N-dealkylation with IC50 ranges of 0.019~0.123 μM. In addition, the biflavonoids—selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone—noncompetitively inhibited CYP2C8 activity with respective Ki values of 0.018, 0.083, 0.084, 0.103, and 0.142 μM. As selamariscina A showed the strongest effects, we then evaluated it against six UGT isoforms, where it showed weaker inhibition . Returning to the P450 activities, selamariscina A inhibited CYP2C9-mediated diclofenac hydroxylation and tolbutamide hydroxylation with respective Ki values of 0.032 and 0.065 μM in a competitive and noncompetitive manner. However, it only weakly inhibited CYP1A2, CYP2B6, and CYP3A with respective Ki values of 3.1, 7.9, and 4.5 μM. We conclude that selamariscina A has selective and strong inhibitory effects on the CYP2C8 and CYP2C9 isoforms. This information might be useful in predicting herb-drug interaction potential between biflavonoids and co-administered drugs mainly metabolized by CYP2C8 and CYP2C9. In addition, selamariscina A might be used as a strong CYP2C8 and CYP2C9 inhibitor in P450 reaction-phenotyping studies to identify drug-metabolizing enzymes responsible for the metabolism of new chemicals.Like flavonoids, biflavonoids, dimeric flavonoids, and polyphenolic plant secondary metabolites have antioxidant, antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. However, there is limited data on their effects on cytochrome P450 (P450) and uridine 5′-diphosphoglucuronosyl transferase (UGT) enzyme activities. In this study we evaluate the inhibitory potential of five biflavonoids against nine P450 activities  in human liver microsomes (HLMs) using cocktail incubation and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The most strongly inhibited P450 activity was CYP2C8-mediated amodiaquine  Flavonoids are polyphenolic secondary metabolites that are common in the plant kingdom and are ingested by humans in their food . FlavonoHaving flavonoids in your diet may reduce the risk of atherosclerosis, cardiovascular disease, diabetes mellitus, osteoporosis, and certain cancers ,5. BecauKi value as low as 68 nM in human liver microsomes (HLMs) [Cmax and International Normalized Ratio values than when used alone, indicating the inhibition of CYP2C8, 2C9 and 3A4, major warfarin-metabolizing enzymes [Ginkgo biloba, Selaginella species, Hypericum perforatum, and Garcinia kola [Ginkgo biloba extract [Cupressus funebris, against P450 and UGT enzymes was only recently reported [Several flavonoids are substrates for cytochrome P450 (P450) and uridine 5′-diphosphoglucuronosyl transferase (UGT) enzymes , suggests (HLMs) . It alsos (HLMs) . The flas (HLMs) . Animal s (HLMs) . These rs (HLMs) . Alnaqee enzymes . Biflavo enzymes . They arnia kola . Befittinia kola . In cont extract ). The inreported ,19.Ki) were determined for selamariscina A and compared with those of montelukast, a well-known selective CYP2C8 inhibitor [In this study, we evaluate the inhibitory effects of five biflavonoids—selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone —on nine nhibitor .We purchased acetaminophen, alamethicin, amodiaquine, bupropion, chenodeoxycholic acid, chlorzoxazone, dextromethorphan, estrone glucuronide, glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), hydroxybupropion, magnesium chloride (MgCl2), N-acetylserotonin, β-nicotinamide adenine dinucleotide phosphate (NADP+), N-desethylamodiaquine, omeprazole, phenacetin, trifluoperazine, trimipramine, and uridine 5′-diphosphoglucuronic acid (UDPGA) from Sigma-Aldrich . 4-Hydroxydiclofenac, 4-hydroxytolbutamide, 5-hydroxyrosiglitazone, coumarin, diclofenac, midazolam, montelukast, mycophenolic acid, rosiglitazone, and tolbutamide came from Toronto Research Chemicals . We obtained 1′-hydroxymidazolam from Cayman Chemical , while 7-ethyl-10-hydroxycomptothecine (SN-38) was provided by Santa Cruz Biotechnology . All solvents were LC–MS grade . All the other reagents were of analytical or LC–MS grade and are commercially available. We purchased the pooled human liver microsomes (XTreme 200) from XenoTech . In this study, we used selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone identified from Selaginella tamariscina (Beauv.), which were collected at Yen Tu Mountain, Uong Bi town, Quang Nihn province, Vietnam. The information regarding the identification of their chemical structures was described in our previously published paper ,22.We isolated selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone from Selaginella tamariscina (Beauv.), which were collected at Yen Tu Mountain, Uong Bi town, Quang Nihn province, Vietnam. The five compounds were purified and examined by HPLC to get 95% purity. Their chemical structures were identified by analyzing their NMR data, which were in good agreement with those published in a previous report ,22.v/v). We used these P450 probe substrates: phenacetin for CYP1A2, coumarin for CYP2A6, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6, chlorzoxazone for CYP2E1 and midazolam for CYP3A (+), 3.3 mM MgCl2, and 3.3 mM G6P was added to initiate a reaction, and further incubated for 10 min at 37 °C. The reaction was stopped by adding 50 μL of ice-cold acetonitrile containing 7 nM trimipramine . After centrifugation at 18,000 g , aliquots of supernatants were analyzed by LC–MS/MS . All microsomal incubations were conducted in triplicate.The inhibitory potential of the five biflavonoids on the metabolism of nine P450 probe substrates was evaluated using previously developed methods with minor modifications ,24. Biflor CYP3A . The incTo determine the inhibition mechanism and constants  of the five biflavonoids against CYP2C8 activity, different concentrations of biflavonoids  were added to reaction mixtures containing different concentrations of amodiaquine . The other conditions were the same as in the cytochrome P450 inhibition study.We used HLMs to determine the mechanisms and constants  for selamariscina A inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A. The selamariscina A (0~50 μM) was added into the reaction mixtures, each of which contained concentrations of phenacetin , bupropion , amodiaquine , rosiglitazone , diclofenac , tolbutamide , and midazolam . The substrates were used at concentrations approximately near to their respective Km values ,26,27. TThe time-dependent inhibition of selamariscina A against CYP2C8 and CYP2C9 enzymes was evaluated using an IC50 shift method. Selamariscina A was pre-incubated at six concentrations  with HLMs in the presence of an NDAPH generation system for 30 min at 37 °C. The reaction was initiated by adding 1 μM amodiaquine or 10 μM diclofenac and further incubated for 10 min. Incubation was terminated by adding 50 μL of ice-cold acetonitrile containing 7 nM trimipramine. After centrifugation, aliquots of supernatants were analyzed by LC–MS/MS.The ability of selamariscina A to inhibit the metabolism of six UGT enzyme probe substrates was evaluated using previously developed methods with minor modifications . The micAll metabolites and the IS were separated on a Kinetex XB-C18 column  and analyzed using a Shimadzu LCMS 8060 triple-quadrupole mass spectrometer coupled with a Nexera X2 ultra high-performance liquid chromatography system  equipped with an electrospray ionization interface. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The elution condition was set as 8% B (0–0.5 min), 8%→60% B (0.5–5 min), 60% B (5–6 min), 60%→8% B (6–6.1 min) and 8% B (6.1–9 min) for the analysis of metabolites of P450 probe substrates and set as 0%→40% B (0–1 min), 40%→50% B (1–5 min), 50%→0% B (5–5.1 min), and 0% B (5.1–8 min) for the analysis of metabolites of UGT probe substrates. The flow rate was 0.2 mL/min. Electrospray ionization was performed in positive-ion mode at 4000 V or in negative-ion mode at −3500 V. The optimum operating conditions were determined as follows: vaporizer temperature, 300 °C; capillary temperature, 350 °C; collision gas (argon) pressure, 1.5 mTorr. Quantitation was conducted in selected reaction monitoring (SRM) modes with the precursor-to-product ion transition for each metabolite .Ki) were determined by following several criteria: visual inspection of Dixon plots, Lineweaver–Burk double reciprocal plots, and secondary plots of Lineweaver–Burk plots versus biflavonoid concentrations, the size of the sum of squares of the residuals, Akaike Information Criteria values, the S.E. and 95% confidence interval of the parameter estimates from the nonlinear regression analysis [We analyzed the data with Shimadzu LabSolution LC–MS software. The IC50 values were calculated by WinNonlin software . The type of inhibition and the apparent kinetic parameters for inhibitory activity  . FurtherIn addition, several P450 inhibitors including azamulin, clopidogrel, methoxalene, and ticlopidine ,38,39 haThe inhibitory potential of selamariscina A against uridine 5′-diphosphoglucuronosyl transferase (UGT) activity was evaluated using HLMs . SelamarMontelukast has been used to inhibit CYP2C8 in reaction-phenotyping studies . We re-eCmax/Ki exceeded one and would be possible if it was between 0.1 and 1.0 [Selaginella doderleinii Hieron extracts  [Selaginella doderleinii Hieron extracts (600 mg/kg) [Cmax/Ki were 0.49 and 0.76 from the data of pooled HLMs (Ki = 0.083 μM), indicating that amentoflavone has possible drug interaction potential with CYP2C8 substrate drugs [Cmax (0.22 μM) of amentoflavone after administration of liposome-based Selaginella doderleinii Hieron extracts (200 mg/kg) were 5.4 times higher than those of the control [Cmax/Ki value of 2.65, indicating that amentoflavone has drug interaction potential. In the case of selamariscina A, the present study provides the first published data on its pharmacokinetics in animals and humans. Therefore, it is difficult to estimate the drug interaction potential of selamariscina A for humans. However, selamariscina A might have drug interactions with CYP2C8 substrate drugs such as cerivastatin [It was estimated that an in vivo interaction potential via the inhibition of P450 would likely occur if the ratio of inhibitor  and 1.0 ,41. Baseflavone)  and Sela0 mg/kg) , the reste drugs . The val control , resultivastatin , and rosvastatin  because O-deethylation and diclofenac hydroxylation inhibitory activity against CYP2C8 and CYP2C9, as well as seven other P450s, it exhibited above 50-fold selectivity. Like montelukast and sulfaphenazole, selamariscina A could be useful as a strong CYP2C8 and CYP2C9 inhibitor in P450 phenotyping studies when HLMs are the enzyme source. Additionally, selamariscina A might cause clinically relevant pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP2C8 or CYP2C9. These in vitro findings provide primary data for future in vivo animal and clinical studies on risk prediction related to the interaction of drugs with herbs.In conclusion, we report that selamariscina A is a strong CYP2C8 and CYP2C9 inhibitor. When evaluated for amodiaquine


Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects. Methylxanthines are phytochemicals found at high concentrations in tea, coffee, and chocolate. Among them, theophylline, theobromine, and caffeine are the best known and are often described as mild psychostimulants ,2,3. In AOC3 gene  [The mechanisms of action of methylxanthine psychoactive activity are thought to involve phosphodiesterase inhibition and antagonism of adenosine receptors  . Indeed,ining 3) ,14. Thisining 3) . Pharmacining 3) ,17.The hypothesis that natural methylxanthines can inhibit human SSAO/VAP-1 has been proposed , but not2–C=O–NH–NH2), is classified as toxic since it behaves as an endocrine disruptor [AOC3 gene) will be used hereafter, although semicarbazide was used as a reference inhibitor throughout the study.Semicarbazide is nowadays considered as an “historical” inhibitor of SSAO/VAP-1 because its potency and selectivity have been surpassed by various recently designed inhibitors ,23. In aisruptor , and is isruptor . Consequisruptor . Anotherisruptor . In thisisruptor  has promIn mammals, PrAO is highly expressed at the surface of inflamed blood vessels, but also in vascular smooth muscles and in adipocytes ,27. In lAll the experiments in this study were carried out in homogenates of human subcutaneous adipose tissue (hScAT). This is a unique biological material in that it has high levels of PrAO, while it also contains high amounts of fat, and some sequestration of lipid soluble substances such as methylxanthines is expected. This can make the accurate estimation of kinetic constants difficult and comparison with previous studies using pure recombinant enzyme must be approached with caution. Nonetheless, the following results will show that, among the methylxanthines tested, theobromine and caffeine were significant inhibitors of human PrAO. Furthermore, caffeine, theobromine, and IBMX substantially impaired the MAO activity of human adipose tissue.Benzylamine, methylamine, tyramine, semicarbazide, 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO), hydrogen peroxide, horseradish peroxidase, semicarbazide, all the tested natural methylxanthines and routinely used chemicals were from Sigma-Aldrich-Merck , unless otherwise specified. Amplex Red (from FluoProbes) was purchased from InterChim .Samples of human subcutaneous adipose tissue were obtained from a total of 27 overweight women undergoing reconstructive surgery at Rangueil Hospital , who provided informed consent before inclusion in the study. Their mean age was 47 years, and mean body mass index was 27.5 ± 0.8 kg/m2. Samples of abdominal subcutaneous adipose tissue (ScAT) were obtained from the surgically removed pieces of fat tissue and immediately stored, without any medium, at −80 °C in 35 mL plastic tubes . The study was in compliance with the INSERM guidelines and was approved by the French Ministry of Education, Research and Innovation under the document number DC-2008-452. This is approval to harvest elements of the human body obtained from surgery  with the informed consent of ("absence of opposition to the use of adipose tissue") the donors who are strictly anonymised .Just before the amine oxidase activity assays, human ScAT samples were thawed, then 1–2 g was homogenized for 30 seconds with a homogenizer  and sonicated for 10 s  at room temperature in 10 volumes of 200 mM phosphate buffer (pH 7.5). Homogenization at room temperature was mandatory to avoid the formation of a fat cake, which at colder temperatures becomes solid and thereby hampers sample distribution and enzymatic reactions.2O2). As previously reported [p << 0.001), the majority of the inhibition studies were performed at this benzylamine concentration unless otherwise stated.Homogenates were immediately distributed into 96-well dark microplates (at approximately 50 µg protein/well) and pre-incubated for 10 min without (control) or with 1 mM semicarbazide (abolishing the SSAO activity), or with the tested methylxanthines and their solubilizing vehicles. Incubation was initiated by the addition of 50 µL of substrate at the indicated final concentrations. A fluorimetric method developed for the MAO assay  was sligreported , maximal2O2 as 1 mM benzylamine, mostly in a semicarbazide-sensitive manner, tyramine reached only 40% of the benzylamine-induced signal and was inhibited by pargyline.The same method as above was used for 0.1–1 mM methylamine, another known PrAO substrate , and for2O2 production by hScAT (not shown), the hypothesis that they behaved as amine oxidase substrates was discarded, and they were further tested as potential inhibitors of PrAO or MAO.Furthermore, the fluorimetric amine oxidase assay activity also permitted verification of whether the methyxanthine derivatives on their own were oxidized, or not, by human ScAT. Since none of the methylxanthines tested was capable of increasing the baseline of HApart from caffeine, which was readily soluble in water, 7-methylxanthine, paraxanthine, and theophylline were dissolved in 50% v/v DMSO, which permitted testing at a higher final concentration of 2.5 mM in the presence of 12.5% v/v DMSO . Theobromine could be adequately dissolved in pure DMSO only and was the lone agent tested at 2.5 mM with DMSO at 3700 mM (25% v/v) final. IBMX was dissolved in 10% ethanol (EtOH), and when present in the assays at 2.5 mM, its vehicle was at a level of 2.5% v/v . Uric acid was solubilized in NaOH 1 M, and its higher dose was tested in the presence of 100 mM NaOH final concentration. For all agents, no reprecipitation was observed in the subsequent dilutions made in water or phosphate buffer. No difference was observed between the extemporaneously made solutions and those thawed after less than two weeks of freezing at −20 °C. The influence of vehicles on both amine oxidase activity and hydrogen peroxide detection is detailed in the Results section. When necessary, a correction was applied to crude data to distinguish the effect of high doses of a methylxanthine from that of its vehicle.p < 0.05. Non-linear regression analysis was performed with Graph Pad Prism 5.0.Experimental data are given as mean ± SEM of the indicated number of independent observations and were analyzed with Prism 6 for Mac OS X  by a one–way ANOVA followed by Dunnett’s multiple comparisons. Statistical significance was assumed when 2O2, while DMSO was without any notable effect up to 3700 mM , the final DMSO concentrations present in the assays ranged from 3700 mM (for compounds dissolved in pure DMSO such as theobromine) down to 1850 mM (for xanthines dissolved in 50% v/v DMSO). This occurred when methylxanthines were tested at higher final concentrations of 2.5 mM. Typically, lower DMSO concentrations (<< 925 mM) were routinely used in the assays when solubilized xanthines were diluted for tests at millimolar or sub-millimolar doses. In spite of its recommended use for the dilution of uric acid , NaOH waAll inhibition studies were carried out with 1.0 mM of benzylamine as the substrate since the Km of the PrAO activity found in hScAT was 126 µM . When prThe fact that inhibition was greatest with caffeine and theobromine indicates that the presence of N-methylation of the xanthine core at positions 3 and 7 is required for maximum inhibition. The same pattern was obtained with bovine PrAO . It is pTo further characterize the capacity of methylxantines to inhibit a PrAO-catalyzed reaction, we determined their influence on the oxidation of another substrate: methylamine. This molecule is a physiologically relevant PrAO substrate present in mammals and comes either from the oxidation of adrenaline, or from exogenous sources, since it is naturally present in foods and beverages . Since tAs expected, semicarbazide totally inhibited the oxidation of methylamine , which was resistant to pargyline inhibition , confirming that methylamine oxidation in hScAT is entirely due to PrAO, as recently reported . The oxica. 80%. This may be due to the lower bulk of this substrate, which may allow for greater access to an inhibitor binding site than benzylamine. Differences in patterns of inhibition between benzylamine and methylamine have been previously observed for the bovine enzyme [Inhibition of PrAO catalyzed methylamine oxidation followed a broadly similar pattern to that seen when benzylmine was a substrate with the exception that the non-natural methylxanthine, IBMX, was found to be highly effective, inhibiting activity by e enzyme .In an attempt to analyze the nature of the inhibition by methylxanthines on PrAO activity, the oxidation of increasing doses of benzylamine was explored at: 0.1, 0.2, 0.5, and 1.0 mM, with theobromine at 0, 0.1, 1.0, and 2.5 mM. A Lineweaver–Burk plot of these inhibition curves  illustra50 of 0.27 mM for theobromine  Thus, ttor, see ,45. If btor, see .Besides PrAO, human adipose cells also express MAO, an enzyme located at the outer mitochondrial membrane, mainly of the MAO-A form . It was As with PrAO activity, verification of vehicle influence indicated that DMSO significantly impaired tyramine oxidation when present at ≥12.5 % v/v (≥1850 mM), in other words, the concentrations used as the vehicle for 2.5 mM paraxanthine, 7-methylxanthine, or theophylline . When usThe Km for the oxidation of tyramine by hScAT homogenates was 66 µM  and the To circumvent the problem of methylxanthine solubility, especially in the case of theobromine, we tested for inhibition at a lower concentration of substrate and observed that the oxidation of 0.1 mM tyramine was significantly inhibited with 1 mM theobromine as well as by 7-methylxanthine, theophylline, or caffeine, irrespective of their different vehicles. Tyramine oxidation was not altered by semicarbazide or by lower doses of DMSO .At 0.1 mM tyramine, the reaction rate was equivalent to 63.3% of the rate at saturating tyramine. At this lower concentration, we clearly observed inhibition by all the methylxanthines tested. One mM caffeine diminished activity from 63.3% to 26.7% of the reference value. Taken together, all these data showed a clear impairment of tyramine oxidation by methylxanthines, but did not provide evidence of selectivity for PrAO vs. MAO inhibition.In the present study, the oxidation of benzylamine, methylamine, and tyramine by crude homogenates of adipose tissue obtained from overweight women confirmed that the oxidizing enzymes PrAO and MAO were abundantly expressed in this tissue and their activities were readily detectable without prior protein purification. The use of crude human biological material offered the advantage of determining the activity of the human native forms of PrAO and MAO, allowing for possible interplay with naturally occurring partners, regulators, or repressors to be examined. The clear-cut sensitivity to semicarbazide, which abolished benzylamine and methylamine oxidation (while having no effect on tyramine oxidation), clearly confirmed that the hydrogen peroxide released in the presence of these substrates was dependent on PrAO activity. In turn, tyramine oxidation was largely obliterated by pargyline, thereby showing it was predominantly MAO-dependent.in vivo conditions, the methylxanthines are less likely to be trapped in adipocyte lipid droplets and are, therefore, more available to interact with membrane enzymes such as PrAO. Thus, in the present case, the effect of methylxanthine concentration on enzyme activity will be more complex than for a purified enzyme preparation. The true concentration reaching the active site cannot be known with certainty.A known limitation when using such crude biological material arises due to the presence of large amounts of fat in adipose tissue that can influence enzyme accessibility for methylxanthines that are poorly soluble in aqueous solutions. Under in vitro studies are only indicative of what can occur upon ingestion of the dietary compounds or drugs, and our observations on the amine oxidase inhibitory properties of methylxanthines need to be confirmed by pharmacokinetics and in vivo studies before drawing definitive conclusions. In this regard, it is important to note that the PrAO activity found in human adipose tissue is approximately one thousand times higher than that found in the plasma of obese subjects [Obviously, our subjects . It can subjects , and shoSince we were aware of the limited bioavailability of the methylxanthines, we included in our approach a test of several vehicles that included as least two criteria: their capacity to readily solubilize these compounds at millimolar doses, but also their ability to produce minimal interference in the fluorescent assay used for quantification of sub-micromolar doses of hydrogen peroxide. The latter issue could have been avoided by using other more reliable detection methods to determine amine oxidase activity such as that using vanillic acid , though 50 values of methylxanthines found in the present study, our approach has shown that the nature of the inhibition exhibited by theobromine is not simply competitive. This may indicate that methylxanthine binding sites exists not only in the active site of the enzyme, but also in other locations involved in allosteric regulation. This is consistent with the observations by Holt et al., who suggested multiple binding sites [Large concentrations of vehicles were required since the methylxanthines were inactive at micromolar doses (at which they are water soluble), whereas they were inhibitory between 1.0 and 2.5 mM. It must be specified that, lowering the substrates below their Km values would have allowed us to use less inhibitors and vehicles but would dramatically reduce the signal-to-noise ratio. In spite of the high apparent ICng sites ,46 Alterng sites ,44. It iAlthough initially confusing, such in vitro effects for high doses of methylxanthines are neither unexpected nor artifactual. A requirement of millimolar concentration of methylxanthines has already been observed for the inhibition of phosphodiesterases, the antagonism of adenosine receptors  or the i50 values in the micromolar range, they have been successfully used to develop novel selective MAO inhibitors [When we examined inhibition of MAO, we showed that several methylxanthines inhibited MAO in humans. It is important to note that, by comparison to resveratrol and related stilbenes, theobromine inhibits PrAO to the same extent as MAO, while the stilbenes are reported to inhibit MAO more than PrAO. Since the stilbenes exhibit IC50 values ranging between 5 and 25 µM [Obviously there are other plant-derived products that inhibit PrAO at much lower concentrations than theobromine such as the hydrolyzable tannin geraniin, which is reported to have an apparent inhibition constant, Ki, in the micromolar range . Eugenolnd 25 µM . Neverthnd 25 µM . It can nd 25 µM , and cannd 25 µM , indicatnd 25 µM , as obseAnother important finding of the study was that caffeine almost totally abolished the capacity of hScAT to oxidize tyramine. Such inhibition by caffeine of the native MAO found in human adipose depots (mainly of the A form ) is in tin vitro in this study are not relevant for nutritional or pharmacological trials in humans, in view of the high concentrations required, structural modifications of methylxanthines can likely lead to more potent inhibitors of MAO-A and MAO-B or PrAO. Thus, for example, targeting the imidazole binding site on PrAO using xanthine analogs is a worthwhile avenue of research indicated by this work. The increasing number of patents related to the anti-inflammatory properties of agents blocking PrAO activity, and thereby limiting the lymphocyte extravasation at the sites of inflammation, leads us to suppose that methylxanthine-derivatives may have the required properties to improve this novel therapeutic approach.Finally, two aspects need to be discussed in view of the observations reported by our preclinical approach. The first one deals with phytotherapy, traditional medicine, or personalized nutrition. Since methylxanthines coexist in various medicinal plants, their individual moderate inhibitory properties can be cumulative in consumers, leading to a relevant and sustained PrAO/MAO inhibition upon ingestion of coffee, chocolates, or food supplements. Indeed, one has to not only take into account the plasma concentrations of each methylxanthine , but the sum of these and their metabolites achieved in normal or excessive consumers or in subjects with altered PrAO or MAO expression. The notion of a “matrix effect” and synergism should be taken into consideration when further analyzing the beneficial and adverse effects of methyxanthine-rich foods, beverages, or supplements. The second aspect deals with the use of methylxanthines as a scaffold for the design and screening of novel inhibitors of either PrAO or MAO of the A and B forms ,30. EvenMeanwhile, the PrAO and MAO inhibition provoked by methylxanthines should be considered in ongoing and future investigations using these drugs for in vitro experiments where high concentrations are often used to block phosphodiesterases or adenosine receptors such as, for instance, the inclusion of IBMX in differentiation media for preadipocyte cell lines or in prolonged treatments of rodent  or humanin vivo studies with human volunteers. The only in vivo study reported to date was carried out in rats [The levels of methylxanthines attained in vivo (bioavailabilty) have been the subject of research and there is evidence of some variation by gender and by age . However in rats . That stThis study confirms that the bulk of benzylamine and methylamine oxidizing activity in hScAT is carried out by PrAO, and the bulk of tyramine oxidation is carried out by MAO. We have also shown, for the first time, clear evidence that human adipose PrAO activity is strongly inhibited by some methylxanthines (theobromine and caffeine) and not others, albeit at high concentrations. Among the naturally occurring methylxanthines, methylation at positions 3 and 7 of the xanthine nucleus appears to be important for inhibition, identical to the pattern observed for the bovine enzyme . Surprisin vivo study reported to date showed that caffeine, when administered to rats, inhibited PrAO in adipose tissues [It was not possible to clearly deduce the effect of dietary levels of theobromine and caffeine on PrAO from this work due to the difficulty in measuring a Ki in crude hScAT homogenates. However, it is clear that when administered as drugs, the resulting amounts of some methylxanthines could inhibit this enzyme. The only  tissues . Our stuIt is also of considerable interest that roughly 75% of the tyramine oxidation by hScAT was inhibited by pargyline. The non-natural compound IBMX inhibited this activity by an extraordinary 80%. This was followed by caffeine, which inhibited tyramine oxidation by around 75%. These findings show that it is important to consider inhibition of MAO when examining the effect of therapeutic doses of some methylxanthines and raises the intriguing possibility that a xanthine scaffold could be the basis for design of novel PrAO and MAO inhibitors.In summary, the clinical significance of this work is that it will provide the first baseline data relating the methylxanthine structure to inhibition efficacy for PrAO and MAO in hScAT. It will act as a stimulus to further investigate the combined action of methylxanthines on human PrAO and MAO. It will also prompt an exploration of methylxanthine derivatives as possible therapeutics for a wide range of disorders. Finally, it suggests that the large number of existing xanthine derivatives currently in use as therapeutics should be tested for their effect on PrAO and MAO.


APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit. New cells are made when a single cell duplicates its DNA and divides into two cells, distributing the DNA equally between them, in a process called mitosis. The splitting of the two copies of DNA happens through a series of controlled events known as mitotic exit. Previous research has suggested that mitotic exit relies on both the destruction of specific proteins and the removal of tags called phosphate groups from other proteins. Phosphate groups modify how proteins behave and their removal can trigger changes in a protein’s activity.Although protein destruction and phosphate group removal were known to be important to mitotic exit, it was not understood how they are coordinated in the cell to ensure the correct order of events. Holder et al. have used a technique called mass spectrometry to monitor the level of thousands of proteins, and any tags attached to them, during mitotic exit in human cells grown in the laboratory.The experiments revealed that the destruction of a single protein, known as cyclin B, plays a major role in triggering subsequent events. The removal of cyclin B activates enzymes known as phosphatases, which remove phosphate groups from proteins. Phosphatases then act on a wide range of proteins in a specific order that depends on the environment surrounding the phosphate group. This ‘chain’ of phosphatase activity determines the order of events during mitotic exit.The findings of Holder et al. contribute to the basic understanding of how mitotic exit works. Errors in the process can affect the stability of a cell’s genome, contributing to diseases such as cancer. In the future, this may help to identify what goes wrong in these cases and potential avenues for developing treatments. Each cell cycle concludes with mitotic exit, wherein aligned chromosomes are segregated into two newly forming cells. This process has to be executed faithfully, since errors adversely impact the longevity and genomic stability of these new cells, thus contributing to a variety of pathologies, including cancer and neurological disorders . MitoticPP1 and PP2A-B55, both members of the PPP family, are thought to be the major phosphatases responsible for dephosphorylation during mitotic exit in human cells . ConsequPP1 and PP2A share highly similar catalytic subunits and individually do not demonstrate different substrate specificities, necessitating the presence of regulatory subunits to target their activities appropriately . PP1 catCDK1-inactivation and the loss of chromosome cohesion are required to initiate mitotic exit. This is achieved through the APC/C-mediated destruction of cyclin B and securin, respectively . The APCCDC20 becomes active, leading to ubiquitination and proteasomal destruction of many substrates. It has been proposed that continual synthesis and destruction of CDC20 is required for rapid APC/CCDC20 activation upon checkpoint satisfaction  for destruction , triggers a graded series of dephosphorylations whose rate is encoded into the system at the level of individual phospho-sites. We find that one important general feature of this system is that PPPs will more readily dephosphorylate phospho-threonine in a basic environment, both at the level of an individual phosphatase, PP1, or the system as a whole. Significantly, these dephosphorylations proceed normally in the absence of APC/C activity or protein translation following CDK inhibition, demonstrating that ordered proteolysis and protein synthesis alone are insufficient to co-ordinate the events of mitotic exit. Instead we suggest that the main orchestration of mitotic exit events is mediated by regulated dephosphorylation.To gain a more detailed quantitative understanding of mitotic exit it was important to be able to follow changes in both protein abundance and phosphorylation state in vivo with high temporal resolution. To achieve this, HeLa cells were synchronised in mitotic prometaphase by washout from nocodazole arrest for 25 min to allow spindle formation and chromosome alignment. To promote entry into anaphase with a high degree of synchrony, CDK1 was then inhibited using flavopiridol and samples taken every 30 s until 10 min and at increasing intervals up to 60 min . MarkersFour repeats of these time course samples were processed for mass spectrometry using quantitative di-methyl labelling followed by phospho-peptide enrichment . This reStrikingly, given the number of APC/C substrates and proteins synthesised for mitosis, the vast majority of the total proteome was stable during the time window observed . In factIn this data set CCNB1 destruction followed more typical delayed sigmoidal kinetics during mitotic exit , whilst By contrast, when similarly analysed, dephosphorylations clustered neatly into four groups; early, intermediate, late and stable . SequencA clustering analysis of data generated following MPS1 inhibition yielded the same trends in sequence characteristics seen after CDK1 inhibition . Only twAlthough over 2000 proteins were reliably quantified across the time course, it remained possible that lower abundance proteins were being degraded but could not be detected due to instrument limitations. Therefore, high pH fractionation was used to increase the depth of the total-proteome, using samples taken every 10 min across two repeats of the time course. With this approach over 5600 proteins were identified, with 3748 being reliably quantitated . The proIt has been proposed that continued synthesis of key mitotic proteins including CDC20 and CCNB1 is essential for sustained spindle checkpoint function . ProteinHowever, due to the number of CHX treated cells analysed the possibility of small differences being present cannot be excluded . ImportaCheckpoint function was then interrogated directly under conditions of inhibited protein synthesis by treating these cells simultaneously with nocodazole and either DMSO or CHX. In control cells, CCNB1 fluorescence increased until mitotic entry followed by a small plateau, after which the fluorescence gradually decreased , this deChanges in protein level were then followed by western blot in cells arrested in mitosis using either nocodazole or taxol treatment to activate the spindle checkpoint. BUBR1 and MAD2 were both stable for two hours following inhibition of protein synthesis . During We then asked if protein synthesis was required to complete anaphase A and initiate cytokinesis using time lapse imaging. In both control and CHX-treated cells, the mean time for furrow ingression following chromosome separation was ~ 9 min . To provThe data show that beyond the initial destruction of CCNB1, GMNN and securin, protein level homeostasis is not responsible for regulating the events of anaphase. If regulation of anaphase is indeed dominated by phosphatase activity then substrate dephosphorylation should be unaffected by the absence of proteolytic activity . Owing tCDH1 activity. Predictably, the levels of CDH1 were stable throughout the time course, as CDH1 is necessary for APC/C activity into the next G1 phase.An initial low-resolution time course was carried out to assess dephosphorylation under conditions of APC/C inhibition. Cells were prepared as before, and pre-treated with either DMSO, APC/C inhibitors alone or APC/C and proteasome inhibitors (40 µM MG-132) prior to CDK inhibition. The functional consequences of these treatments are highlighted in Importantly, under all conditions dephosphorylation of PP1 and PP2A-B55 substrates could be seen to occur as rapidly as in control cells, as indicated by the dephosphorylation kinetics of PPP1CA-pT320 and PRC1-pT481 . These rIt was found that five clusters gave rise to the most sensible groupings of phospho-site behaviour across both time courses . This alPrevious work has demonstrated that the preference of PP2A-B55 towards basic substrates impacts the order of events in anaphase . In the The clustering comparison of 2734 phospho-sites from control and PPP1CA/C depleted samples was empirically found to require eight clusters corresponding to early, intermediate, late and stable phosphorylation sites defined in Depletion of PP1 has been reported to lead to hyperphosphorylation of Histone H3-T3 in prometaphase and metaphase . IncreasThis approach identified a sub-set of ~300 phosphorylation sites where the steady state level of phosphorylation more than doubled following depletion of PP1 . These sWe conclude that the preference for phospho-threonine in the absence of downstream negative charge may be a general property of PPP holoenzymes with relevance for the ordering of events during mitotic exit. Together, these results support the view that the cascade of dephosphorylation reactions mediated by a combination of PP1 and PP2A provides a mechanism to determine the order of events during anaphase.The combination of high temporal-resolution mass spectrometry with the cell biological techniques used in this research provides insights into cell cycle control. These data show that proteolysis plays a crucial role initiating the mitotic exit transitions whereas phosphatase activity is required for downstream regulation of order within the system. Therefore, the following model is proposed with regard to how CDK-inhibited phosphatases and the APC/C co-operate to ensure orderly progression through mitotic exit . ProteolHere, we have primarily focused on CCNB1 in the context of driving mitotic exit, however both CCNA2 and CCNB2 play important roles during mitosis. CCNA2 is sufficient to trigger mitotic entry in the absence of CCNB1, and can form active CDK1-CCNA2 complexes . Pro-metThe role of CCNB2 is less clear, with CCNB2 deficient mice developing normally into fertile adults . Some stThe very different rates of proteolysis between early and late APC/C substrates described here supports the view that CDC20 and CDH1 globally satisfy two different requirements of mitotic exit  and 8. FXenopus eggs which found that wide-spread dephosphorylation was triggered by the destruction of a small number of proteins, typified by the APC/CCDC20 substrates: CCNB1/2, securin and GMNN  to allow cells to rebuild bipolar mitotic spindles. CDK inhibition, using 20 µM flavopiridol, was used to trigger mitotic exit. Where necessary, drugs were added at the start of the 25 min incubation to ensure maximal inhibition prior to the addition of flavopiridol. MG-132, a proteasome inhibitor, was used at 40 µM while apcin and proTAME, APC/C inhibitors, were used at 400 µM and 25 µM, respectively. Cycloheximide (CHX) was used at 100 µg/ml.For high-resolution time courses HeLa cells were arrested in mitosis using 100 ng/ml nocodazole treatment for 20 hr. Mitotic cells were harvested by shake-off, washed twice in 1x PBS, and once in Opti-MEM, both of which had been pre-equilibrated to 37°C, 5% CO0 sample were required, therefore twelve 300 μl aliquots were taken at this time and each added to a tube pre-aliquoted with 600 µl ice cold urea buffer. Samples were thawed on ice for 20 min with intermittent vortexing and the concentration determined by Bradford assay. A 50 µl aliquot of lysate was taken for western blotting, added to 25 µl of 3x sample buffer and boiled for 5 min prior to loading for western blots. Typically, 6 µg was loaded on each western blot but when necessary increased to 10 µg. Western blot data from time course samples depleted of PP1 subunits were published previously ) and immediately snap frozen. The urea buffer was supplemented with protease inhibitors , phosphatase inhibitors  and 100 nM okadaic acid. For the subsequent di-methyl labelling protocol greater quantities of teviously , here thTime-course samples were reduced using 4 mM dithiothreitol (DTT) (Fluka) for 25 min at 56°C followed by alkylation, using 8 mM iodoacetamide (IAA) incubation, in the dark for 30 min. Excess IAA was quenched by addition of DTT to a final concentration of 8 mM. Proteins were digested first with lysyl endopeptidase (Lys-C)  for 4 hr at 37°C in 8 M urea. The sample was then diluted to 2 M urea, with 50 mM ammonium bicarbonate. Trypsin was then added for 12 hr at 37°C. Both enzymes were used at 1:50 ratio relative to the starting amount of protein. Digestions were quenched by acidification with 5% (vol/vol) formic acid (FA).Digested peptides, equivalent to 0.4–1.5 mg of total protein, were bound to SepPak reverse-phase C18-columns (Waters) and subjected to on-column dimethyl labelling as previously described . Peptideav, for 5 min at room temperature (Eppendorf Centrifuge 5427 R). Columns were washed with 65 µl elution buffer (5% ammonia solution in water), then three times with 65 µl loading buffer (5% [vol/vol] trifluoroacetic acid (TFA), 1 M glycolic acid, 80% [vol/vol] acetonitrile (ACN)). An equal volume of loading buffer was added to the dimethyl labelled peptide mixtures. Phospho-peptides were then bound to the column 65 µl at a time. Following loading, columns were washed once each with loading buffer, 0.2% [vol/vol] TFA acid in 80% [vol/vol] ACN, followed by 20% [vol/vol] ACN. Phospho-peptides were eluted with three washes of 20 µl elution buffer (5% ammonium solution) into 20 µl of 10% [vol/vol] FA and 10% [vol/vol] DMSO. This gave a total volume of 120 µl of which 20 µl was loaded onto the mass spectrometer.Phospho-peptide enrichment was performed using microspin columns packed with titanium dioxide . All spin steps were performed at 550 rpm, equivalent to 34x gav, 3 min unless otherwise specified. Tips were conditioned once each with methanol, 20 mM NH3/80% [vol/vol] ACN and 20 mM NH3. Next 25 µg of peptides, previously resuspended in 5% FA, 5% DMSO [vol/vol], were loaded on each tip; 1500x gav, 3 min. The flow-through was reapplied twice more and the final flow-through (FT) retained. Bound peptides were washed with 20 µl 20 mM NH3 and the flow through was then pooled with FT to create the FT/F1 fraction. Peptides were then eluted with 20 µl of 20 mM NH3 buffers of increasing ACN composition (4–80%), which formed fractions F2-7. Samples containing <10% ACN were diluted 1:1 whereas samples with higher concentrations of ACN were diluted to 10% [vol/vol] ACN and 80% of the sample loaded directly on the mass spectrometer. These dilutions were performed with 5% [vol/vol] FA, 5% [vol/vol] DMSO. Fractions FT/F1 and F6-7 were run on 30 min gradients whilst fractions F2-5 were run on 120 min gradients.Home-made in stage tips, constructed from five 4G C18 discs (Empore) were used for fractionation, all spins were carried out at 3000x g6, maximum injection time 250 ms. The 20 most abundant peaks were fragmented using CID  or ETD  in a data-dependent decision tree method for titanium dioxide-enriched peptides. Peaks from other samples were fragmented using CID alone. Peptide identification and quantification of ‘heavy’ to ‘light’ phospho-peptide ratios was then performed using MaxQuant (LC was performed using an EASY-nano-LC 1000 system (Proxeon) in which peptides were initially trapped on a 75 µm internal diameter guard column packed with Reprosil-Gold 120 C18, 3 µm, 120 Å pores (Dr. Maisch GmbH) in solvent A (0.1% [vol/vol] FA in water) using a constant pressure of 500 bar. Peptides were then separated on a 45°C heated EASY-Spray column  using a 3 hr linear 8–30% (vol/vol) acetonitrile gradient and constant 200 nl/min flow rate. Titanium dioxide-enriched peptides were introduced via an EASY-Spray nano-electrospray ion source into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Other samples were injected into Q-exactive mass spectrometers (Thermo Fischer Scientific), using linear 8–30% [vol/vol] acetonitrile gradients between 1 and 3 hr in length. For non-fractionated total protein samples, 6 µg was loaded onto the mass spectrometer for a 3 hr gradient. Spectra were acquired with resolution 30,000, m/z range 350–1,500, AGC target 10MaxQuant .0 for each timepoint was averaged across repeat experiments, presuming at least two valid quantification values were observed. Protein groups and phospho-peptides were then filtered for the number of values observed over time, using CCNB1 and PRC1-pT481 as reference profiles of proteolysis and dephosphorylation, respectively. While there were a small number of dephosphorylations that were more rapid than the PRC1-pT481, these were less well understood and would not serve as a reliable reference. At this point any remaining missing values were replaced with the constant value, 0, to enable the clustering algorithm to function correctly. The hierarchical clustering algorithm within Perseus was used, grouping rows into four clusters and outputting a heat map. For clustering a single condition, ten iterations and one restart were allowed, this was increased to 25 iterations and two restarts when comparing two conditions.The MaxQuant  search o0 sample from different experimental conditions were mixed together, for example control t0 light label mixed with the t0 heavy label from a perturbed condition. The ratios were extracted from the cross-mixed samples and used to adjust the protein/phospho-peptide ratios relative to control. Following this, a more iterative approach to clustering was required; starting from three, the number of clusters was gradually increased. This was continued until the settings were found which yielded the most clusters without producing random groupings containing very few identifications. Taking this approach allowed the comparison of complex trends found within the large data sets from the two conditions. The heatmap shown was derived from the majority result of the clustering analysis in each case. In the case of PPP1CA/C depleted time course, an error occurred during mixing heavy and light label resulting in all ratios being up-shifted. To account for this, the average ratio from total proteome siControl and siPPP1CA/C cross-mixed samples was used to adjust the ratios from the corresponding phospho-proteome samples. These corrected ratios were then used to adjust siPPP1CA/C timecourse ratios relative to siControl as described above.When comparing time-courses obtained under two or more different conditions it was necessary to account for changes in the steady state level of a protein or phospho-peptide using a crosswise comparison. To do this, samples comprised of equal quantities of the starting light and heavy labelled tClusters were exported separately from Perseus. The phospho-site sequences of each individual cluster were then aligned in IceLogo 1.2 software , using tTo calculate the half-lives of individual proteins from fractionated time course samples the Maxquant output was filtered and missing values replaced, as above. The ratios for each time point were averaged across experimental repeats and then median subtracted. A correction of 0.85 was added to all ratios to remove any negative values. To account for technical variation in detection between the samples at 50 and 60 min, where the latter value was consistently higher across all proteins, these ratios were averaged. This endpoint value was then used to calculate a percentage of protein remaining after 60 min and converted to a half-life.0 were collected by shake-off and pelleted 400x gav, 60 s and then resuspended in 1 ml 1x PBS, 37°C and spun 1000x gav, 30 s. The washed pellet was lysed in 50 µl of 1.5x sample buffer and diluted 1:3 with 1x samples buffer and 5 µl loaded for western blotting. Time was then started following addition of either 100 µg/ml cycloheximide or an equal volume of DMSO to the remaining wells. Samples were harvested, as above, at the remaining timepoints.HeLa cells were seeded at 50,000 cells/well on six well plates in DMEM/FBS and grown for 72 hr. These cells were arrested in mitosis using either a 100 ng/ml nocodazole, 20 hr or 85 ng/ml taxol, 16 hr. Cells for t2, and imaging was started 9 hr later. Cells were imaged in a chamber maintained at 37°C, 5% CO2, using an Olympus Ix81 inverted microscope, 60 × 1.42 NA oil immersion objective and an Ultraview Vox spinning disk confocal system (Perkin Elmer). A C9100-EM-CCD camera was used for image capture. Volocity software was used for experimental set up, image projection and exporting. Cells were excited with 488 nM lasers with 100 ms exposures at 4% laser power. Cycloheximide and nocodazole were diluted in 100 µl of pre-equilibrated media before being added dropwise when necessary during the experiment, to a final concentration 100 µg/ml and 100 ng/ml, respectively.Live cell imaging was carried out in 35 mm dishes with a 14 mm No. 1.5 thickness coverglass window (MatTek Corporation). HeLa CCNB1-GFP cells were seeded at 50,000 cells/imaging dish and grown for 48 hr before being treated with 2 mM thymidine for 18 hr. The thymidine was removed by washing twice with 2 ml 1x PBS and twice with 2 ml DMEM, both pre-equilibrated at 37°C, 5% CODensitometric quantification of western blots was also performed in ImageJ. Intensity was measured in a 60 × 30 px region of interest around each band. An adjacent region was also measured to account for background intensity. Microscopy images were processed in ImageJ. For the CCNB1-GFP fluorescence measurements over time, raw image data were opened in ImageJ using the BioFormats plug-in. Images were then Z-projected to sum the intensity across the volume of the whole cell. A region of interest was drawn around each cell in interphase, this shape was copied to a vacant region of the field of view for a measurement of background signal. The integrated density was then measured in these regions over time and the background subtracted. The corrected values were plotted as raw intensity. Graphs and statistical analysis were performed in GraphPad Prism. All figure layouts were created using Adobe Photoshop and Illustrator (Adobe CC). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper provides key insights into the cell cycle controls that direct exit from mitosis. Through a high-resolution proteomics approach, the authors determine the relative contributions of proteolysis and protein dephosphorylation to mitotic exit. The findings show that cyclin B destruction initiates sequential waves of dephosphorylation, dependent on the phosphatase PP1.Decision letter after peer review:eLife. Your article has been reviewed by Anna Akhmanova as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Paola Vagnarelli (Reviewer #1); Helder Maiato (Reviewer #3).Thank you for submitting your article "Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The Authors have addressed in this paper the important question of how mitotic progression is temporally regulated. The contribution of proteolysis and protein de-phosphorylation are well accepted as main mechanisms driving the exit from mitosis. However, the relative contribution and the temporal regulation are still in need of clarification. Here, the authors have set up a system to analyse and compare both protein degradation and protein de-phopshoryation in a relatively short timeline upon induction of exit form mitosis. They have presented interesting datasets that allowed the author to map the proteome during mitotic exit and the de-phosphorylation kinetics of several proteins during this stage of the cell cycle. Based on these analyses and on several manipulations of the system which included inhibiting protein degradation / protein synthesis or protein de-phosphoryaltion, they concluded that mitotic exit is temporally driven mainly by de-phosphorylations that are triggered by CDK1 inactivation and that proteolysis (apart for cyclin B) is not the main contributor to mitotic exit. This study is important and provides some provocative ideas on how exit from mitosis is regulated. This paper will be of exceptional interest after addressing a few points.Essential revisions:1) More information about the stages of populations of cells were analysed should be provided. With the presented data it is not clear how mitotic/cell cycle stage is inferred from the time course. The time points are very close to each other and having an idea of how many cells are really going through the process in a synchronous matter is very important. This information is missing in the paper. For example, the labels at the top of Figure 1 indicate timepoints that reflect anaphase and telophase. Time-lapse microscopy of GFP-cyclin B1 in HeLa cells showed GFP-cyclin B1 degradation starts at the metaphase-anaphase transition, but the labels in Figure 1 indicate different timing. Similarly, at the top of Figure 3B, "G1" is stated and it is not clear what this means. The timing of the forced mitotic exit system used here may not be comparable with an unperturbed mitosis. It would be useful to know, using a live cell imaging approach, how the population behaves after releasing the cell from nocodazole and then from the addition of the CDK1 inhibitor. This would also allow comparing the time course of mitotic exit to the ones for cells that are normally cycling. It has been reported that there are differences and some of them could explain the "noise" of a few datasets. A phenotype-proteome correlation could help to clarify.2) Some statements are too strong and should be rephrased or discussed more critically.One example is the experiments in Figure 4:Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition”: “…There was no statistically significant difference in the mean time spent in mitosis following addition of CHX . This suggests that neither mitotic progression nor SAC function is perturbed in the absence of protein synthesis”. The number of cells analysed for the experiment in 4C is quite small for the treated ones (n=7), this explains why the statistic is not strong enough. If we look at the distribution, the majority of the cells (n=5) show actually a shorter NEB-Anaphase timing. This could indicate that there is a difference. To make this statement, more cells need to be analysed or the statement needs to be removed.3) Similarly, in Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition” the statement " the CCNB1-GFP cells treated with CHX did not display any apparent structural abnormalities during this time window " does not represent what the image shows. Just as gross analysis, it is quite visible that the cell does not properly round up, the spindle is not really well formed/shaped. This is just looking at the DIC and CCNB1-GFP, but no other characterisation has been provided. Therefore, as it stands, this conclusion is not supported by the evidence provided and more analyses are required to really demonstrate that mitotic progression does not require protein synthesis.4) Some western blots on the kinetics and relative quantifications are based on n=1 or 2 experiment . A repeat would be appropriate since there is quite some noise in the set up as shown by the error bars in Figure 4F.0 of the experiment. This is fine in a control situation. However, it is well described that repression of phosphatases in mitosis causes hyper-phosphorylation of the substrates. If a protein accumulates as phosphorylated in double the amount, then of course, regardless of the phosphatase, it would take double the time (or more time) in order to be de-phosphorylated. So, the apparent delayed kinetic is not a reflection of the reduced de-phosphorylation but just of the extra number of molecules that need to be processed. For example, if normally there are 10 phospho-molecules to get to 50% the cell needs to process 5 and if the capacity of the cell is 1 molecule/minute it will take 5 minutes. The time will change enormously if the starting point is 100 molecules. The prolonged time occurs even if the capacity of the cell does not change.5) The experiment from Figure 7 needs some adjustments because the method to inactivate PP1 could cause indirect effects, accounting for the observations. siRNA for the catalytic subunit (more than 100 holoenzymes affected) for a few days is expected to cause a lot of defects in the phospho-proteome. If this experiment is to be included, the data should be re-evaluated. The authors have calculated the level of phosphorylation based on a normalisation to the t0 first. This would provide a normalisation baseline and allow the authors to distinguish between a problem of delayed de-phosphorylation in mitotic-exit or a problem of early mitosis hyperphoshorylation.It is therefore important to compare the level of phosphorylation for each protein in the control and siRNA samples at t6) The Abstract concludes with the following statement, "selective proteolysis of cyclin B[1] drives the bulk of changes observed during mitotic exit". Treatment with flavopiridol will inhibit all CDK complexes, not just cyclin B1-CDK1. The data clearly suggest that one of the major roles of the APC/C is to reduce CDK activity. But the data do not exclude phosphorylations (possibly key ones) mediated by other cyclin-CDK complexes during mitosis that are also inhibited by flavopiridol. Do cyclin B2 and cyclin A2 levels decrease during the time course? This point should be discussed.eLife. Your revised article has been evaluated by Anna Akhmanova (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Overall, the reviewers are satisfied with the responses to their comments, however, there is one exception which relates to the re-analysis of the phosphoproteome in Figure 7, which we request is further documented. The point under discussion is the fold change in phosphorylation upon PP1 depletion. While you report in the rebuttal letter that on average the difference is 1.4-fold and argue that this level of fold-change is not major, others might have a different interpretation. Depending on the distribution of fold-changes and how the average is calculated , a 1.4-fold global change  could be viewed as major. This average value could reflect a massive effect on a subset of proteins  and very little impact on other, non-PP1-regulated proteins, which when averaged together lead to a "modest" average global fold change of 1.4. To address this, it would be more informative to show a distribution of phosphorylation fold changes for all detected phosphorylation sites, potentially highlighting specific substrates or sites that show very high fold change. Readers then can make their own judgment about if this change is major or not. This is important because there are examples in the literature of substrates that change drastically . Essential revisions:1) More information about the stages of populations of cells were analysed should be provided. With the presented data it is not clear how mitotic/cell cycle stage is inferred from the timecourse. The time points are very close to each other and having an idea of how many cells are really going through the process in a synchronous matter is very important. This information is missing in the paper. For example, the labels at the top of Figure 1 indicate timepoints that reflect anaphase and telophase. Timelapse microscopy of GFP-cyclin B1 in HeLa cells showed GFP-cyclin B1 degradation starts at the metaphase-anaphase transition, but the labels in Figure 1 indicate different timing. Similarly, at the top of Figure 3B, "G1" is stated and it is not clear what this means. The timing of the forced mitotic exit system used here may not be comparable with an unperturbed mitosis. It would be useful to know, using a live cell imaging approach, how the population behaves after releasing the cell from nocodazole and then from the addition of the CDK1 inhibitor. This would also allow comparing the time course of mitotic exit to the ones for cells that are normally cycling. It has been reported that there are differences and some of them could explain the "noise" of a few datasets. A phenotype-proteome correlation could help to clarify.To answer this question, we first need to explain our methodology and the way we have assigned the different stages of mitotic exit using specific biochemical markers. This approach allows us to more accurately compare single cell data, for example of GFP-cyclin B levels with the ensemble biochemical data obtained using populations of millions of cells. To be able to make such a comparison we first need to produce highly synchronous anaphase on a scale large enough to perform biochemical experiments. The methodology uses a mitotic arrest to create a defined starting cell population, collection of mitotic cells by shake-off and then triggering synchronous anaphase entry using chemical inhibition of either CDK1-cyclin B or the checkpoint kinase MPS1 is well-established within the lab and has been published previously . That work has used a combination of live cell imaging and biochemistry to establish thresholds for CDK1-cyclin B and PP2A-B55 phosphatase activities and shown timing of events is comparable in this system. Additionally, this experimental strategy has been used within the wider literature and shown to be comparable to normal anaphase . Our work builds on those studies, and to repeat that here is not necessary. We should note that it is inevitable that the timing comparisons become less exact at later time points due to the heterogeneity in cell behaviour and biochemical data.To assign the specific stages of mitotic exit in the biochemical analysis shown in Figure 1, we marked anaphase as the point at which the anaphase-specific phosphorylation, PRC1-pT602, begins to accumulate to detectable levels. PRC1 pT602 phosphorylation is prevented during mitosis by the CDK1-dependent phosphorylation of PRC1 at T481 which is removed by PP2A-B55 in anaphase . We don’t differentiate anaphase A and B in this case, since sister chromatid separation can’t be easily followed in our biochemical assays. Telophase was marked as the point at which the anaphase PRC1-pT602 signal began to decline and CCNB1 signal was no longer detectable. These events are indicative of separate biochemical states defined by levels of kinase activity. In anaphase cyclin B levels are still rapidly falling, whereas in telophase they have reached a low level. By comparison to the loss of global CDK1-cyclin B activity, the activity of Aurora and PLK1 persists in anaphase and the proteolytic inactivation of these accessory kinases commences in telophase. These profiles of mitotic kinase activity and stability are critical hallmarks of directionality during mitotic exit and seemed appropriate to use as both indicators of our experimental system reproducing the expected behaviour and of approximate cell staging to aid interpretation of the results.With respect to destruction of CCNB1, we do not claim that destruction of CCNB1 begins after anaphase onset. The relative timing of anaphase dephosphorylations and metaphase proteolytic destruction of CCNB1, securin and GMNN are altered as a direct consequence of our experimental set up. Importantly, the use of CDK1 inhibition to drive cells in mitosis uncouples CDK activity from CCNB1 level, which thus follows APC/C activity as we have modelled previously . This changes the order of events, allowing early anaphase dephosphorylations to precede destruction of CCNB1.Subsection “Ordered dephosphorylation, not proteolysis, is the dominant mode of anaphase regulation”: “Of all the detected proteins only CCNB1 and securin (Western blot only) exhibited rapid enough destruction to impact the events of anaphase. Importantly, in unperturbed cells, these degradation events occur prior to the onset of anaphase.”To increase clarity regarding this matter we have now altered the text describing the interpretation of Figure 1.Subsection “Ordered dephosphorylation, not proteolysis, is the dominant mode of anaphase regulation”: “The use of chemical CDK inhibition to trigger mitotic exit uncouples CDK1 activity from the level of CCNB1, allowing cells to exit mitosis prematurely prior to CCNB1 proteolysis. Consequently, some dephosphorylations take place earlier than under physiological conditions ”.The labels used in Figure 3B illustrate the large difference in proteolytic rate of early and late APC/C substrates by drawing an approximate correlation between experimentally derived protein half-lives and progression through mitotic exit. They are not a definitive assignment of the cell cycle stage during which protein levels reach their minimum value. Indeed, whilst many of these proteins were stable during our window of observation, we cannot exclude accelerated rates of proteolysis occurring beyond the timescale of our analysis, leading to an over-estimation of half-life. However, we believe these qualitative assignments are appropriate, based on the wealth of published live cell data of cells exiting mitosis. In particular, 15-20 minutes after anaphase onset occurs nuclear pores have reassembled and nuclear import has restarted . Furthermore, Cep55 begins to replace PLK1 at the midbody  and the bulk of PRC1-pT602 signal  is lost 60 minutes following anaphase entry and CDK inhibition, respectively. Finally, by two hours, chromosomes have decondensed, cells have flattened and re-adhered.2) Some statements are too strong and should be rephrased or discussed more critically.One example is the experiments in Figure 4:Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition”: “…There was no statistically significant difference in the mean time spent in mitosis following addition of CHX . This suggests that neither mitotic progression nor SAC function is perturbed in the absence of protein synthesis”. The number of cells analysed for the experiment in 4C is quite small for the treated ones (n=7), this explains why the statistic is not strong enough. If we look at the distribution, the majority of the cells (n=5) show actually a shorter NEB-Anaphase timing. This could indicate that there is a difference. To make this statement, more cells need to be analysed or the statement needs to be removed.We have revised the text and rephrased these statements. We accept that the sample size for the analysis of time spent in mitosis following blockade of protein synthesis with cycloheximide (CHX) is small. This is a consequence of the technically challenging experimental setup where we live image single cells and add CHX. If CHX is added too early, then cells fail to produce enough cyclin B to enter mitosis. It is extremely difficult to predict which late G2 cells have synthesized sufficient CCNB1 for mitotic entry prior to addition of CHX.Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition”: “Cells which had accumulated adequate levels of CCNB1 before the addition of CHX entered mitosis , and spent a similar time in mitosis to the control cells . […] Importantly, unlike MAD2 depletion , cells treated with CHX did not collapse out of mitosis, suggesting that cells were able to support spindle checkpoint function and enter anaphase following destruction of CCNB1.”3) Similarly, in Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition” the statement " the CCNB1-GFP cells treated with CHX did not display any apparent structural abnormalities during this time window " does not represent what the image shows. Just as gross analysis, it is quite visible that the cell does not properly round up, the spindle is not really well formed/shaped. This is just looking at the DIC and CCNB1-GFP, but no other characterisation has been provided. Therefore, as it stands, this conclusion is not supported by the evidence provided and more analyses are required to really demonstrate that mitotic progression does not require protein synthesis.We have removed the statement in question. The revised text now describes the observations more simply.Subsection “Protein synthesis is not required for progression through the metaphase-anaphase transition”: “We then asked if protein synthesis was required to complete anaphase A and initiate cytokinesis using time lapse imaging. […] Destruction of proteins during mitotic exit was then followed by Western blotting and was found to be unaffected by inhibition of protein synthesis .”4) Some western blots on the kinetics and relative quantifications are based on n=1 or 2 experiment . A repeat would be appropriate since there is quite some noise in the set up as shown by the error bars in Figure 4F.We have now added additional data to Figure 4D-4F and included this in the quantification so that n=3. We have not included an additional repeat of Figure 5, since Figure 6—figure supplement 1 already repeats the two main conditions from this experiment (DMSO and APC/C inhibition) using a higher rate of sampling. The quantification of the relevant timepoints has now been included in Figure 5 to more fully address this point.0 of the experiment. This is fine in a control situation. However, it is well described that repression of phosphatases in mitosis causes hyper-phosphorylation of the substrates. If a protein accumulates as phosphorylated in double the amount, then of course, regardless of the phosphatase, it would take double the time (or more time) in order to be de-phosphorylated. So, the apparent delayed kinetic is not a reflection of the reduced de-phosphorylation but just of the extra number of molecules that need to be processed. For example, if normally there are 10 phospho-molecules to get to 50% the cell needs to process 5 and if the capacity of the cell is 1 molecule/minute it will take 5 minutes. The time will change enormously if the starting point is 100 molecules. The prolonged time occurs even if the capacity of the cell does not change.5) The experiment from Figure 7 needs some adjustments because the method to inactivate PP1 could cause indirect effects, accounting for the observations. siRNA for the catalytic subunit (more than 100 holoenzymes affected) for a few days is expected to cause a lot of defects in the phospho-proteome. If this experiment is to be included, the data should be re-evaluated. The authors have calculated the level of phosphorylation based on a normalisation to the t0 first. This would provide a normalisation baseline and allow the authors to distinguish between a problem of delayed de-phosphorylation in mitotic-exit or a problem of early mitosis hyperphoshorylation.It is therefore important to compare the level of phosphorylation for each protein in the control and siRNA samples at t0 samples of different conditions, e.g. t0 heavy siControl mixed with t0 hight siPPP1CA/C. These values were used to adjust the observed H/L phospho-peptide ratios from the siPPP1CA/C condition relative to control. The average ratio of total protein from siCon/siPPP1CAC cross-mixed samples, which was not the expected ratio of 1, was used to account for a technical error in quantifying amounts of protein between different conditions. Following cross-mixing, the average t0 phospho-peptide ratio was 1.01 and 1.42 in the control and PP1 depleted conditions, respectively. This modest increase in phosphorylation state is inconsistent with the large increase in phosphorylation occupancy required to lead to delayed dephosphorylation in the way described by the reviewers. The majority of mitotic phosphorylations are CDK-dependent and are typically high occupancy, due to the maximal CDK activity during mitosis. It has been shown that more than half of the mitotic phospho-proteome reaches >70% occupancy . This reduces the potential for hyper-phosphorylation to develop for CDK substrates in the absence of opposing phosphatase activity.Analysis of shifts in the steady state levels of phosphorylation for individual peptides in the mitotic phospho-proteomes following depletion of catalytic subunits is possible using our methodology because of the crosswise mixing approach we use. We have now provided a revised analysis of the data set in Figure 7 which should address the concerns. To do that we used data from cross-mixed samples containing equal quantities of phospho-peptides from equivalent tTo explain this data, one has to consider the way PP1 and PP2A-B55 are regulated. Because both PP2A-B55 and PP1 are inhibited during mitosis, in a CDK1-dependent manner, there is minimal global activity of these phosphatases. Consequently, although local pools of PP1 exist, inactivation of these phosphatases has little effect on the global mitotic phospho-proteome. It therefore follows that our global analysis is unaffected by the point raised in the reviews. Inactivation of CDK-opposing phosphatases will have much larger effects on the dynamics of dephosphorylation in anaphase, and potentially the G2/M transition, but not the level of steady-state mitotic phosphorylation. This was previously observed when global levels of mitotic phosphorylation did not increase following depletion of PP2A-B55 . The scenario described by the reviewers may be more applicable when the mechanisms required for inhibition of PP1 and PP2A-B55 have been removed, namely PP1-T320 mutants or B55-inhibitors ENSA/ARPP19 and MASTL. Consequently, CDK would have to continually counteract the activity of PP1 and PP2A-B55, potentially shifting steady-state phosphorylation levels slightly downward. Additionally, PP2A-B56 and PP6 are active throughout mitosis and dynamically oppose locally regulated Aurora kinase activities. Therefore, inactivation of PP2A-B56 and PP6 would likely result in an upshift in steady-state phosphorylation because these substrate phosphorylation occupancies are normally limited by activity of the respective PPP.6) The Abstract concludes with the following statement, "selective proteolysis of cyclin B[1] drives the bulk of changes observed during mitotic exit". Treatment with flavopiridol will inhibit all CDK complexes, not just cyclin B1-CDK1. The data clearly suggest that one of the major roles of the APC/C is to reduce CDK activity. But the data do not exclude phosphorylations (possibly key ones) mediated by other cyclin-CDK complexes during mitosis that are also inhibited by flavopiridol. Do cyclin B2 and cyclin A2 levels decrease during the timecourse? This point should be discussed.Our work has to be viewed alongside the large body of published work showing that Cyclin B1 is the only major cyclin remaining immediately prior to the onset of anaphase and is the major cyclin needed to sustain mitosis during a spindle checkpoint arrest. Cyclin A2 (CCNA2) is rapidly destroyed following nuclear envelope breakdown, independent of active checkpoint signalling . We can therefore conclude that CCNA2 is unlikely to contribute to regulation of mitotic exit in this system. Our approach using a mitotic arrest thus gives a biochemical state where cyclin B persists, but cyclin A levels are very low, and mimics the conditions preceding a normal metaphase-anaphase transition. In agreement with this view, CCNA2 is barely detectable during the observed experimental window during mitotic exit (Author response image 1).Levels of CCNB2 do decrease during the course of the experiment. However, mice lacking CCNB2 are fertile and develop normally. Furthermore, CCNB2 is expressed at lower levels than CCNB1 so may not be able to provide sufficient levels of CDK activation to compensate for all functions of CCNB1 . This latter point is supported by the inconsistent detection of CCNB2 across the time course, even in fractionated total proteome samples . Taking into account the prior degradation of CCNA2 and the low levels of CCNB2, we would therefore argue that degradation of solely CCNB1 is the driving factor for mitotic exit. We accept it is important to address the roll of other cyclins in this context and the following text has been added to address these topics in subsection “Ordered proteolysis of cyclins during mitosis and mitotic exit”:“Here, we have primarily focused on CCNB1 in the context of driving mitotic exit, however both CCNA2 and CCNB1 play important roles during mitosis. […] We would therefore propose that it is the destruction of solely CCNB1 which drives mitotic exit.”The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Overall, the reviewers are satisfied with the responses to their comments, however, there is one exception which relates to the re-analysis of the phosphoproteome in Figure 7, which we request is further documented. The point under discussion is the fold change in phosphorylation upon PP1 depletion. While you report in the rebuttal letter that on average the difference is 1.4-fold and argue that this level of fold-change is not major, others might have a different interpretation. Depending on the distribution of fold-changes and how the average is calculated , a 1.4-fold global change  could be viewed as major. This average value could reflect a massive effect on a subset of proteins  and very little impact on other, non-PP1-regulated proteins, which when averaged together lead to a "modest" average global fold change of 1.4. To address this, it would be more more informative to show a distribution of phosphorylation fold changes for all detected phosphorylation sites, potentially highlighting specific substrates or sites that show very high fold change. Readers then can make their own judgment about if this change is major or not. This is important because there are examples in the literature of substrates that change drastically .0 ratios in Control and PPP1CA/C conditions, as well as specific examples of phosphorylation sites from proteins with key mitotic functions; BUB1, INCENP and Ki-67 .To clarify the point in question we have included an additional figure within the revised manuscript, Figure 7—figure supplement 1, with the relevant data included in Figure 7—source data 1. This extended figure shows a comparison of the starting tFrom the distribution of starting ratios, it can be seen that the majority of phospho-sites show a modest, or no, increase in steady-state mitotic phosphorylation. We suggest that these can be separated into two categories, the first are phosphorylation sites that are not subject to PP1-dependent dephosphorylation. The second category would consist of phosphorylation sites which are dephosphorylated in a PP1-dependent manner during anaphase. Importantly, the pool of PP1 responsible for the anaphase dephosphorylation of these sites would be inhibited during mitosis, meaning the mitotic abundance of these phosphorylation sites is independent of PP1. Therefore, depletion of PP1 would not result in a large change in steady-state levels of these phosphorylations rather it would delay the kinetics of dephosphorylation during anaphase.A sub-set of ~300 phosphorylation sites demonstrate a steady state level of phosphorylation which more than doubled following depletion of PP1 . This behaviour is indicative of PP1-dependent regulation during mitosis, marking these phosphorylation sites as candidate mitotic substrates of PP1 . This is consistent with published data regarding H3-pT3 highlighted by the reviewers . Furthermore, for these sites it would be difficult to distinguish whether any observed delay in anaphase dephosphorylation is due to the absence of anaphase-PP1 activity or due to the presence of more molecules to dephosphorylate, as previously stated by the reviewers in response to our initial submission. However, this does not impact our global analysis of dephosphorylation in the absence of PP1 as, in order to enable efficient clustering of the data, phosphorylation sites with a starting ratio of greater than 2.5 are excluded from the clustering analysis.To address these points the following text has been added to subsection “PP1 and PP2A-B55 share a preference for basic phospho-threonine sites”:“Depletion of PP1 has been reported to lead to hyperphosphorylation of Histone H3-T3 in prometaphase and metaphase . […] Importantly, these PP1-dependent changes occurred on both phospho-threonine and phospho-serine.”Our global analysis of mitotic exit in the absence of PP1 activity will provide a useful list of potential PP1 substrates. However, our methodology only provides evidence of a dependence on PP1 activity not of direct dephosphorylation by PP1, nor the holoenzymes responsible for these dephosphorylations. This information, whilst important, is beyond the scope of the question we were addressing and should instead form the basis of future studies.


Left ventricular non-compaction (LVNC) is a rare congenital morphogenetic abnormality and occurs as a result of an arrest in the compaction of the embryonic myocardium during development resulting in excessive trabeculations in the left ventricle (LV). The relationship of the papillary muscle (PM) development and LVNC has not been previously described.To assess the correlation between LVNC and both the degree of partitions as well as the insertion site  of PM as compared to patients with normal compacted myocardium.We enrolled 297 consecutive patients referred to our cardiac magnetic resonance (CMR) center for cardiac evaluation. We assessed the non-compacted (trabecular region) and compacted myocardium by drawing diameters of each at the left ventricular apex and averaging from two long axis cine views. LVNC was defined as non-compacted to compacted ratio 2.3. The PM insertion site was determined by dividing the long axis of the LV into 3 equal regions and determining in which region  the papillary muscle inserted into the LV. The number of partitions of the PM was determined by counting the number of separate PM visualized 10 mm apical from the mid plane of the LV on short axis cine view.The mean age of the patients was 58.1 years, 60% were males. The patients were referred to CMR for the following reasons: evaluation of left ventricular function (37%), viability (30%), valvular diseases (21%), and other (12%). The average LVEF was 51.2 ± 14.6%. The mean number of PM partition in the LVNC group was 5, and in the normal group was 6 (p = 0.41). The LVNC group had a significantly higher apical insertion of the PM compared to the normal group, 87% (67 of 77 patients) vs 67.3% .Patients with LVNC have a higher incidence of apical insertion of the papillary muscle into the myocardium, without a higher number of partitions.


In patients with ST-elevation myocardial infarction (STEMI), percutaneous coronary intervention (PCI)-mediated reperfusion is preferred over pharmacoinvasive reperfusion with fibrinolysis if transfer to a PCI centre can be ensured in ≤120 min. We evaluated the ambulance driving time to primary PCI centres in the Netherlands and assessed to what extent ambulance driving times were impacted by the expansion of off-site PCI centres.p < 0.001). In postal code areas that were impacted by the initiation of off-site PCI, the median driving time decreased from 25.4 (18.2–33.1) to 14.7 min (8.9–20.9) (p < 0.001). Ambulance driving times of >120 min were only seen in non-mainland areas.We calculated the driving routes from every Dutch postal code to the nearest PCI centre with (on-site) or without (off-site) surgical back-up. We used data from ambulance records to estimate the ambulance driving time on each route. There were 16 on-site and 14 off-site PCI centres. The median (interquartile range) time to on-site PCI centres was 18.8 min (12.2–26.3) compared with 14.9 min (8.9–20.9) to any PCI centre (Based on a computational model, timely ambulance transfer to a PCI centre within 120 min is available to almost all STEMI patients in the Netherlands. Expansion of off-site PCI has significantly reduced the driving time to PCI centres. European guidelines recommend transfer to a percutaneous coronary intervention (PCI) centre if primary PCI can be ensured in ≤120 min.The expansion of off-site PCI centres in the Netherlands has significantly reduced the driving time to PCI centres.Timely ambulance transfer to a PCI centre within 120 min is available to all patients with ST-elevation myocardial infarction living on the Dutch mainland.Percutaneous coronary intervention (PCI) is a commonly used treatment strategy for coronary revascularisation in both stable coronary artery disease and acute coronary syndrome (ACS) . In the In its early days, PCI was only performed in hospitals with on-site back-up for emergency cardiac surgery . HoweverCurrently, there are 30 PCI centres in the Netherlands, of which 14 are off-site PCI centres. However, it remains unknown to what extent the expansion of off-site PCI centres has impacted ambulance driving times for STEMI patients on a national scale. We evaluated the ambulance driving time to a centre for primary PCI care in the Netherlands. In addition, we investigated to what degree off-site PCI centres have contributed to a reduction in ambulance driving times.We conducted a study assessing the accessibility of primary PCI care in the Netherlands, measured by ambulance driving time. We compared ambulance driving time in two settings: (1) a setting where PCI is only performed at on-site PCI centres, and (2) the current setting that includes on-site and off-site PCI centres.https://www.gps-coordinates.net) (NH). We visually checked the hospitals’ coordinates using a satellite map (https://www.google.com/maps).We obtained a dataset containing the number of inhabitants per four-digit postal code in the Netherlands in 2016 from Statistics Netherlands (CBS). We then used four-digit postal code polygon data describing the geographical coordinates of every Dutch postal code in the same year. We obtained a list of all on-site and off-site PCI centres from the Netherlands Society of Cardiology (NVVC) and the Dutch Association for Thoracic Surgery (NVT). Of note, all PCI centres in the Netherlands perform primary PCI on a 24/7 basis. We collected geographical coordinates of the listed hospitals by entering their addresses into an online coordinate converter  of four-digit postal codes in the Netherlands in 2015. The calculations were based on a shortest-route algorithm. To estimate the ambulance driving time, we used historical data from the ambulance transportation records and the known speed limit for each road section. We then calculated the ambulance driving time from each four-digit postal code centroid to the centroid of the nearest PCI centre. We assumed that ambulances transporting STEMI patients operate with the highest priority . The ambulance driving time was estimated for both on-site and off-site PCI centres. The dataset was produced by an external party  on behalf of the National Institute for Public Health and the Environment (RIVM).t-test to compare means and standard deviations. We assessed the fraction of the population living ≤120 min from a PCI centre. Additionally, we assessed the fraction of the population living ≤60 kilometre (km) from any PCI centre. We used 60 km as a cut-off, because a previous Dutch study demonstrated that a distance of >60 km from a PCI centre is associated with an increased total ischaemic time in STEMI patients [We displayed accessibility to PCI care by plotting the ambulance driving time to the nearest PCI centre from each four-digit postal code centroid on a map. In addition, we combined the ambulance driving time to the nearest PCI centre from each postal code with the number of inhabitants per postal code area. We then plotted the ambulance driving time and the corresponding fraction of the population nearest to an on-site or off-site PCI centre. We compared median driving distances and corresponding interquartile ranges (IQRs) using the Wilcoxon signed rank test. In addition, we used the paired patients . All datOn 1 January 2019, there were 30 PCI centres in the Netherlands: 16 on-site and 14 off-site centres (listed in the Appendix). We calculated 4046 centroids for the Dutch four-digit postal code areas.p < 0.001). In postal code areas with a reduction in ambulance driving time after the expansion of off-site PCI, the median ambulance driving time decreased from 25.4 (18.2–33.1) to 14.7 min (8.9–20.9) (p < 0.001). The reduction in estimated driving time after the establishment of a nearby off-site PCI centre ranged from 0.1–67.0 min. Differences in driving time to an on-site or any PCI centre for mainland and non-mainland areas (Wadden Islands) are displayed in Tab. The median ambulance driving time was 18.8 min (IQR 12.2–26.3) when only on-site PCI centres were included and 14.9 min (8.9–20.9) to any PCI centre  lived ≤60 km from a PCI centre. The fraction of the Dutch population with access to PCI care in the Netherlands based on ambulance driving time is displayed in Fig. n = 17,068,005) of the Dutch population could be transported to an on-site PCI centre <90 min. After the expansion of off-site PCI centres, 99.94%  of the population could be transported to any PCI centre <90 min . This indicates PCI-mediated reperfusion rather than a pharmacoinvasive strategy can be provided to all Dutch STEMI patients, in accordance with European Society of Cardiology guidelines . MoreoveA large Swedish study of >10,000 STEMI patients demonstrated that a first medical contact-to-PCI delay of >1 h is associated with an increase in mortality  . TherefoWe observed that 99% of the population lived ≤60 km from a PCI centre. This cut-off was based on a study of >4000 STEMI patients from a large on-site PCI centre in the Netherlands . This stp < 0.001). Moreover, a Danish nationwide study could not demonstrate an association between distance to a PCI centre and improved outcome in out-of-hospital cardiac arrest patients, supporting the centralised strategy [The number of off-site PCI centres has increased, and these centres are regarded as safe when compared with on-site PCI centres , 20, 21.strategy .Ten years ago, the expansion of off-site primary PCI centres in the Netherlands sparked reactions, from both supporters and opponents , 26. OurOne of the strengths of this study is the use of reliable data from government institutions to display a simple structure indicator reflecting one aspect of the quality of a healthcare system .However, there are some limitations. First, our study focused on the impact of off-site PCI expansion on time delays in STEMI patients. However, other local initiatives that were concomitantly introduced, such as prehospital ECG transmission or a home-to-hospital feedback dashboard, have also led to shorter time delays , 30. SecBased on a computational model, the expansion of off-site PCI centres in the Netherlands has reduced the ambulance driving time to PCI centres in this country. It seems unlikely that additional expansion will further improve accessibility to PCI. Future efforts to improve PCI and ACS care should focus on strengthening the collaboration between general practitioners, ambulance services, and off-site and on-site PCI centres, and on further development of the Netherlands Heart Registry.