NBS). TMS pulses were delivered every 2000 ms (jitter 1900–2200 ms). The EEG amplifier gates the TMS artefact and prevents saturation by means of a proprietary sample-and-hold circuit that keeps the analog output of the amplifier constant between 100 μs before and 2 ms after the stimulushttp://www.fil.ion.ucl.ac.uk/spm/), and analyzed with MATLAB® . Data were visually inspected for artifacted channels and trials. Trials corresponding to vigilance lapses based on the simultaneous vigilance tasks were excluded. EEG signals were re-referenced to the average of all good channels. Continuous EEG recordings were low-pass filtered at 80 Hz, downsampled from 1450 to 1000 Hz and high-pass filtered at 1 Hz. They were split into epochs −100 and 300 ms pre and post TMS pulses. Baseline correction was computed using −100 to −1.5 ms pre TMS pulse data. Robust averaging was applied to compute the mean evoked response of each sessionTMS-hdEEG data were preprocessed and analyzed with Statistical Parametric Mapping 12  and gamma range (30–50 Hz) were computed for the electrode nearest to the TMS stimulation at the individual level, during the time window of 5–50 ms.+, Na+, Cl−, Ca2+), axonal delays from superficial to deep layers, and mean post-synaptic firing rateDynamic Causal Modeling (DCM) is a physiologically-driven approach to investigate how brain connectivity is affected by context, based on a realistic generative model36Here we used a single fully-free parameter DCM, whereby all 20 parameters of the NMM were allowed to vary in the experimental setup across TMS/EEG sessions. These parameters were chosen to allow for the model to recreate the multifaceted physiological and synaptic changes under sleep deprivationTo test whether time-of-day impacts on excitatory/inhibitory drive, two proxies for these drives were used. First, neurotransmitter system parameters were identified, which do not account for the synchronicity but simply the shape of the EPSP/IPSP. This index was therefore a model-based metric of GABA/Glutamate receptor function balanceminus sum of fast AMPAR and NMDAR receptor time constants, where larger values mean that I/EPSPs have faster, shorter dynamics.GABA/Glutamate balance: GABAaR receptor time constants, where larger values mean that I/EPSPs have slower, more prolonged dynamics. minus sum of inhibitory connections from inhibitory interneurons to stellate and superficial pyramidal cells.Neuronal cell-to-cell excitation/inhibition balance: sum of excitatory connections from stellate and deep pyramidal cells to inhibitory interneurons The indices were constructed as follows:Detailed results of the analyses of cortical excitability changes with wakefulness extension and their relationships with other aspect of brain function and physiology and behavior have already been reported® Channels and epochs with artefacts  were rejected after visual inspection. Continuous EEG recordings were downsampled from 1450 to 500 Hz. Power spectral densities were computed using Welch’s methods (pwelch function in MATLAB 7.5), which consist in splitting the data segment into overlapping windows, processing these, then averaging the spectra across them. Power density of artifact-free 4-s epochs was averaged over the 2 min epoch. EEG activity was computed over frontal region  for delta (0.75–4 Hz), theta (4.5–7.5 Hz), alpha (8–12 Hz), sigma (12.5–18 Hz) and beta (18.5–30 Hz) frequency bands over the entire 2-min recording.Data were analyzed with SPM12 implemented in MATLABSaliva samples were first placed at 4 °C, prior centrifugation and congelation at −20 °C within 12 h. Salivary melatonin was measured by radioimmunoassay , as previously describedEstimation of circadian phase  was determined based on raw values. The first 4 samples were disregarded and maximum secretion level was set as the median of the 3 highest concentrations during the constant routine. Baseline level was set to be the median of the values collected from wake-up time +5 h to wake-up time +10 h. DLMOn was computed as time at which melatonin level reach 20% of the baseline to maximum difference (following linear interpolation).z-scored to provide normalized amplitudes. TMS vigilance task was normalized by dividing performance to the duration of task and then z-scored. Frontal waking EEG activity in the different frequency bands were normalized by dividing power in a given band by the sum of frequencies within 0.75 and 30 Hz over the same region. To examine the time-course of the DCM excitation/inhibition drive, comparisons were made with mixed-model analyses of variance for repeated measures (PROC Mixed), with within-subject factor “time”. Contrasts were assessed with the LSMEANS statement. All p-values were based on Kenward-Roger’s corrected degrees of freedom. Post hoc p-values sought for significant least square mean different, i.e. time course values significantly different from the mean, and were corrected for multiple post hoc using Tukey-Kramer correction. Estimation of circadian phase  was determined based on raw values. The 4 first samples were disregarded and maximum secretion level was set as the median of the 3 highest concentrations during the constant routine. Baseline level was set to be the median of the values collected from wake-up time +5 h to wake-up time +10 h. Dim Light Melatonin Onset (DLMOn) was computed as time at which melatonin level reach 20% of the baseline to maximum difference (following linear interpolation).All statistical analyses were performed with SAS version 9.3 . All data were realigned according to circadian phase determined from individual melatonin profiles. All DCM-parameters and TMS/EEG evoked responses were To estimate how excitatory-inhibitory parameters  were associated to TMS-evoked EEG response , spontaneous waking EEG power bands, subjective sleepiness and vigilance task behavioral responses, Spearman correlation analyses were performed to estimate the relationship across these variables. Furthermore, Analyses of Covariance  were performed to estimate the relationship between neuronal cell-to-cell connectivity and GABA/Glutamate balance and these macroscopic system-level changes across circadian phases.How to cite this article: Chellappa, S. L. et al. Circadian dynamics in measures of cortical excitation and inhibition balance. Sci. Rep.6, 33661; doi: 10.1038/srep33661 (2016).


Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found.In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients’ IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs.Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients’ IgE.Acid-HWPs with medium (30%) and high (50–60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients’ IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay.Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins. A large proportion of food allergies are IgE mediated. In these patients, the immune system is unbalanced towards a Th2 profile and allergens induce the production of immunoglobulins E (IgE), which bind to high-affinity IgE receptors (FcεRI) expressed on effector cells . Subsequent crosslinking of IgE by allergens induces clustering of FcεRI and subsequent effector cell degranulation with the release of mediators such as histamine, leukotrienes and cytokines, resulting in allergic symptoms.Wheat proteins, of which at least 80% are gluten proteins, have been shown to trigger IgE-mediated food, respiratory and contact allergies ,2. The ghttp://www.allergyvigilance.org/). More dramatically, in Japan, a facial soap containing the acid-HWP Glupearl19S® (GP19S) was involved in more than 2000 cases of severe allergic reactions [Beginning in the 2000s, case reports have described allergic reactions to hydrolyzed wheat proteins (HWPs) in several European countries –8. In 20eactions .The same IgE-binding consensus peptide, QPQQPFPQ, was identified for the French and Japanese patients ,12 and cDuring acid hydrolysis of gluten proteins, deamidation does not occur on all glutamines. Acid-HWPs undergo variable rates of deamidation and hydrolysis depending on treatment intensity  ,14, resuThe potency of acid-HWP products to trigger reactions, i.e., their capacity to activate allergic effector cells in the presence of allergen-specific IgE, can be addressed with basophils and IgE. Humanized rat basophilic leukemia (RBL) cell lines, which express at least the α-chain of human FcεRI, can be used as a functional assay in the presence of human sera ,20. IndeRecombinant monoclonal IgEs have been used as serum substitutes for studying the molecular properties of allergens and IgE/allergen interactions ,22. In pThe present work aimed to clarify how the extent of deamidation affects allergenicity by analyzing the reactivity of IgE from acid-HWP-allergic patients towards HWP products showing various deamidation levels. A chimeric mouse/human IgE directed to the neo-epitope, QPEEPFPE, was also studied for its capacity to be used in assessing the triggering potential of these products.Human sera were collected from 2001 to 2014. All sera were collected into three distinct clinical trials corresponding to 3 successive research projects on the characterization of wheat allergens before their inclusion into our bio-collection (DC-2008-809). The first clinical trial was funded by the INRA call “Food, quality and Security: Identification of wheat allergenic proteins”. It was approved by the Ethical Committee of the Cochin hospital . The second was funded by the INRA Food Research Program: Study of feasibility of wheat based products for some wheat allergic patients. It was approved by the Ethical Committee of Biomedical Research from Lorraine and by the Health Ministry (DGS2007-0066). The third was funded by the National Research Agency: PREDEXPITOPE . It was approved by the Ethical Committee of Ile de France III and AFSSAPS (authorization number 2008-A01565–50). These 3 clinical trials received ethical approvals for serum collection and their use for research purposes and written consents were obtained from the patients or their parents.Laboratory and industrial samples of native and deamidated gliadins and glutens were used in this study. Native gluten and native gliadins were previously prepared from wheat flour  according to Battais et al. . BrieflyIndustrial samples of HWP (HWP1 to HWP4 and GP19S) have been commercialized for food or cosmetic applications in Europe and in Japan and were previously described in former papers ,13,14,28Sera were obtained from nine subjects with food allergy to acid-HWP . The pat2HPO4, 1 mM KH2PO4; pH 7.2). Gluten and Acid-HWP were solubilized in 0.1 M carbonate buffer (pH 9.6) with 0.1% SDS and 0.2% 2-mercaptoethanol. All antigens were then diluted in 0.1M carbonate buffer (pH9.6) at 5μg/mL for coating. Human sera (diluted 1:10) and chIgE-DG1 (1:1000) were analyzed in triplicate. Concentrations in specific IgE were calculated by reference to a standard curve included in each microtiter plate . The standard curve was fit using four-parameter logistic regression. For each serum, results were normalized in comparison to the laboratory D-gluten sample by determining the ratios of antigen-specific IgE concentrations to the D-Gluten-specific IgE concentration. To determine differences in antigen recognition, matched ratios per serum were analyzed by repeated-measures one-way ANOVA and subsequent Bonferroni’s multiple-comparisons test (p<0.05) using GRAPHPAD PRISM version 5.02 .Sera were analyzed for specific IgE by F-ELISA on white NuncMaxiSorp 384 well microtiter plates  with a Biomek NXP automated workstation  as described . AntigenTGC ATT CCG ATG TGC AGC TTC AGG; 3’ VH primer: CGT ACG ACT CAC CTG AGG AGA CGG TGA CCG T) and VK  primer pairs respectively. These primers contained bsmI (bold) or bsiWI (underlined) restriction sites to allow cloning of the VH PCR product into plasmid pLNOH-2IgE and of the VK PCR product into plasmid pLNOK. In a first step, PCR products were ligated into pGEMTeasy  to facilitate digestion with bsmI and bsiWI and subcloning onto pLNOH-2IgE and pLNOK.Mab mDG1  was raised in mouse against the peptide LQPEEPFPEQC containing a dominant IgE-binding epitope for patients allergic to acid-HWP. Variable domains of heavy and light chains (VH and VL) of mDG1 were cloned in pMD18 and sequenced . In order to express in mammalian cells the chimeric mouse/human IgE specific for this deamidated epitope we used two plasmids: pLNOH-2IgE and pLNOK . pLNOH and pLNOK had been designed to express IgG3 heavy chain and kappa light chain , pLNOH was further modified to give pLNOH-2IgE in order to express IgE heavy chain . pLNOH and pLNOK DNA were kindly provided by Pr Lund . cDNA of mDG1 variable domains VH and VL were cloned in pMD18 and amplified by PCR with VH (5’VH primer: ® CRL-1573) with pLNOH2IgE-DG1VH and pLNOK-DG1VL plasmids. To this end, adherent HEK293 cells were cultured at 5% CO2 and 37°C in DMEM containing 4.5 g/L L-glucose (ThermoFisher) and supplemented with 10% decomplemented FBS , 4 mM L-glutamine, 100 U/mL penicillin  and 100 μg/mL streptomycin . The day before transfection, 3 x 105 cells were seeded into 24-well culture plates and grown for 24 h in 293 FreeStyle™ expression medium (ThermoFisher). Co-transfection with 1 μg of each heavy and light chain-encoding plasmid was performed using 293fectin™  and Opti-MEM  according to the manufacturer’s instructions. ChIgE-DG1 containing supernatant was collected 4 days after transfection and cleared by centrifugation for 10 min at 2500 g before 0.22 μm filtration and dialysis against PBS by ultracentrifugation on an Amicon® 50 kDa MWCO Ultra cell Unit  and stored at -20°C.Full-length recombinant chimeric human IgE (chIgE-DG1) containing the mDG1 paratope was expressed by transient co-transfection of HEK293 cells  and separated by SDS-PAGE. Proteins were transferred onto a 0.22 μm nitrocellulose membrane  using the semi-dry transfer system . After saturation with PBS containing 4% BSA , the membrane was incubated with peroxidase-conjugated anti-human IgE  or peroxidase-conjugated anti-human kappa light chains  diluted in PBS containing 0.1% BSA. Detection was performed using ECL reagent  and a cooled digital camera .The activity and specificity of chIgE-DG1 were controlled and compared with those of mDG1 via indirect ELISA on native or deamidated gliadins coated at 5 μg/mL. ChIgE-DG1 was revealed with peroxidase-conjugated anti-human IgE, while mDG1 was revealed with peroxidase-conjugated goat anti-mouse IgG .Degranulation assays were performed either with stripped human basophils as previously described  or with 6 cells per condition was performed for 30 min at 37°C. The reaction was stopped with EDTA, and cells were stained by incubation at 4°C with labeled anti-CD123-phycoerythrin-cyanin5 , CD63-fluorescein isothiocyanate , and CD203c-phycoerythrin-cyanin 7  followed by flow cytometric analysis. Basophils were gated as side scatter–low, CD123+, and CD203c+ cells. Cells were measured with a FACS LSRII flow cytometer (BD Biosciences), and data were analyzed using BD FACS Diva software (BD Biosciences). CD63++ basophils were determined after stimulation with D-gluten and after stimulation with anti-IgE. Basophil activation was expressed as the percentage of CD63++ basophils induced by D-gluten to CD63++ basophils induced by the anti-IgE antibody.For the stripped basophil assay, buffy coats were obtained from healthy, de-identified blood donors at the Massachusetts General Hospital Blood Donor Center . The study was approved by the Institutional Review Board of Partners Healthcare . Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll centrifugation. After washing, IgEs were stripped from the cell surface with a lactic acid buffer as previously described . The cel4 or 2.5x104 cells/well in 96-well flat-bottom culture plates were grown for 24 h before sensitization with patient sera (diluted 1:25) or with chIgE-DG1 (100 ng/mL) for 24 h. Sensitized RBL-SX38 cells were washed 3 times with PBS before stimulation with either mouse anti-human IgE  or with antigens (0.1 ng/mL to 10 μg/mL) diluted in Tyrode buffer with 50% D2O. β-hexosaminidase release induced by the crosslinking of the FcεRI was measured with pNAG  as previously described [50) were then determined using GraphPad Prism 5.02 for Windows . The EC50 values of different samples were compared by two-way ANOVA followed by Bonferroni’s multiple-comparisons test.For the humanized RBL assay, sera were decomplemented by heating for 45 min at 56°C in the presence of 4 M glucose to reduce cytotoxicity . RBL-SX3escribed . For eac2O (v/v) and stimulation of sensitized RBL-SX38 cells. Data were analyzed with a Wilcoxon matched-pairs signed rank test for HWP2 or GP19S (*: p ≤ 0.05).For inhibition experiments, cells were sensitized with 6 patients' sera and mDG1  was used50 [Products with known and unknown levels of deamidation were used in the study. To enable a comparison among them, a competitive ELISA with mAb mDG1 was performed on all samples and the relative abundance of deamidated sequences was estimated using a comparison of their IC50 Table 1)50 [TableThe sera from nine acid-HWP-allergic patients were used for this study. Native and deamidated gliadins, gluten and industrial HWP-specific IgE concentrations were measured by F-ELISA. Patients’ IgEs were almost exclusively directed towards deamidated proteins since they displayed no or very low reactivity to native gliadin or gluten extracts . To comp50 values (approximately 10 ng/mL) were found for the 7 sera, although both the concentration and the proportion of specific IgEs to D-gluten of the total IgE varied considerably among the sera  and were stimulated with D-Gluten (from 1 to 1000 ng/mL) (data not shown). Wheat-allergic serum-sensitized RBL cells did not induce any β-hexosaminidase release upon stimulation with any doses of D-Gluten. However, acid-HWP-allergic sera (n = 7 of 9) -sensitized RBL cells resulted in the release of β-hexosaminidase to D-gluten in a dose-dependent manner. Similar ECthe sera .50 revealed that HWP2, HWP3 and GP19S were not significantly different (p<0.05), while HWP4 displayed a lower triggering capacity (p<0.01). These results suggest that patient IgE preferentially binds to highly deamidated products, which possess strong RBL activation abilities.Two sera (#285 and #299) were used to investigate the capacity of the different HWP samples to induce RBL activation. These 2 sera were selected because they bound differently to weakly deamidated samples in ELISA . Native gluten did not induce cell degranulation . The proTo further explore the contribution of highly deamidated sequences to IgE binding and RBL cell activation, mDG1 was used for inhibition experiments. This mouse mAb specifically binds the highly deamidated sequences E/QPEEPFPE and more weakly to the sequences E/QPQEPFPE . The abiSince the highly deamidated sequences bound by mDG1 notably contributed to RBL cell degranulation, this mAb was used to produce a chimeric mouse/human recombinant IgE. cDNAs encoding the variable regions of mDG1 were cloned into plasmids containing sequences encoding constant regions of IgE heavy and kappa light chains in order to produce full-length IgE. HEK293 cells were co-transfected with the two expression vectors to transiently express a chimeric human IgE (chIgE-DG1) harboring the mDG1 paratope. After transfection, expression of chIgE-DG1 in the culture supernatant was assayed by Western blotting with anti-human Fcε (anti-hFcε) and anti-human κ light chain (anti-hkL) antibodies. A single band was revealed at approximately 190 kDa with both anti-hFcε and anti-hkL antibodies. After reduction, this band shifted to two bands, resolving at approximately 70 kDa with anti-hFcε and 25 kDa with anti-hkL , as expe50 was graphically estimated at 200 ng/mL  in the RBL-SX38 degranulation assay. RBL-SX38 cells were passively sensitized with either the pool of sera or with chIgE-DG1 and stimulated with serially diluted native or deamidated samples. Native gliadins and gluten did not induce cell degranulation, whereas all deamidated samples induced β-hexosaminidase release of the cells sensitized either with the serum pool or with chIgE-DG1 . All HWPtable in . The indIn vivo models have been used to investigate the allergenic potency of different HWPs in comparison to native gluten proteins [in vitro evaluation tool of the antigenic and triggering potency of functionalized glutens. The effect of the deamidation level of the products on the reactivity of IgEs from patients allergic to acid-HWP was first explored. Then, patients' and chimeric IgE responses were compared via ELISA and a functional basophil degranulation assay.Acid-HWPs have been shown to trigger numerous cases of contact or food allergies in Europe and Japan. proteins ,33,34. HA set of laboratory and industrial deamidated wheat proteins were used in the study. According to analysis, the samples could be classified and ranked into 3 groups, characterized by their weak (approximately 15%), medium (approximately 30%) or high (50–60%) level of deamidationIgEs from HWP-allergic patients reacted with all deamidated industrial and laboratory samples, while they did not bind or bound only very weakly to the native gliadin or gluten samples. They reacted markedly more strongly with the samples with high and medium deamidation rates than with the weakly modified samples. Variability in IgE binding among patients’ sera was sample dependent; it was particularly high for D-GLIA 15 and HWP3 and 4. For HWP3, this variability may be linked to the heterogeneity of the composition of this product, which is both deamidated and extensively hydrolyzed.The products with medium and high deamidation rates also possessed the strongest ability to activate RBL-SX38 cells sensitized with patient IgE. In addition, mAb mDG1, presenting a restricted reactivity to the heptapeptides E/QPEEPFPE, was able to markedly inhibit the activation of HWP-induced RBL cells. These results highlight that the allergenicity of deamidated products was mainly borne by epitopes with a relatively high modification level.2, Q3 or Q4.These results are in accordance with the sequential epitopes identified in our previous work . Using sDue to the significant contribution to IgE-binding and basophil activation of the deamidated epitopes bound by mDG1, this mAb was used to generate a chimeric mouse/human recombinant IgE specific for these sequences. The mAb was produced in the HEK293 cell line using an expression vector obtained by inserting cDNA encoding variable domains of mAb mDG1 into plasmids containing the sequences encoding the human IgE scaffold according to Christensen et al. (2008). The resulting recombinant chIgE-DG1 retained the activity and the specificity of the original mouse IgG and was able to bind to the human FcεRI receptor on both human basophils and RBL-SX38 cells. More interestingly, human basophils and RBL-SX38 cells sensitized with this unique chIgE-DG1 were activated by deamidated gluten. This ability of deamidated gluten to induce the degranulation of basophils sensitized with a single monoclonal IgE has to be related to the repetition of the mAb epitopes within γ and ω2-gliadin sequences. The repetition of linear epitopes in the allergen sequence is a distinctive feature of the repeated domains of gluten proteins ,36. We pWe established that the chIgE-DG1 possesses an IgE-binding capacity equivalent to that of patients’ sera on laboratory and industrial samples with medium and high deamidation rates and did not react with native gliadin and gluten fractions. As expected, because the reactivity of its paratope is limited to a few highly deamidated epitopes, the chIgE-DG1 possessed a more restricted specificity than did most of the patients’ sera.The chIgE-DG1 also mimicked patient sera when used in the functional RBL assay. In this assay, the various deamidated products were ranked in the same order according to their triggering potential, irrespective of the sensitizing antibodies (chIgE-DG1 or human IgE pool). Regarding the five industrial products, the products with the highest potency were the same in the two sensitizing conditions, i.e., HWP2, 3 and GP19S. The HWP1 product displayed an intermediate response; depending on the antibodies, it was 3 to 10 times less potent for cell activation than the former products; HWP4 was less active, approximately 100 times less active than HWP2. The ranking of products in the RBL assay matched the classification performed on the basis of the deamidation levels. Although HWP3 presented a higher degree of proteolysis than HWP2, this did not affect its triggering potential.in vitro alternative to the use of human sera or animal models. This test can be used to assess the allergenicity of functionalized wheat proteins. In the same way, Knipping et al. recently proposed an RBL degranulation assay based on a pool of β-lactoglobulin-specific chimeric IgE for the in vitro assessment of whey hydrolysate [Functionalized glutens are among ingredients derived from plant proteins usable as animal protein substitutes in the context of sustainable food production. Safety assessment of modified glutens is therefore required with respect to the significant allergenic risk of acid-HWPs, which has been highlighted by the numerous Japanese cases and by the severity of reactions in both Europe and Japan. Although, no cases of allergy to acid-HWPs have been reported by the French Allergy Vigilance Network from 2014, Nakamura recently showed that such ingredients were still released on the market by European companies . Human srolysate . A ring-rolysate .Of note, for the group of French sera evaluated in this study, the HWP GP19S commercialized in Japan displayed IgE-binding and triggering capacities matching those of two products released in Europe. Although the sensitization mode and the duration of exposition differed in these populations, cases of allergy to acid-HWPs presented high similarities, notably in the type and severity of symptoms. The larger number of cases in Japan probably resulted from a combination of factors such as the wide exposure to the GP19S-containing soap, the presence of a detergent that was shown to favor sensitization by gluten proteins , and repThe comparison between patients’ and chimeric IgE antibodies enables investigations of the composition of patients' IgE repertoire. The similar reactivity of patients’ IgEs and of the chimeric anti-peptide IgE suggests that continuous epitopes play an important role in this type of allergy. The equivalent triggering activity of the IgE pool with that of the monoclonal chIgE-DG1 suggests that a limited IgE repertoire might be involved in allergy to acid-HWPs, with IgE preferentially binding highly deamidated sequences. However, we observed that the patients’ sera displayed a somewhat larger reactivity than the chIgE-DG1, notably towards low deamidated products or epitopes ,13. ChemTo conclude, a chimeric mouse/human IgE was produced against the deamidated epitope QPEEPFPE involved in allergy to acid-HWP products. This recombinant antibody was equivalent to patient sera in its ability to classify and rank industrial deamidated glutens in a basophil activation assay and could be a relevant tool in the allergenicity evaluation of functionalized glutens. This reagent also demonstrates that epitope organization in the repeated domains of some gluten proteins allows a single monoclonal IgE to efficiently trigger effector cells. This study also shows that the IgE repertoire of French acid-HWP-allergic patients might be restricted and that effector cells are mainly activated by highly deamidated epitopes.S1 FigHydrolyzed wheat protein (30μg) or native gluten (10 μg) were separeated on StainFree 4–20% gels (Bio-Rad). Proteins were revealed by 5 min. photocativation of in gel-flurorochrome which bind to tryptophan . GP, Glupearl19S. Glut, native gluten. D-Glut, deamidated gluten.(DOCX)Click here for additional data file.S1 TablePatient’s reactivity towards native proteins (Wheat or Gluten) or Deamidated Gluten (D-Gluten from ALK-Abello) was tested by skin prick test. Specific IgE concentrations to albumins/globulins fraction (A/G), gliadins, ω5 gliadins, LTP and D-Gluten (laboratory made) were determined by F-ELISA.–negative; nd, not determined; U, Urticaria; EIA, Exercise induced anaphylaxis; AS, Anaphylaxis; AD Atopic dermatitis.(DOCX)Click here for additional data file.S2 Table®.Concentrations of total and specific IgE were determined by F- ELISA. D-GLIA, Deamidated Gliadins. HWP, Hydrolyzed Wheat Proteins. GP19S, GluPearl 19S(DOCX)Click here for additional data file.