Pbcn-type distortion which includes the tilting and squishing of hexameric clusters. This type of phase transition may provide an explanation for previously observed ferroelectric signatures in dielectric spectroscopy of ice VI and could be relevant for other icy materials.Ice is a material of fundamental importance for a wide range of scientific disciplines including physics, chemistry, and biology, as well as space and materials science. A well-known feature of its phase diagram is that high-temperature phases of ice with orientational disorder of the hydrogen-bonded water molecules undergo phase transitions to their ordered counterparts upon cooling. Here, we present an example where this trend is broken. Instead, hydrochloric-acid-doped ice VI undergoes an alternative type of phase transition upon cooling at high pressure as the orientationally disordered ice remains disordered but undergoes structural distortions. As seen with in-situ neutron diffraction, the resulting phase of ice, ice XIX, forms through a  Water’s phase diagram exhibits several hydrogen-disordered phases which become ordered upon cooling, but the behavior of ice VI is still debated. The authors, using high-pressure neutron diffraction, identify structural distortions that transform ice VI into ice XIX, here identified as a hydrogen disordered phase. The H2O molecule is a versatile molecular building block capable of forming at least eleven different types of four-fold connected crystalline networks through hydrogen bonding1. For a given network topology, the hydrogen-bonded water molecules can either display orientational disorder or order resulting in so-called hydrogen-disordered and hydrogen-ordered phases of ice, respectively. The general trend is that hydrogen-disordered phases of ice crystallise from the liquid phase which are then expected to undergo hydrogen-ordering phase transitions at low temperatures to their hydrogen-ordered counterparts in line with the third law of thermodynamics 6 clusters in the gas phase11. Upon cooling pure ice VI, the hydrogen disorder is frozen-in as observed by neutron diffraction12. However, dielectric spectroscopy measurements have indicated a very slow transition to a ferroelectric state below ~125 K14. This discrepancy has so far not been clarified. A break-through in hydrogen ordering ice VI came with using hydrochloric acid (HCl) as a dopant which led to the discovery of the antiferroelectric hydrogen-ordered ice XV6. The transition from ice VI to ice XV is accompanied by an increase in volume which means that the most ordered ice XV samples were obtained upon slow-cooling at ambient pressure15. Upon cooling HCl-doped ice VI at pressures above ~1 GPa, the hydrogen-ordering phase transition from ice VI to ice XV is suppressed progressively even though the glass transition temperature of the molecular reorientation dynamics has been lowered significantly by the acid dopant18. These unusual doped samples have been called deep-glassy ice VI19. Upon heating at ambient pressure, deep-glassy ice VI shows the phenomenon of transient ordering as the metastable ice VI state first undergoes irreversible hydrogen ordering to ice XV and then reversible hydrogen disordering to ice VI17.Ice VI is a hydrogen-disordered phase of ice that crystallises from liquid water in the 0.6–2.2 GPa pressure rangesee Fig. 9. The nesee Fig. . These c20. As proposed by the ice β-XV scenario, the low-temperature endotherm arises from a new hydrogen-ordered phase of ice that is more hydrogen-ordered, differently hydrogen-ordered and more stable than ice XV21. In contrast to this, it has been pointed out that the new endotherm is consistent with a kinetic overshoot effect associated with the underlying glass transition of the deep glassy ice VI19. Upon increasing the pressure, more relaxed states are obtained that give more pronounced endotherms. The kinetic origin of the endotherms was illustrated very clearly by varying the heating rate: Depending on the state of relaxation, fast heating could make the endotherm appear whereas slow-heating leads to its disappearance19. It has also been argued that the X-ray diffraction and dielectric spectroscopy data in ref. 20 are consistent with the deep glassy ice VI scenario19. Following a Raman spectroscopic study21, combined inelastic neutron spectroscopy and neutron diffraction data of an HCl-doped ice VI slow-cooled at 1.7 GPa were presented22. Both the spectroscopic and the diffraction data showed that the sample was structurally very similar to standard pure ice VI. The deep glassy ice VI scenario requires the samples to decrease slightly in entropy as the pressure is increased. It was speculated that local and orientationally uncorrelated hydrogen ordering may be occurring as the pressure is increased22. This could lead to increased levels of local distortions that could eventually even reduce the space group symmetry of ice VI22.Recently, it was shown that HCl-doped ice VI samples cooled at pressures above 1.4 GPa display an endothermic feature before the exothermic transient ordering upon heating at ambient pressure and at certain heating ratesIn this work, using in-situ pressure powder neutron diffraction we find evidence for such distortion phenomena of the hydrogen-disordered structure and a systematic analysis of the symmetry changes is presented. The mechanically most meaningful type of distortion is also the one that provides the best fit to the diffraction data. As a final point, we highlight similarities between the pressure-induced ice VI distortion and those observed in perovskite materials.2O ice sample doped with 0.01 molar DCl at ~1.6 GPa. The diffraction pattern at 230 K is consistent with fully hydrogen-disordered ice VI with P42/nmc space group symmetry. Remarkably, slow-cooling to 100 K at ~0.5 K min−1 and ~1.6 GPa leads to the appearance of two very weak additional Bragg features at ~2.14 and ~2.21 Å, as shown in Fig. P42/nmc ice VI model fits the low-temperature diffraction data reasonably well. However, the additional peaks and the intensity increase at ~1.77 Å cannot be reproduced. This means that a new phase of ice has formed that is structurally closely related to ice VI but with a lower space group symmetry. The tickmarks of a corresponding P1 structure with the lattice constants of ice VI in Supplementary Fig. Figure P42/nmc space group of ice VI. This shows that the increase of the unit cell to a √2 × √2 × 1 supercell is quite frequently encountered. Here, a more intuitive crystal-chemical approach is presented that considers local distortions of the hexameric units as the origin for the change in space group symmetry in line with the conclusions of our spectroscopic study22. As it turns out, a √2 × √2 × 1 supercell is the smallest cell needed for describing meaningful distortions of the individual networks. Even the visual appearance of the ice VI structure with its linked hexameric clusters suggests that such a structure could be prone to local distortions .In a next step, the fully hydrogen-disordered Pbcn model clearly shows distortions of the hexameric clusters  approach using the RMCProfile softwareg(r), shown in Fig. g(r) functions look quite similar for the 230 and 100 K data respectively. Close inspection of the O-D g(r) functions shows that the covalent O–D distances at ~0.9 Å are broadened toward lower and higher distances in the 100 K data and the same applies to the hydrogen-bonded O···D distances at ~1.7 Å. The hydrogen-bonded O–O distances at ~2.7 Å in the O-O g(r) are also slightly broadened in the 100 K data. Cooling ice VI to low temperatures without local and asymmetric distortions would lead to sharpening of these features due to the reduction in the thermal displacements. The observed broadening of the short-distance features in the O-D and O-O g(r) functions are therefore consistent with a distorted structure at the low temperature. Interestingly, at larger distances, the O-O g(r) function at 100 K is somewhat more defined than at 230 K. This implies that despite the local distortions, at larger distances the effects of the lower temperature and hence smaller thermal displacements become visible. Overall, the RMC analysis has shown that the low-temperature diffraction data can be fitted with a fully hydrogen-disordered ice VI model that allows local distortions.The experimental diffraction data could be easily fitted with the RMC approach, as shown in Fig. P1 if no tolerances in the atomic positions are allowed. However, upon increasing the search distance within the FINDSYM software24, all the previously discussed subgroups of Pbcn, which resulted from further distortions, were identified before Pbcn was found above 0.36 Å. In summary, all three approaches including the systematic search for crystallographic subgroups, the analysis of meaningful mechanical distortions, and the symmetry-free RMCProfile refinements point towards Pbcn-type structural distortions.In the next step, the large simulation box was analysed for symmetry as shown in Supplementary Fig. Pbcn-type distortions at low temperatures and ~1.6 GPa that make sense both from the crystallographic point of view and the mechanics of the crystal structure. These distortions represent an alternative type of phase transition in icy materials in addition to hydrogen ordering at low temperature. While the resulting structure is related to the hydrogen-disordered ice VI, given its lower space group symmetry, it seems justified to assign the Roman numeral XIX to this phase of ice25. In line with our earlier work, ice XIX is a deep glassy state of ice VI that has experienced distortions under pressure extensive enough to lower the space group symmetry. In ref. 22 we already suspected that something like this may be possible. Accordingly, the reduction of volume by distorting the hexameric clusters achieves the reduction in entropy of the sample. It is also emphasised that a distorted hydrogen-disordered ice XIX structure with local stress elements is consistent with the Raman21 and INS data22 collected earlier for corresponding H2O samples at ambient pressure. The distortion-induced stress can also be seen from increased widths of the Bragg peaks in X-ray20 and neutron diffraction of recovered H2O samples22.DCl-doped ice VI undergoes 26. Consistent with our distortion model, they found that the sample decreases in volume upon the ice VI to ice XIX transition. No crystallographic model was presented and in analogy with previous work6, the additional Bragg features were interpreted in terms of a new hydrogen-ordered structure. To compare the diffraction data collected by us and Yamane and coworkers, the relative intensities of the Bragg peaks at 2.10 and 2.14 Å were determined (see inset in Fig. 26, this ratio is 0.059 compared to 0.197 in our data. This suggests that the pressure in our study must have been somewhat higher leading to more extensive distortions.While analysing this data, two related manuscripts have appeared on preprint servers. Firstly, Yamane and co-workers also collected in-situ pressure diffraction data and found very similar trends in the diffraction data at 1.6 GPa and low temperature including the additional Bragg features and the increase in intensity of the ~1.77 Å peak27. The peak intensity ratio of their data is 0.062 which may suggest that the decompression has led to partial removal of the distortions. Like Yamane and co-workers, they also interpreted their data in terms of a new hydrogen-ordered structure and suggested Pcc2 as the space group symmetry. Using the Pbcn model which permits full hydrogen order. The details of this analysis are shown in Supplementary Fig. Gasser and coworkers recently studied a corresponding recovered sample at ambient pressure and 70 K with neutron diffraction27, the ice β-XV scenario21, which suggested that ice XIX is more hydrogen-ordered than ice XV, can now be firmly excluded. The appearance of the new Bragg features upon cooling doped ice VI at high pressures has now been firmly established by three independent studies. However, the most intense additional Bragg features were found in our study.To conclude, ice XIX may be very weakly hydrogen ordered. However, as argued here, the main structural characteristics of ice XIX are the distorted hexameric units of its hydrogen-bonded networks. As evident from the analysis here and also the structural data in ref. 28. It, therefore, remains an open question what happens to ice XIX once compressed to higher pressures. As previously discussed, considering the many different interactions between neighbouring hexameric units in ice XIX arising from the hydrogen disorder, the actual space group symmetry of ice XIX may be best described by P1 which is a polar space group. Hence, it seems possible that the different local distortions of the hydrogen-disordered hexameric clusters, which all have different structures at the local level because of the hydrogen disorder, lead to weak ferroelectricity. Such subtle effects may be difficult to pick up in diffraction but they would provide an explanation for the weak ferroelectric behaviour of ice VI found with dielectric spectroscopy at low temperatures14. If the distortion of hydrogen-disordered phases at low temperatures and high pressures leads to ferroelectric ice in general will need to be investigated in future studies. In this context, it would be interesting to investigate if the hydrogen-disordered ice VII obtained by low-temperature compression28 is crystallographically fully consistent with the space group symmetry of the corresponding high-temperature phase.The distortion of subunits in ice under pressure opens up an interesting line of future research. It has been shown that ice VI can be compressed up to 3.8 GPa at 95 K before it transforms to ice VII29. As can be seen in Fig. c direction. P4/mbm octahedral tilting, which is the equivalent of the Pbcn tilting in ice XIX, and the closely related I4/mcm tilting have been observed for a range of perovskites under pressure31. If other types of distortions of the ice VI structure are possible at even higher pressures will be the focus of future studies.Finally, it is interesting to note that pressure-induced structural distortions of materials containing cluster units are very common. A prime example is the perovskite structure which contains octahedral units linked to one another at the corners2O was filled into a null-scattering a TiZr gasket and mounted between sintered diamond anvils of a Paris-Edinburgh press on the PEARL beamline32 at the ISIS Neutron and Muon Source, Didcot, UK. Ice VI was prepared by compression at room temperature to ~1.6 GPa. The temperature of the press was controlled with a reservoir of liquid nitrogen at the bottom of the containment vessel and a heating element. Two long data collection runs were conducted at 230 and 100 K as indicated in Fig. A 0.01 molar DCl solution in D33. For the RMCProfile simulations, 4 × 4 × 5 supercells of the √2 × √2 × 1 ice VI unit cell were constructed using the lattice constants obtained from the Rietveld refinements. These structures were fully hydrogen-disordered and harmonic potentials of equal strength were defined between the oxygen and hydrogen sites as well as between the two hydrogen sites along with each hydrogen bond. The equilibrium distances for this were 0.92 and 0.85 Å, respectively. The contributions from the diamond anvils in the diffraction data were subtracted using the calculated profiles from the Rietveld refinement. The background function was also taken from the converged Rietveld refinements.Rietveld fits were carried out using the GSAS softwareSupplementary InformationPeer Review File


Artemisia or Scopolia, and its effects have shown to be promising in areas of anticancer and Alzheimer’s disease. However, the role and mechanisms by which artesunate affects the aggregation of platelets and the formation of a thrombus are currently not understood. This study examines the ways artesunate affects the aggregation of platelets and the formation of a thrombus on platelets induced by U46619. As a result, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production were increased significantly by artesunate relative to the doses, as well as phosphorylated vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP3R), substrates to cAMP-dependent kinase and cGMP-dependent kinase, in a significant manner. The Ca2+, normally mobilized from the dense tubular system, was inhibited due to IP3R phosphorylation from artesunate, and phosphorylated VASP aided in inhibiting platelet activity via αIIb/β3 platelet membrane inactivation and inhibiting fibrinogen binding. In addition, MAPK and PI3K/Akt phosphorylation was inhibited via artesunate in a significant manner, causing the production of TXA2 and intracellular granular secretion (serotonin and ATP release) to be reduced. Therefore, we suggest that artesunate has value as a substance that inhibits platelet aggregation and thrombus formation through an antiplatelet mechanism.Normal activation of platelets and their aggregation are crucial for proper hemostasis. It appears that excessive or abnormal aggregation of platelets may bring about cardiovascular diseases such as stroke, atherosclerosis, and thrombosis. For this reason, finding a substance that can regulate platelet aggregation or suppress aggregation will aid in the prevention and treatment of cardiovascular diseases. Artesunate is a compound extracted from the plant roots of  Worldwide, the most prevalent cause of death is cardiovascular disease (CVD). The United States reported that one in seven deaths were due to coronary heart disease and one in nine deaths were due to heart failure . The rolThrombotic events are dramatically lowered through the inhibition of platelets via pharmacological agents, and CVD treatment and prevention uses such clinical drugs. Unfortunately, complications and side effects such as gastric bleeding, aplastic anemia, or even thrombocytopenia can arise from these drugs, often minimizing the benefits. Therefore, they require the development of minimal prophylactic or therapeutic alternatives . In addi2+]i needed for proper platelet aggregation via an increase in cyclic adenosine monophosphate (cAMP) levels i. 2+]i levels were increased from 100.3 ± 0.5 nM to 611.3 ± 29.4 nM by U46619. However, artesunate (50~200 μM) significantly decreased [Ca2+]i increased by U46619  significantly reduced the TXA2 production to 39.44 ± 1.28, 22.30 ± 6.20, 13.90 ± 0.89, and 10.70 ± 2.89 ng/108 cells, respectively  reduced the release of serotonin increased by U46619 to 139.70 ± 3.63, 111.87 ± 12.94, 95.39 ± 2.90, and 35.66 ± 3.57 ng/108 cells, respectively  increased ATP release from 0.16 ± 0.02 μM in intact cells to 7.81 ± 0.15 μM B; howeveectively C. These Artesunate’s effect on PI3K/Akt phosphorylation, a phosphoprotein involved with platelet granule release, was confirmed. As demonstrated in 2, was determined. As demonstrated in Artesunate’s effect on MAPK  phosphorylation, involved in the release of granules in platelets and the production of TXAPlatelet activation and aggregation occurs through platelet inducers, and over time, signal transduction via the external pathway causes the formation of fibrin clot. Accordingly, the present study investigated artesunate’s effect on platelets-mediated fibrin clot retraction by thrombin induction. As demonstrated in 2) is hydrolyzed by phospholipase C-γ2 (PLC- γ2), producing diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). The IP3 from PIP2 degradation causes intracellular Ca2+ mobilization ([Ca2+]i) from the dense tubular system into the cytosol and is activated by DG-dependent protein kinase C (PKC) [2+]i results in myosin light chain and pleckstrin  being phosphorylated during platelet aggregation i and inhibit the aggregation of platelets through cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) [2+]i inhibition. The results indicate that artesunate-increased cAMP plays a vital part in platelet activation inhibition via down-regulation of [Ca2+]i. The cAMP, when increased, phosphorylates multiple substrates through the PKA activation, and, more specifically, is known to affect the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) i. In addition, increased production of cAMP with artesunate led to vasodilator-stimulated phosphoprotein (VASP) being phosphorylated through activation of PKA. VASP is an important substrate of the cAMP/cGMP-dependent kinase (PKA/PKG) that helps with platelet activation regulation through regulating adhesion properties and platelet secretion, and VASP phosphorylation has shown to cause inhibition of the aggregation of platelets via inhibiting activation of integrin αIIb/β3 [Having the opposite effect, cyclic nucleotides (cGMP and CAMP) have shown the ability to lower [Case (PKA) . With thr (IP3R) . As demo αIIb/β3 ,35.3 in platelets induced with U46619. It is thought that the increased cAMP production by artesunate caused the activation of PKA and phosphorylated PKA-dependent VASP Ser157, which caused fibrinogen binding to αIIb/β3 to be inhibited. In the future, further research will need to explore the mechanism by which artesunate caused a cAMP production increase. cAMP and cGMP are known to depend on adenylyl cyclase/guanylyl cyclase or cyclic nucleotide phosphodiesterase (PDE) activation [With this study, artesunate strongly inhibited fibrinogen binding to αIIb/βtivation . Being ttivation . As a ma2) and TXA2 generation, which leads to the aggregation of platelets [2 generation, due to its ability to act as a powerful autacoid which causes additional platelet activation and aggregation. Accordingly, a substance that inhibits TXA2 production, such as ozagrel or aspirin, makes for a helpful antiplatelet compound [In addition, the pathway for PI3K/Akt is noted among phosphoproteins that act during intracellular signaling when platelets are activated, and their phosphorylation plays a major role during platelet function regulation, such as platelet aggregation and dense granule secretion . In addilatelets . One indcompound ,38.2 and secretion of granules (serotonin and ATP release), which are important markers of platelet aggregation, were measured. Further, we attempted to illuminate artesunate’s relationship with PI3K/Akt and MAPK phosphorylation. As a result, it was established that artesunate suppressed the strongly increased TXA2 production with U46619, and the release of serotonin and ATP (intracellular granule secretion indicators) was strongly reduced due to artesunate (2 and intracellular secretion of granules (serotonin and ATP release).With this study, artesunate showed a significant concentration-dependent inhibi-tory effect on the aggregation of platelets induced by some agonists , especially by U46619. In particular, in the aggregation induced by U46619, artesunate showed an IC50 of 105.26 µM. However, since this is a result obtained when the washed platelets are treated, there is a limit to explain how much is required when actually administered. Furthermore, considering the case of intranasal administration of artesunate to mice for the treatment of malaria, it can be seen that almost all of it is absorbed and present in the blood for 15 min, but it is reduced by half after 15 min . In facttesunate . Additio3-mediated signal transduction and granule release generally alter the platelet cytoskeleton, affecting the aggregation of platelets and the formation of a thrombus. Thrombus formation is the utmost vital step while repairing blood vessels that have been damaged, and during this time, activated platelets accumulate and a meshwork of fibrin and platelets develops. When a fibrin clot is forming it begins to contract for 30 to 60 min and ultimately results in a thrombus plug. The retraction of a fibrin clot relies heavily on the fibrinogen and αIIb/β3 interaction, and inhibitors of αIIb/β3 activity have been shown to prevent the formation of a thrombus [3, coagulation and platelet αIIb/β3 activation are induced by thrombin, leading to clot thrombus formation. As demonstrated in Additionally, increased integrin αIIb/βthrombus . To incr3R and VASP phosphorylation, which significantly inhibited the recruitment of Ca2+ and activation of integrin αIIb/β3 into the cytoplasm. In addition, artesunate has been shown to exhibit antiplatelet function by inhibiting granule release by regulating phosphorylation of phosphoproteins acting on signaling transduction such as PI3K/Akt and MAPK. Finally, the production of a thrombin-induced fibrin clot was significantly inhibited. Therefore, we suggest that artesunate has value as a substance that inhibits platelet aggregation and thrombus formation through an antiplatelet mechanism.In conclusion, artesunate increased cAMP in human platelets to induce IP2 enzyme immunoassay (EIA) kit, serotonin EIA kit, ATP assay kit, and the cAMP and cGMP EIA kit are available at Cayman Chemical Co. . Invitrogen Molecular Probes  supplied Fibrinogen Alexa Fluor 488 conjugates and Fura 2-AM. Sigma Chemical Corporation  provided the other reagents. Western blotting lysis buffers and antibodies were supplied by Cell Signaling . Thermo Fisher Scientific  provided enhanced chemiluminescence solution and polyvinylidene difluoride membrane.Avention Corporation  provided artesunate . U46619 g to collect platelets, then washed (twice) with a buffer . Suspension buffer  suspended the platelets . Platelet aggregation was avoided by performing all procedures at 25 °C.Human platelet-rich plasma (PRP) collected from healthy volunteers, who provided informed consent, was obtained from the Korean Red Cross Blood Center , and its experimental use was approved by KRBC and the Namseoul University Institutional Review Board . The method previously performed was used to prepare the suspended platelets [8 cells/mL) was carried out while adding artesunate at different concentrations. Then, 2 mM CaCl2 and U46619 (0.5 μM) were added for stimulation at a duration of 5 min. The aggregometer  measured the experiment with a stirring speed of 1000 rpm, and the rate of aggregation was calculated with an increase in the light transmittance. The suspension permeability of suspension buffer 0% was used as the reference value. Artesunate was dissolved with a concentration of 0.1% dimethyl sulfoxide (DMSO), and all tests were performed by adding the same dose of DMSO.For 3 min and at 37 °C, incubation of the suspended platelets (108 cells/mL) occurred at room temperature with the addition of different artesunate concentrations, and then it was centrifuged for 2 min at 12,000× g. A synergy HT multi-reader  measured the supernatants with an LDH EIA kit.Confirming the release of Lactate dehydrogenase (LDH) from the cytoplasm determined the cytotoxicity. For two hours, incubation of the suspended platelets (108 cells/mL) occurred at 37 °C while adding different artesunate concentrations, then 2 mM CaCl2 was added with U46619 (0.5 μM) for stimulation at a duration of 5 min. Termination of the reaction occurred by adding 1 M HCl, and a Synergy HT Multi-Reader  measured cGMP/cAMP using the cAMP or cGMP EIA kit.For 3 min, incubation of the suspended platelets (108 cells/mL) were prepared based on the steps specified above. The incubation of washed platelets occurred for 3 min at 37 °C with 2 mM CaCl2 and U46619 (0.5 μM) stimulation for a duration of 5 min. A spectrofluorometer  from BioTeck Instrument  measured the Fura 2 fluorescence. The fluorescence was analyzed with excitation wavelengths at 340 and 380 nm and emission at 510 nm. The calculation of the amount of Ca2+ mobilized was performed using the method of Grynkiewicz [For 60 min, incubation of PRP occurred with Fura 2-AM (5 μM) at 37 °C, and the suspended platelets (10nkiewicz .2 to the suspended platelets (108 cells/mL), U46619 (0.5 μM) was used for stimulation at a duration of 5 min. The addition of phosphate-buffered saline  containing 0.5% paraformaldehyde terminated the reaction. Light blocking was used during this process and a flow cytometer (FACS) from BD Bioscience  measured fibrinogen binding. The software, Cell-Quest (BD Biosciences), was used to analyze.After treatment with Alexa Fluor 488-human fibrinogen (30 μg/mL) by adding 2 mM CaCl8 cells/mL) occurred at 37 °C while adding different artesunate concentrations, then 2 mM CaCl2 was added with U46619 (0.5 μM) for stimulation at a duration of 5 min. A synergy HT multi-reader  measured TXB2 (a stable TXA2 metabolite) production with the use of the TXB2 EIA kit.For 3 min, incubation of the suspended platelets (108 cells/mL) occurred at 37° with different artesunate concentrations, then 2 mM CaCl2 were added with U46619 (0.5 μM) for stimulation at a duration of 5 min. Once the reaction was stopped with ice-cold 2 mM EDTA, a synergy HT multi-reader  and serotonin or ATP EIA kit measured the released serotonin/ATP in the upper layer due to centrifuging. The degree of secretion of granular substances in platelets out of the cells was confirmed by measuring ATP or serotonin in the upper layer by centrifugation after an aggregation reaction.For 3 min, incubation of the suspended platelets  was used to measure the concentration of proteins from platelet lysates. Protein (20 μg) was isolated via 4–20% SDS–PAGE and transferred to PVDF membrane. The primary antibody was treated with a dilution factor of 1:1000 and the secondary antibody was treated with a dilution factor of 1:2000. Visualization was performed using ECL reagent .2 at a temperature of 37 °C and a time of 15 min. A digital camera then took photographs of the platelets-mediated fibrin clots. The software, ImageJ , calculated the area of coagulation.To avoid sticking, a polyethylene tube contained the PRP (500 μL) which was then stimulated with thrombin (0.05 U/mL) and 2 mM CaClp < 0.05 was considered statistically significant.All data are presented as mean ± standard deviation with varying number of observations. A repeated experiment was performed with platelets from four normal adults. ANOVA was performed to identify major differences between groups, and Scheffe’s method was used. Statistical analysis was performed using SPSS 21.0.0.0 software , and


Morus alba root bark and the compounds therein. In our continuous study of other parts of this plant, the ability of the branch of Morus alba to inhibit α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), and advanced glycation end products (AGEs) formation was evaluated. Moreover, there are no previous studies that have performed enzyme kinetics and molecular docking analyses, along with assessments of peroxynitrite (ONOO−) inhibitory activities. Since the Morus alba branch exhibited favorable inhibitory effects, repeated column chromatography was performed to obtain eight compounds, including four flavonoids , one arylbenzofuran (2), one stilbene (5), one Diels–Alder-type adduct (7), and one sterol (4). Among them, compounds 1–3 and 5–7 were mixed-type inhibitors of α-glucosidase, sharing the same catalytic residues with acarbose and the same allosteric sites with (Z)-3-bytylidenephthalide. On the other hand, kuwanon C (1) and oxyresveratrol (5) interacted with residues of the allosteric site (α3 and α6 helices) of PTP1B, indicating their use as non-competitive inhibitors. Interestingly, kuwanon G (7) directly bound the catalytic site, or interrupted the binding between the substrate and the active site, as a mixed-type inhibitor. Moreover, most of the compounds exhibited greater activity against AGE formation and ONOO− than positive controls. The IC50 values required to inhibit ONOO− using compounds 1, 3, 5, 6, and 7 were reported for the first time, and range from 1.08 to 12.92 μM. Based on the structure–activity relationship, the presence of hydroxyl, resorcinol, and prenyl moieties was important in the prevention of diabetes’ pathological mechanisms, and these findings have been further supported by molecular docking analysis. These computational and experimental results will be useful in the development of therapeutic candidates to prevent/treat diabetes and its complications.Previously, we reported the anti-diabetic effect of  Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia, which results from carbohydrate metabolic disorders associated with impaired insulin production type 1) or developed insulin resistance type 2) -1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound A) (PDB ID: 1NNY), with a resolution of 2.4 Å [To investigate the protein–ligand interactions at the molecular level, molecular docking analyses were conducted with compounds of 1.6 Å ) and theof 2.4 Å , and a sof 2.4 Å , were obMorus alba branch extract, the MeOH extract and its fractions were tested in inhibition assays. The EtOAc fraction showed a significantly greater inhibitory activity against α-glucosidase and AGEs than the positive controls, with IC50 values of 2.74 ± 0.15 and 6.40 ± 0.31 μg/mL, respectively. This fraction showed moderate inhibitory activity against PTP1B and ONOO− compared with the positive controls, with IC50 values of 8.09 ± 0.08 and 6.74 ± 0.15 μg/mL, respectively. This fraction showed more promising activity than the MeOH extract and other fractions  exhibited the most potent activity against α-glucosidase, with an IC50 value of 1.44 ± 0.11 μM, followed by oxyresveratrol (5), kuwanon C (1), norartocarpetin (6), moracin M (2), and dihydromorin (3). The PTP1B inhibition results showed a similar tendency. Oxyresveratrol (5) showed the most potent activity against PTP1B, with an IC50 value of 2.85 ± 0.30 μM, followed by kuwanon G (7), kuwanon C (1), dihydromorin (3), and moracin M (2). Moracin M (2) and dihydromorin (3) showed weaker inhibitory activities toward PTP1B than ursolic acid, and norartocarpetin (6) showed no PTP1B-inhibitory activity at 100 μg/mL.Compounds 1–3 and 5–7 isolated from the EtOAc fraction were examined using AGE formation inhibition and ONOO− scavenging assays. As shown in 2) exhibited the most potent activity against AGE formation and ONOO−, with IC50 values of 2.10 ± 0.22 μM and 1.08 ± 0.04 μM, respectively. On the other hand, kuwanon C (1) showed moderate scavenging activity in the ONOO− assay, but no inhibitory activity on AGE formation at 100 μg/mL.Compounds α-glucosidase and PTP1B inhibition demonstrated by the test compounds, enzyme kinetic analyses were conducted at various concentrations of pNPG and pNPP, respectively. Each line of inhibitors intersected on the xy-side of the Lineweaver–Burk plots, indicating mixed-type inhibition. On the other hand, each line of inhibitors also intersected at the same point plots on the x-axis, indicating non-competitive inhibition [Ki value for an enzyme–inhibitor complex is determined via the value of the x-axis, which implies −Ki [1–3 and 5–7) appeared to be mixed-type inhibitors for the α-glucosidase enzyme, with Ki values of 6.85, 3.32, 10.22, 1.14, 19.90, and 2.03 μM, respectively. Kuwanon (7) also exhibited a mixed-type inhibition mode for PTP1B, with a Ki value of 12.41 μM. In contrast, kuwanon C (1) and oxyresveratrol (5) appeared to be non-competitive inhibitors for the PTP1B enzyme, with Ki values of 39.43 and 2.16 μM, respectively , are listed in 1)–α-glucosidase complex at the catalytic site presented a −6.66 kcal/mol binding energy, with four H-bonds formed via interactions between three hydroxyl (OH) groups and one carbonyl group and the Gln279, Arg315, Arg442, and Asp307 residues. In addition, kuwanon C (1) showed the same hydrophobic interacting residues, Tyr158 and Phe303, as acarbose, the catalytic inhibitor (1) and the Glu296, Ser298, Leu297, Glu271, and Arg270 residues. The alkyl groups and aromatic ring structure exhibited hydrophobic interactions with Ala292, Lys13, Ile263, Ile272, Ile262, and Arg263 residues, and a π–anion interaction with the Glu271 residue, respectively, sharing Ala292 and Lys13 residues in the allosteric site with BIP (2) gave rise to five H-bonds between three OH groups and oxygen in the benzofuran structure and the Arg442, Asp69, Gln182, Asp215, and Glu411 residues . Moracin M (2) also exhibited a hydrophobic interaction with the Tyr72 and Tyr158 residues, and showed a π–cation interaction between the aromatic ring and the Arg442 residue (2)–α-glucosidase complex at the allosteric site displayed four H-bonds between the 6-, 3′-, and 5′-OH groups and the Lys16, His295, Asn259, and Thr274 residues, and exhibited hydrophobic interactions with Trp15 and Ala292 residues (3) and the Gln279, Arg315, Arg442, Asp69, Glu277, and Asp352 residues. Dihydromorin (3) also interacted with the Tyr72 residue, via hydrophobic interaction, and showed a π–cation (Arg442) and two π–anion (Asp352 and Glu411) interactions (3) also showed hydrophobic interactions with Ala292 and Arg263 residues (5) showed the same amino acid residues as acarbose, such as Asp352 (H-bond and π–anion interaction), Arg442 (hydrophobic and π–cation interactions), and Asp69 (π–anion interaction) residues at the catalytic site (5) formed six H-bonds with the Thr274, Thr290, Cys342, Ile272, Asn259, and Glu296 residues, showing −6.98 kcal/mol binding energy. In addition, oxyresveratrol (5) displayed hydrophobic interactions with Ala292, Trp15, and Ser291 residues (6)–α-glucosidase complex at the catalytic site showed a −6.64 kcal/mol binding energy, with an H-bond between the OH group and the Gln353 residue, and a hydrophobic interaction with the Val216 residue. In addition, the aromatic rings of norartocarpetin (6) showed a π–cation and two π–anion interactions with the Arg442, Glu277, and Asp352 residues, and also with acarbose (6) showed a hydrophobic interaction with the Ala292 and Lys13 residues, and its aromatic rings showed a π–anion interaction with the Glu271 residue (7) showed a −5.99 kcal/mol binding energy, forming six H-bonds between four OH groups and the Asn350, Gln353, Glu277, Asp352, Asp242, and Glu411 residues. In addition, kuwanon G (7) hydrophobically interacted with the Phe303 residue (7) formed seven H-bonds with the Ser298, Asn259, Ile272, Asp341, Thr290, Ala292, and Arg270 residues at the allosteric site. Kuwanon G (7) also exhibited a hydrophobic interaction with the Ala292, Trp15, His295, Ile262, Ile272, and Arg263 residues, as well as with BIP. Moreover, the aromatic rings of kuwanon G (7) showed one π–cation (Lys13) and one π–anion interaction (Glu271)  l.1), oxyresveratrol (5), and kuwanon G (7). The ligand–enzyme complexes of the three test compounds, and of compound A and compound B, were rigidly placed in the same pocket as PTP1B using AutoDock 4.2. The binding energies of the test compounds, along with their H-bond, hydrophobic, and electrostatic interactions , are listed in Molecular docking analyses with PTP1B were conducted for kuwanon C (1) and the Gln200, Gly277, and Ala189 residues. Additionally, kuwanon C (1) showed the same hydrophobic interacting residues, Phe196, Phe280, Lys197, and Leu192, as compound B, the allosteric inhibitor (5) displayed a hydrophobic interaction with Phe280, Phe196, and Leu192 residues, and formed six H-bonds between its four OH groups and the Asn193, Lys197, Glu200, Glu276, and Ala189 residues  (7) interacted with both the catalytic site and the allosteric site of PTP1B, indicating it as a mixed inhibitor. The kuwanon G (7)–PTP1B complex at the allosteric site exhibited five H-bonds with the Asn193, Gly277, Phe280, and Glu200 residues . Kuwanon G (7) also showed the same hydrophobic interacting residues as compound B—Phe196, Phe280, Ile281, and Leu192—and showed a π–cation interaction with the Lys197 residue (7) and the Ser216, Gln266, Asp48, Met258, Gln262, and Tyr46 residues, similarly to compound A, a catalytic inhibitor. The aromatic rings of kuwanon G (7) showed a π–cation interaction with the Arg221 residue  b. On the residue c. In the residue d.Morus alba branches for the in vitro assay, as well as the kinetic study and molecular docking analysis, of two enzymes involved in DM and its complications. All test compounds showed stronger inhibitory activity against α-glucosidase than the positive control, especially oxyresveratrol (5) and kuwanon G (7). Among them, kuwanon C (1), which possesses a prenyl moiety, showed a more potent α-glucosidase inhibitory activity than dihydromorin (3) and norartocarpetin (6). A similar result was reported in a recent study, which found that the presence of a prenyl moiety enhanced the α-glucosidase inhibitory activity. Similarly, the prenylation of C-8 can enhance the hydrophobic interactions between compounds and α-glucosidase, leading to an increase in inhibitory activity [3) and norartocarpetin (6) showed similar IC50 values, but the difference between the structures was only a C2–C3 double bond or an OH group on C-3. Tadera et al. [5) showed the most potent activity against PTP1B, with an IC50 value of 2.85 μM, followed by kuwanon G (7), kuwanon C (1), dihydromorin (3), and moracin M (2). Our data are similar to those reported in previous studies, except for norartocarpetin (6), and we are the first to report the IC50 value of dihydromorin (3) (180.22 μM). Zhao et al. [1) previously showed higher inhibitory activity than norartocarpetin (6) at the same tested concentration (58% inhibition for kuwanon C (1) and 20% inhibition for norartocarpetin (6) at 20 μg/mL). Contrary to our results, norartocarpetin (6) exhibited potent activity against PTP1B (5.18 μM vs. >100 μg/mL) [5), kuwanon G (7), and kuwanon C (1) were likely caused by an increase in bioavailability due to the presence of a prenyl group and multiple aromatic rings.Many natural products, characterized by enormous structural and chemical diversity, show potent bioactivity, with lower cytotoxicity and side effects than synthetic drugs . New theactivity . On the a et al.  reporteduwanon G . Among to et al.  reported0 μg/mL) , possibl0 μg/mL) ,46. Thus5) and kuwanon G (7). We conducted enzyme kinetic studies of the enzymes and compounds 1–3 and 5–7. All compounds were mixed-type inhibitors of α-glucosidase, binding to the catalytic and allosteric sites of the free enzyme and enzyme–substrate complex. Kuwanon G (7) was also a mixed-type inhibitor of PTP1B, as was found in the results of Paudel et al. [1) and oxyresveratrol (5) were non-competitive inhibitors of PTP1B. The Ki value defines the affinity between an enzyme and its inhibitors, i.e., lower Ki values indicate a better affinity between enzyme and inhibitor. Oxyresveratrol (5) showed the lowest Ki value (1.14 μM) against α-glucosidase, which implied that it had the strongest affinity with the enzyme, followed by kuwanon G (7), moracin M (2), kuwanon C (1), dihydromorin (3), and norartocarpetin (6). Similarly, the Ki value of oxyresveratrol (5) against PTP1B was the lowest (2.16 μM), followed by those of kuwanon G (7) and kuwanon C (1).Although most of the compounds tested here have been subjects of previous reports on the inhibition of α-glucosidase and PTP1B, no studies have examined enzymes’ kinetics, or performed molecular docking simulations, except on oxyresveratrol  and its catalytic ligand α-d-glucose (PDB ID: 3A4A), because the structure of α-glucosidase derived from Saccharomyces cerevisiae is not crystallized [1–3 and 5–7 interacted with the residues similarly to those used by acarbose, a known competitive inhibitor, as well as its surrounding residues. On the other hand, compounds 1–3 and 5–7 became bound to a different pocket in the catalytic site, but interacted with residues similarly to those used by BIP, a known non-competitive inhibitor. A recent study reported four allosteric sites for α-glucosidase (sites 1–4) [1–3 and 5–7 and BIP showed allosteric inhibition by interacting with site 4. Moreover, kuwanon C (1), dihydromorin (3), norartocarpetin (6), and kuwanon G (7) showed lower binding energies for allosteric inhibition than catalytic inhibition, indicating that these compounds preferentially bind to allosteric sites. On the other hand, moracin M (2) and oxyresveratrol (5) showed lower binding energies for catalytic inhibition than allosteric inhibition, indicating that these compounds preferentially bind to catalytic sites.Based on the results of our enzyme kinetic study, we conducted molecular docking analyses of compounds tallized . Isomalttallized . The thrtallized . The restes 1–4) , and we 1), oxyresveratrol (5), and kuwanon G (7), using the complex structures of PTP1B, along with compound A (PDB ID: 1NNY) and compound B (PDB ID: 1T49). Kuwanon C (1) and oxyresveratrol (5) became bound to allosteric sites, whereas kuwanon G (7) bound to both catalytic and allosteric sites, although it had a lower binding energy for allosteric inhibition than catalytic inhibition, indicating its preference for allosteric sites. In addition, the results of the PTP1B docking analysis show that kuwanon C (1), oxyresveratrol (5), and kuwanon G (7) interacted with residues similarly to those used by compound B, a known non-competitive inhibitor. The allosteric site is found on the C-terminal portion of PTP1B, and on the side with α3 (residues 186–200), α6 (264–281), and α7 helices (287–295). Some residues of α3, α6, α7, and loop 11 are involved in the regulation of PTP1B function [1), oxyresveratrol (5), kuwanon G (7), and compound B exhibited interactions with residues of the α3 and α6 helices. The residues interacting with kuwanon C (1), oxyresveratrol (5), kuwanon G (7), and compound B were Glu200, Ala189, Phe196, Lys197, Leu192, and Asn193 on the α3 helix; and Gly277, Phe280, Ile281, and Glu276 on the α6 helix. Therefore, those compounds were identified as non-competitive inhibitors. On the other hand, kuwanon G (7) also exhibited interactions with residues similarly to those used by compound A. The base of the active site is formed by residues from His214 to Arg221, which belong to the β12 sheet, loop 15, and the α4 helix. This site is surrounded by loop 1, loop 4, loop 13 (WPD loop), and loop 17 [7) and compound A were Ser215, Ala217, and Gly218 (loop15); Ile219, Arg221, and Gly220 (α4 helix); Asp48, Tyr46, and Val49 (loop 1); Trp179 (WPD loop); and Met258 and Gln262 (loop 17). Among the interactions displayed by kuwanon G (7), the Asp48 and Tyr46 residues of the α4 helix were related to the tyrosine moiety of the substrate. Additionally, the WPD loop, including residues 177–185, is essential for catalysis into a substrate, after which it converts to its closed state. However, this state can be prevented by the allosteric inhibitor in the α3-α6-α7 helices [7) likely interrupts the binding between the substrate and the catalytic site by interacting with residues from the α3 and α6 helices. Therefore, our results indicate that kuwanon G (7) interacted with both catalytic and allosteric sites as a mixed-type inhibitor.We used the results of our enzyme kinetic study to conduct a molecular docking analysis of kuwanon C  required to inhibit AGE formation using norartocarpetin (6), and the IC50 values  required to inhibit ONOO− using kuwanon C (1), dihydromorin (3), oxyresveratrol (5), norartocarpetin (6), and kuwanon G (7), have been reported for the first time. In the present study, kuwanon C (1) showed moderate activity in the ONOO− scavenging assay, while its activity in the AGE formation inhibition assay could not be determined at the tested concentration up to 100 μg/mL. On the other hand, moracin M (2) exhibited the most potent bioactivity against both AGE formation, with an IC50 value of 7.15 μM, and ONOO−, with an IC50 value of 2.42 μM, which are similar to the results of a previous study [− scavenging activity in flavonoids. The 3-OH group on ring C was positively influenced by a substituent 5- or 7-OH. Moreover, the 4-keto group can weaken the intramolecular H-bond, resulting in the optimal reactivity of the 3-OH group. The monophenol structure showed less activity than resorcinol [3) could be due to the presence of 3-OH and a resorcinol group. Furthermore, the IC50 values of flavones  and flavonols  do not show significant differences [3) and norartocarpetin (6), which also showed no significant difference in IC50 value. On the other hand, the difference between kuwanon C (1) and norartocarpetin (6) comprises the presence of a prenyl moiety, and norartocarpetin (6) showed more potent activity than kuwanon C (1), indicating that the OH group could be more influential in ONOO− scavenging than the prenyl moiety. This trend is supported by research suggesting that prenylation does not significantly affect the inhibition of lipid peroxidation by ONOO− [− scavenging activity of flavonoids 1, 3, and 6 mirrored those derived for AGE formation inhibitory activity, the effect of the relationship between structure and inhibitory activity on AGE formation can be similarly explained.In the two antioxidant mechanism-related assays, the AGE formation inhibitory and ONOOus study . The presorcinol . The strferences , with reby ONOO− . Since tMorus alba branches could be used as significant therapeutic or preventive agents for DM and its complications through their antioxidant activity. However, further studies should be conducted to characterize the relationship of ONOO− with two DM-associated enzymes, and the main mechanism, using an insulin-resistant cell line or a DM animal model.Overall, our results illustrate that the compounds isolated from Morus alba branch extracts and their components exhibit anti-diabetic activity, including α-glucosidase and PTP1B inhibition. We also found inhibitory activity against AGE formation and ONOO−. All tested compounds showed better inhibitory effects against α-glucosidase than acarbose, with IC50 values ranging from 1.44 to 47.35 μM; moreover, oxyresveratrol (5) and kuwanon G (7) showed more potent inhibitory effects against PTP1B. The enzyme kinetic study indicated that all compounds showed a mixed-type inhibition pattern toward α-glucosidase. While kuwanon G (7) exhibited a mixed-type inhibition mode toward PTP1B, kuwanon C (1) and oxyresveratrol (5) showed a non-competitive inhibition pattern. These results are further supported by our molecular docking analysis, which showed that all compounds interacted with similar residues known to be important in catalytic and allosteric sites. Additionally, most of the compounds, especially moracin M (2), exhibited potent ONOO− scavenging activity and AGE formation inhibition. Thus, proving the inhibition mechanism via computational and experimental studies could be useful in the development of functional foods to treat DM and its complications.Our results demonstrate that


Discovering molecules that regulateclosely related protein isoformsis challenging, and in many cases the consequences of isoform-specificpharmacological regulation remains unknown. RAF isoforms are commonlymutated oncogenes that serve as effector kinases in MAP kinase signaling.BRAF/CRAF heterodimers are believed to be the primary RAF signalingspecies, and many RAF inhibitors lead to a “paradoxical activation”of RAF kinase activity through transactivation of the CRAF protomer;this leads to resistance mechanisms and secondary tumors. It has beenhypothesized that CRAF-selective inhibition might bypass paradoxicalactivation, but no CRAF-selective inhibitor has been reported andthe consequences of pharmacologically inhibiting CRAF have remainedunknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectivelytarget inhibitors to CRAF. Our results suggest that selective CRAFinhibition promotes paradoxical activation and exemplify how BOLTmay be used to triage potential targets for drug discovery beforeany target-selective small molecules are known. In many cases no small molecule exists thatcan selectively target a specific isoform and so the potential ofselective pharmacological regulation remains unknown. Strategies thatdefine the consequence of selective pharmacological regulation forspecific isoforms would provide an approach for triaging moleculartargets and enable efforts to be focused on developing selective smallmolecules for the most valuable and validated targets. However, addressingthis challenge without isoform-selective small molecules in hand presentsan apparent paradox. For protein kinases this paradox has been addressedby the mutation of a gatekeeper residue in the active site to createan active enzyme containing a “hole” that can be selectivelyinhibited by an ATP analogue containing a chemical “bump”;biorthogonal ligand tethering (BOLT). In this approachwe site-specifically and cotranslationally encode a noncanonical aminoacid (ncAA) bearing a bio-orthogonal group (commonly (2S)-2-amino-6--bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino) hexanoic acid (BCNK (l-lysine (CypK)6 into the target protein usinggenetic code expansion.8 We then add a druglike small molecule–tetrazineconjugate to cells. The conjugate reacts with the target protein througha rapid bioorthgonal inverse electron demand Diels–Alder reaction12 and tethers the druglike small molecule to the target  or Nεe target 1B. Such 22 The canonical pathway integrates extracellular signals through transmembranereceptors, switches Ras GTPase to the active GTP-bound form, and recruitsRAF kinases to the membrane, where they are activated.23 Three RAF protein kinase  serveas effector kinases in the RAS-ERK signaling cascade. These kinasesdrive the activating phosphorylation of MEK, which ultimately resultsin an ERK-mediated transcriptional response.24 Key to the activation of RAF kinases is the RAS-mediated disruptionof their autoinhibited conformation25 andthe formation of homo- and heterodimers.28The Ras/ERK signaling pathway controls many cellular processes,including differentiation, proliferation, and survival. Frequent mutationsin the pathway, primarily generating activated forms of RAS and BRAF,are observed in more than 30% of human cancers.32 Understanding the specific mechanism of action of RAF inhibitorshas been the focus of intense research efforts and has challengedthe academic and drug discovery communities for nearly 2 decades.38A greatdeal of effort has gone into generating RAF inhibitors.In cells expressing mutant BRAF , inhibitors suppressRAF activity and ERK signaling, while in cells expressing wild-typeBRAF most inhibitors cause an undesired increase in RAF activity andERK signaling .37 The role of RAF dimerization is central to bothphysiological and inhibitor induced signaling.39 The homo- and heterodimers formed by wild-type BRAF and CRAF areresponsible for phosphorylating MEK. While mutant BRAFV600E is constitutively active and has a limited role in dimerization,40 the BRAF-CRAF heterodimer is believed to bethe primary species in both native signaling and paradoxical activation.42 Genetic and biochemical resultshave repeatedly implicated CRAF as the primary species responsiblefor phosphorylating MEK in paradoxical activation and native signaling.46 Specifically, inhibitor-boundBRAF is implicated in promoting heterodimerization with unbound CRAF,causing transactivation of CRAF through an allosteric mechanism atthe protein–protein interface between protomer kinase domains.32Prevailing models of paradoxical activation center on inhibitorspromoting RAF dimerization and ultimately eliciting MEK-ERK pathwayactivation, an outcome that is amplified by oncogenic RAS mutations.50 Recent genetic ablation of CRAFsuggested that removal of the protein may afford a therapeutic benefit.51 However, as no well-characterized CRAF-selective inhibitors havebeen reported, the consequences of selective CRAF inhibition haveremained unknown. Here, we develop BOLT to selectively target inhibitorsto CRAF. Our results suggest that selective CRAF inhibition promotesparadoxical activation.Decades of genetics research have employed both germline andconditionalallele manipulations of RAF isoforms; these studies have revealedboth redundancy and distinct functions for BRAF and CRAF in differentcell types and stages of cancer progression.in vitro, preclinical, and clinical studies.37 Notably, none are selective to the CRAF isoform in mutant RAS cells.Drawing upon available RAF-selective pharmacophores, we designed andsynthesized a series of potential BOLT ligands composed of three chemicalmoieties: a pharmacophore, a linker, and a tetrazine (Supporting Information). Pharmacophores were chosen from classic and distinctRAF inhibitors, PLX4720  and AZ628 . We created AZ13-tet (containing the AZ628pharmacophore) and the AZ181-tet (containing the PLX4720 pharmacophore).We used the structure of RAF-inhibitor complexes53 to design synthetically accessible BOLT ligands that should notinterfere with pharmacophore binding. We demonstrated that BOLT ligandsexhibited similar cellular responses and paradoxical activation totheir parent inhibitors; as expected, BOLT ligands showed a decreasein potency .There areover 2 dozen well-characterized small-molecule inhibitorstargeting RAF kinases, with characterization spanning Figure S2).20 We encoded BCNK at sites in boththe N and C lobes of CRAF using the MmPylRS54 /MmtRNAPylCUA pair, BCNK, and CRAF (YXXXTAG) . The majority of selected positions showed BCNK-dependentexpression of full length CRAF and were efficiently labeled with afluorescent tetrazine conjugate . We also incorporated BocK .We chose CRAF(S357TAG) and CRAF(Q436TAG)  for further characterization; these mutantsproduce good levels of ncAA-dependent expression and position thencAA, adjacent to the solvent channel, where tethering may enablethe pharmacophores to access their native binding sites within thesame monomer. Distance measurements suggest that tethering acrossthe RAF dimer – in a trans mode– is prevented by therelatively short linker. Analogous positions on ARAF and BRAF werealso amenable to ncAA incorporation  as a BFP-T2A fusion wasassembled and integrated into the genome of HCT116 cells using thePiggyBac Transposase .55 We used FACS selection for BFP fluorescenceto discover clones expressing a high level of the synthetase. We thuscreated the stable cell line: HCT116 4x[MmtRNAPyl ]-BFP-2A-PylRS (henceforthdesignated HCT116*) . We transfected 4x[MmtRNAPyl] CRAF(YXXXTAG)into this cell line and supplemented cells with either BCNK or BocK cells with genetic code expansion machinery to enablethe site-specific incorporation of either BCNK or BocK. A dual expressioncassette encoding nsposase S5A.55 WeFigure S6A,B). CRAF (S357BCNK) showed an increase in MEK phosphorylationwith respect to CRAF (S357BocK) in this assay when both proteins wereimmunoprecipitated from cells to which AZ181-tet had been added .Notably, we observe a similar dose-dependent increase in MEK phosphorylationfor CRAF (Q436BCNK) but not CRAF (Q436BocK), with AZ181-tet . We observe an increase in MEK phosphorylationwithin 20 min of ligand addition . Interestingly, CRAF (Q436BCNK), but not CRAF (Q436BocK), showsa high level of MEK phosphorylation, after immunoprecipitation fromcells treated with 20 nM AZ13-tet, but higher concentrations of AZ13-tetdecrease the MEK phosphorylation mediated by CRAF (Q436BCNK) withoutaffecting the MEK phosphorylation mediated by CRAF (Q436BocK); thelevel of MEK phosphorylation mediated by CRAF (Q436BCNK) from cellstreated with 2 μM AZ13-tet is low and comparable to that mediatedby CRAF (Q436BocK) treated with AZ13-tet (from 20 nM to 2 μM). Crucially, we observe a dose-dependentincrease in MEK phosphorylation for CRAF (S357BCNK), but not CRAF(S357BocK), with both AZ13-tet and AZ181-tet S6C.Nota32 We hypothesized thatwhen CRAF (S357BCNK) and CRAF (Q436BCNK) are immunoprecipitated, theymay coimmunoprecipitate wild-type RAF species. We demonstrated thatkinase-dead CRAF variants, , , , ,57 transactivate associatedRAF monomers in the presence of BOLT ligands; the matched BocK controlsdid not lead to transactivation . This strongly suggested that associated RAF dimers are responsiblefor the tethering-dependent paradoxical activation we observe.Our data are consistent with selective CRAF inhibition, via bio-orthogonalligand tethering, leading to transactivation of associated RAF monomersin a RAF dimer . For all of the ncAA position/BOLTligand combinations tested, the activation is dependent on BOLT ligandconcentration when the protein contains BCNK, but does not show thesame level of activation when the protein does not contain BCNK. Thisindicates that the observed paradoxical activation is selective forCRAF and is dependent on the tethering of the ligand to CRAF. ForCRAF (Q436BCNK) with AZ13-tet this activation occurs at lower concentrationsof BOLT ligand and at higher concentrations we see inhibition; thisis consistent with the second RAF protomer in a dimer being occupiedby a ligand, and with the expected bell-shaped activity concentrationcurve for such systems.31 Upon immunoprecipitating CRAF (S357BCNK) or CRAF (Q436BCNK) fromcells coexpressing BRAF and CRAF nanoluciferase fusions (BRAFnLucand CRAFnLuc), we coimmunoprecipitated both exogenous RAF species;specifically, we detected increasing amounts of BRAFnluc with increasingconcentrations of AZ13-tet .Control experiments using the matched BocK controls suggest this coimmunoprecipitationis stimulated by ligand tethering. These experiments suggest thatwild-type RAF species can be coimmunoprecipitated with immunoprecipitatedCRAF. Thus, the paradoxical activation we observe for CRAF (S357BCNK)or CRAF (Q436BCNK) in our BOLT experiments may result from the dimerizationand transactivation of uninhibited wild-type RAF species.Next, we aimed to confirm that wild-type RAF species can be copurifiedin our immunoprecipitations. AZ628, like many RAF inhibitors, is knownto promote and stabilize RAF dimers in immunoprecipitations and havepronounced effects in wild-type RAF or mutant Ras cells.58 RAF species were expressedwith a Halo tag and CRAF (WT or variants containing BCNK or BocK)were expressed with a C-terminal nanoluciferase fusion .Weused a nanoBRET assay to further investigate BOLT-induced dimerizationof CRAF in cells.e fusion 3A. Sincee fusion 3B–D.We nH) of greater than 1. While many factorsimpact the Hill slope coefficient, values greater than 1 can indicatecovalent and/or bivalent cooperative binding.60 Average calculated EC50 values from independent doseresponse experiments show a clear increase in potency under BOLT conditions. In summary, BOLTled to at least a fourfold decrease in the EC50 for RAFdimerization. Our activity and dimerization data are consistent withselective inhibition of CRAF, leading to enhanced dimerization ofother RAF monomers, and the activation of those monomers to phosphorylateMEK1.For S357BCNK we observe a notablysteep BRET signal versus AZ13-tetconcentration curve. This corresponds to a Hill slope coefficient(nditions3D. Regre61 A decade of research has since deepened ourunderstanding of the unusual pharmacology and complex regulatory mechanismsgoverning RAF biology and MAPK signaling.66 While our improved molecular understanding of RAFtransactivation has guided patient treatment selection and influencedtreatments targeting two or more nodes in the MAPK pathway,62 progress in developing RAF inhibitors effective against mutant RAS-driventumors remains an unsolved challenge. Recent research has indicatedthe potential for selective inhibition of CRAF-driven signaling tobe a more effective and tolerated therapeutic approach for mutantRAS tumors.51 Identifying selective CRAF kinaseinhibitors that would block BRAF-CRAF heterodimer signaling couldthus be a valuable drug for the treatment of mutant RAS-driven tumors,but would present a major challenge for medicinal chemistry. Beforeembarking on such a challenging drug discovery goal, it would be desirableto have greater confidence and confirmation that such an approachwas valid and that selective CRAF inhibitors would not suffer thesame problem of paradoxical activation shown by previous RAF inhibitors.The development of small-molecule inhibitorstargeting RAF proteinkinases has driven advances in biomedical research and delivered drugsfor the treatment of mutant BRAF-driven melanomas, which have improvedpatient outcomes. However, initial clinical success has been temperedby the emergence of resistance mechanisms and development of secondarytumors resulting from “paradoxical activation”.67 Our results exemplify how BOLT may be used totriage potential targets for drug discovery before any target-selectivesmall molecules are known.We have used BOLT to selectively tether inhibitors to CRAF andinterrogate the consequences of selective CRAF inhibition. Our resultssuggest that selective CRAF inhibition will not be spared the liabilitiesobserved with BRAF-selective and pan RAF inhibitors in eliciting transactivation.We suggest that future CRAF-selective pharmacophores may benefit fromconsidering strategies taken for paradox breaking mutant BRAF inhibitors.


G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR–GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models. G protein-coupled receptors (GPCRs) are the largest and the most diverse receptor family found in eukaryotes and are involved in regulating many diverse physiological processes. Upon activation by extracellular stimuli such as hormones and neurotransmitters, GPCRs transmit signals across the membrane to trigger intracellular signaling cascades. The hallmark seven-transmembrane topology of GPCRs was proposed in the 1980s based on the primary structure of bovine rhodopsin and the β-adrenergic receptor ,3,4. To In cells, unliganded GPCRs exist in an equilibrium between inactive and active states, with inactive states being the most prevalent. Upon agonist binding, the equilibrium shifts toward active conformations that couple with downstream transducers such as heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. Heterotrimeric G-proteins are composed of Gα, Gβ, and Gγ subunits, with Gβ and Gγ forming a constitutive heterodimer (Gβγ). The activation of GPCRs induces conformational changes on the Gα subunit that allows the exchange of bound GDP for GTP. As a result, the Gα subunit dissociates from Gβγ subunits, and then the free Gα and Gβγ subunits regulate the activity of downstream effectors such as adenylyl cyclases, phospholipases, ion channels, and Rho guanine nucleotide exchange factors, thereby modulating diverse signaling pathways . MeanwhiThe first atomic resolution GPCR structure determined was that of bovine rhodopsin bound to its intrinsic inverse agonist 11 cis-retinal . Both stThe activated conformation of GPCRs is relatively unstable and difficult to isolate on its own. Therefore, many GPCR structures that have been resolved in an active state have employed protein complexes that recognize this conformation of the GPCR, such as G proteins or nanobodies ,14,15,162+·calmodulin (Ca2+·CaM)  ATP. Reactions were quenched at 8 min with SDS gel loading buffer, and phosphorylated products were separated on a 4–15% Criterion TGX precast gel. Gels were exposed to a phosphor screen overnight, scanned using a Typhoon scanner, and band intensities corresponding to phosphorylated product were quantified using ImageQuant software. For each independent experiment, band intensities were normalized to the wild type, such that the highest wild-type band intensity was set to 1 to account for the day-to-day variability in the phosphor screen intensity. Each experiment was performed in triplicate. Km and normalized Vmax were determined by plotting the normalized band intensity as a function of either ATP or rhodopsin concentration, and fitting it to the Michaelis–Menten equation. Statistical significance was assessed by one-way ANOVA using multiple comparison corrections. All curve-fitting and statistical analyses were performed using GraphPad Prism 7.03.Steady-state parameters for the phosphorylation of tubulin and rhodopsin in rod outer segments (ROS) by GRK2 with variable ATP concentrations were determined at room temperature in the Reaction Buffer  were plated in 6-well tissue culture dishes and transfected the following day with 300-ng pcDNA3.1-FLAG-β2AR and 400 ng each of pcDNA3.1-Gαs, pcDNA3.1-Gβ, and pcDNA3.1-Gγ and, when present, 400-ng pcDNA3-GRK2 using 6- to 7-µL FuGENE-HD. The total amount of DNA was brought to 2000 ng with salmon sperm DNA. For the isoproterenol (ISO) dose-response experiments, 40–44 h after transfection, cells were serum-starved for 1 h before a 5-min treatment with a range of isoproterenol (ISO) concentrations (100 pM–10 µM) or 10-µM ALP as the “zero” concentration. For the assay of β2AR phosphorylation by GRK2-H6-RH domain mutants, cells were transfected as described above, except WT and mutant pcDNA-GRK2-H6 replaced the pcDNA-GRK2. After 40–44 h, cells were serum starved for 1 h and then treated for 5 min with 2-nM ISO. Cells were washed twice with cold 20-mM Tris, pH 7.5 and 150-mM NaCl and scraped in 200-µL/well receptor solubilization buffer . The resulting lysates were solubilized by mixing for 30 min on an orbital shaker at 4 °C and clarified by centrifugation at 16,000× g at 4 °C. Soluble fractions were treated with PNGase (3.3 U/uL) for 2 h at 37 °C and immunoblotted with (a) β2AR phosphosite antibody that recognizes agonist-induced phosphorylation (pSer355/pSer356), (b) an antibody that recognizes the β2AR carboxyl tail and reflects total β2AR, and (c) GRK2 polyclonal antibody, which were carried out as described [2AR and GRK2 immunoblots. Signals were visualized using the Bio-Rad ChemiDoc XRS System, and band intensities were quantified using Bio-Rad Quantity One software. GraphPad Prism v.6  was used for nonlinear regression curve fitting and statistical analysis. To calculate EC50 in the ISO dose-response assay, data were scaled to 100 for the entire dataset, and values obtained with and without GRK2 were fit to a sigmoidal curve (four-parameter logistic equation) with a variable slope. For the assay of the RH domain mutants, the transfected GRK2-dependent signal was normalized for minor variations in the amount of GRK2 and receptor in the cell lysates and scaled to 100 with the GRK2-H6 WT value. Repeated measures one-way ANOVA and Dunnett’s multiple comparisons test were used to compare the statistical significance of differences between WT GRK2-H6 and the mutant derivatives.COS-7 cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) GlutaMAX  supplemented with fetal bovine serum (10%) and penicillin/streptomycin/amphotericin B  at 37 °C with 5% COescribed ,54. EachWe first assessed three leading GPCR–GRK interaction models in light of the approaches used to derive them and their unresolved issues.Like PKA, the GRK kinase domain consists of small and large lobes. The ATP-binding site is found in a deep crevice formed at their interface, whereas peptide substrates are presumed to bind to the large lobe. They feature an AST loop  that, inThe crystal structure of the GRK6–sangivamycin complex  was the 2+·CaM complex, the kinase domain adopts a conformation similar to that of the GRK6–sangivamycin complex [2+·CaM binds to the kinase domain in a manner similar to that proposed for GPCRs in this model , electron microscopy (EM), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics [2ARs [Like the N-terminal helix, the GRK RH domain is not found in other AGC kinases, making it an equally attractive hypothetical GPCR docking site. Moreover, its terminal and bundle subdomains engage the small and large lobes of the kinase domain, respectively, in a way highly reminiscent of SH3 and SH2 domains of Src . Thus, adynamics . Althougdynamics ,66) propcs [2ARs ,67. Howecs [2ARs  and GRK1cs [2ARs  were notcs [2ARs , the GRK2AR-GRK5 mixture, followed by tandem mass spectrometry, suggested three primary interfaces: ICL3 of the β2AR with the NLBD and CLBD of GRK5, ICL2 of the β2AR with the RH bundle subdomain of GRK5, and the C-terminus of the β2AR with the GRK5 kinase domain [2AR and the lipid-binding domains of GRK5  crosslinking of the βe domain . The obsGRK5 see  was propαN helix , where i2AR [2AR, their interface is disrupted, leading to the release of the RH bundle subdomain from the large lobe of kinase domain. A positively charged patch on the RH domain α5 helix then interacts with acidic lipids on the membrane near ICL2, stabilizing the β2AR-GRK5 complex and leading to full activation of the kinase. In the model of this activated complex  and rhodopsin are unique among GRK-GPCR pairs, because they have coevolved since the emergence of vertebrates to regulate their perception of light. Thus, they represent a highly optimized system and a logical place to start in understanding GPCR–GRK interactions. A recent study used a cell-based proximity assay and in vitro binding assays, along with negative-stain EM and HDX-MS, to examine the interactions of GRK1 and GRK5 with rhodopsin . For theHDX-MS was then used to compare the dynamics of surfaces in human apo GRK1 versus a covalently fused rhodopsin-GRK1 protein, which was presumed to be in complex . OverallThere are also unresolved issues with this third model. For example, the human GRK1-R194A mutation, which obliterates enzyme activity in bovine GRK1 (R191A), as do the analogous mutations in GRK2 and GRK6 , did notIn the two newer models , a simil6-tagged bovine GRK2 and purified them from the HEK293F cells. We then determined their steady-state kinetic parameters using either tubulin or rhodopsin in the rod outer segments (ROS) as substrates. The inactive K220R variant was generated as a negative control for background endogenous GRK activity that may copurifyfrom HEK293F cells. When the kinase domain of GRK2 is superimposed onto those of GRK1/GRK5, the proposed receptor interacting regions in GRK1 and GRK5 are in proximity to the unique structures in GRK2 bearing hydrophobic residues that are solvent exposed in all reported crystal structures for the enzyme A. These Km values or normalized Vmax for tubulin phosphorylation, because we did not expect these mutations would affect the binding kinetics of ATP or the ability of the kinase domain to adopt a closed state in the absence of a receptor. Similarly, we did not expect changes in the Km for rhodopsin phosphorylation when ATP was varied, but changes in the normalized Vmax for rhodopsin could be indicative of a receptor binding defect. However, we only observed small changes in the Km, normalized Vmax, or normalized Vmax/Km  using either tubulin or rhodopsin  transfection of GRK2 only stimulated WT β2AR phosphorylation 40% [2AR-Y326A variant, which is not phosphorylated by endogenous COS-7 cell kinases and therefore allowed the measurement of β2AR phosphorylation by transfected GRK2 [2AR phosphorylation by transfected GRK2. To optimize the agonist- and GRK2-dependent phosphorylation of WT β2AR, we measured its phosphorylation as a function of the ISO concentration in the absence and presence of transfected GRK2. The EC50 values for the phosphorylation of β2AR were 51 and 2.5 nM, respectively, in the absence and presence of transfected GRK2  in β-arrestin1 (arrestin2) [To test these variants against a hormone responsive GPCR, we assessed their ability to phosphorylate the β-7 cells . In a prtion 40% . In thatted GRK2 ,75. For ted GRK2 A. The inrestin2)  or β-arrrestin2)  BRET recrestin2) . In combrestin2) .2AR phosphorylation >20-fold more in the presence of GRK2 than in its absence. We therefore tested the ability of WT GRK2-H6 and the L33N, E36A, E532A, L536N, and L547N variants to phosphorylate WT β2AR using 2-nM ISO to stimulate the receptor. β2AR phosphorylation was abolished when alprenolol, an inverse agonist, replaced ISO or when the kinase-deficient K220R was used in the assay , and regardless of whether one measures phosphorylation with purified components in vitro or in living cells. Point mutations of residues in the N-terminus of GRK2, in contrast, have a much more profound impact on GPCR phosphorylation in living cells and in vitro [In contrast, the newer Models 2 and 3 suggest no direct role for the N-terminus in receptor binding. Instead, the supporting data pointed to docking sites in regions within or immediately adjacent to the terminal subdomain of the RH domain. Since this region varies considerably in sequence among the GRK subfamilies, these models spurred us on to test whether the analogous region in the RH domain of GRK2 could play an important role in GPCR binding. However, our data indicated that the GRK2 RH domain and residues in its preceding loop do not play a major role in GPCR binding, regardless of whether the receptor has a short (rhodopsin) or longer ICL3  and the fact that individual receptors, with poor conservation in their ICL regions, can typically be phosphorylated efficiently by multiple GRKs. A more universal mechanism for GPCR engagement by GRKs therefore seems warranted. Differences in the models could also stem from the confounding requirement of GRKs for the presence of both anionic phospholipids and the activated receptor conformation. In other words, the loss of signal being measured in some of the supporting experiments may simply reflect the propensity of the GRK being studied (GRK5 and GRK1) to bind to membranes (or bicelles) rather than the receptor itself. For example, GRK5 binds to negatively charged membranes regardless of the presence of an agonist-bound receptor, as was the case in the studies backing Model 2, where it was pulled down in the presence of an inverse agonist. It should also be noted that GRK4 subfamily members have a greater propensity to phosphorylate inactive GPCRs in intact cells . The Tan


Both allergen-specific IgE and total IgE in serum play a major role in asthma. However, the role of IgE in chronic obstructive pulmonary disease (COPD) is poorly understood. It was the aim of this study to systematically analyze the relationship between serum IgE levels and disease characteristics in large COPD cohorts.COSYCONET is a comprehensively characterized cohort of patients with COPD: total IgE and IgE specific to common aeroallergens were measured in serum of 2280 patients, and related to clinical characteristics of the patients. WISDOM is another large COPD population (2477 patients): this database contains the information whether total IgE in serum was elevated (≥ 100 IU/l) or normal in patients with COPD.Both in COSYCONET and WISDOM, total IgE was elevated (≥ 100 IU/l) in > 30% of the patients, higher in men than in women, and higher in currently than in not currently smoking men. In COSYCONET, total IgE was elevated in patients with a history of asthma and/or allergies. Men with at least one exacerbation in the last 12 months  had higher median total IgE (71.3 IU/l) than men without exacerbations (48.3 IU/l): this difference was also observed in the subgroups of not currently smoking men and of men without a history of asthma. Surprisingly, a history of exacerbations did not impact on total IgE in women with COPD. Patients in the highest tertiles of total IgE  or allergen-specific IgE  were at risk of lung function decline .These data suggest that IgE may play a role in specific COPD subgroups. Clinical trials using antibodies targeting the IgE pathway , especially in men with recurrent exacerbations and elevated serum IgE, could elucidate potential therapeutic implications of our observations.The online version contains supplementary material available at 10.1186/s12931-021-01847-0. Allergen-specific Immunoglobulin E (IgE) is postulated to be a key driver of exacerbations in allergic asthma . ClinicaThe finding that FcεRI is not only upregulated on pDCs in asthma, but also in COPD, led to the hypothesis that anti-IgE treatment might also reduce exacerbations in COPD . However1 < 50% predicted. Data from 2477 patients were available. The WISDOM database contains the information whether patients had elevated (≥ 100 IU/l) or normal (< 100 IU/l) serum IgE levels.The multicenter cohort study COSYCONET (German COPD and Systemic Consequences—Comorbidities Network) investigates the phenotypes and progression of COPD and its comorbidities, as described . In 31 sTotal and allergen-specific (SX-1) IgE concentrations in serum were determined using the ImmunoCAP system . The SX1 screening panel detects 8 allergen-specific IgE antibodies: against house dust mite (Dermatophagoides pteronyssinus), cat, dog, birch, timothy (Phleum pratense), rye, ragweed, Cladosporium herbarum.1 during follow-up, we performed group-based trajectory modeling of FEV1 using the STATA/IC 15 package TRAJ . This approach is based on the SAS PROC TRAJ macro kit and fits a semiparametric (discrete mixture) model for longitudinal data using the maximum likelihood method. We used the Bayesian information criterion to establish the optimal number of groups with at least 5% of participants in the smallest trajectory. Trajectory groups were termed group A (increasing FEV1), group B (stable FEV1), and group C (declining FEV1). Afterwards, logistic regression analyses were performed to assess the association between baseline IgE levels and trajectory group C (declining FEV1) using group B as the reference category. The analysis was adjusted for age, gender, body mass index (BMI), initial FEV1 (% predicted), and smoking status.Statistical analyses were performed using SPSS . IgE in serum was not normally distributed. Therefore, the Mann–Whitney U-test was used to determine differences in IgE between subgroups of the cohort. Correlation analyses were performed using Spearman’s correlation coefficient. Probability values of p < 0.05 were regarded as significant. Box plots display the median (line within the box), interquartile range (edges of the box) and extremes . Restricted cubic splines were used as an unbiased approach to visualize the non-linear association between total or allergen-specific IgE (SX1) and the exacerbation history. Three knots of total or allergen-specific IgE were placed corresponding to the 10th, 50th, and 90th percentile of the IgE levels, respectively. Moreover, to assess the association between total or allergen-specific IgE at baseline and changes of FEV1 < 50% predicted (GOLD grades 3 and 4).In COSYCONET, total IgE at baseline visit 1) was available in 2280, allergen-specific IgE (SX1) in 2326 patients. Both parameters were documented in 2263 patients. Table  was avaiIn COSYCONET, allergen-specific IgE SX1) was elevated (≥ 0.35 IU/ml) in 24.8% of the total cohort . There was a correlation  between total and allergen-specific IgE (SX1) in COSYCONET  was higher in men than in women . Both in females and males, total IgE and allergen-specific IgE SX1) levels were higher in patients with a history of asthma  or self-reported allergies , as compared to patients without a history of asthma or allergies  (data not shown). Total and allergen-specific IgE according to spirometric GOLD grades are detailed in the  revealed that the highest tertiles of total IgE (> 91.5 IU/ml) and allergen-specific IgE (> 0.19 IU/ml) were both associated with a significant risk for lung function decline  displayed higher total IgE (median: 71.3 IU/ml) than men without exacerbations median: 48.3 IU/ml): this difference was not found in women  was found in 31.2% of the patients in COSYCONET. In WISDOM, this proportion was slightly higher (34.2%), most likely due to the higher percentage of males : percentages of men with elevated IgE were similar . These data are confirmed by the COPDGene study , where 34.3% of the patients had elevated total IgE . The larBoth in COSYCONET and WISDOM, there were higher total IgE levels in men than in women, and in currently smoking than in not currently smoking men. This impact of gender and smoking on total IgE is in accordance with the literature. In SAPALDIA , total IExacerbations have a major impact on disease progression and treatment strategies in COPD –35. We f1, both in male and in female patients with COPD. These data suggest that allergen-specific IgE might play a hitherto unrecognized role in the pathogenesis of lung function abnormalities in COPD [COPD may show a wide variation regarding change of lung function over time . We foun in COPD . It need in COPD , 38–40.This study has several strengths. First, it analyzes a large number of patients with COPD . Second, the German COSYCONET cohort is a well characterized cohort, with extensive information on the history and the characteristics of the patients. Finally, lung function data in COSYCONET were available over a period of 3 years, enabling an analysis of the relationship between IgE and lung function trajectories. Therefore, the COSYCONET dataset offered a unique opportunity to analyze the relationship between serum IgE and clinical characteristics of patients with COPD. Moreover, we did not only measure total IgE, but also allergen-specific IgE using the SX1 screening test which comprises 8 common aeroallergens . Thus, we were able to analyze effects of total IgE and allergen-specific IgE separately.There are several limitations. First, due to the large number of serum samples in the COSYCONET cohort, the analysis of allergen-specific IgE was limited to a screening panel of 8 common aeroallergens. Several relevant allergens might have, therefore, been missed in this analysis . In addiElevated serum IgE is associated with the occurrence of exacerbations in men with COPD and with the risk of lung function decline. We speculate that IgE-mediated pathways might be involved in the pathogenesis of exacerbations in men with COPD and in the pathogenesis of progressive airflow limitation in patients with elevated IgE levels. Clinical trials using antibodies targeting the IgE pathway , especially in men with recurrent exacerbations and elevated total serum IgE, could elucidate potential therapeutic implications of our observations.Additional file 1. Supplementary Tables and Figures.


The distribution coefficient “K” (the ratio of the Mg2+ concentration in the ice layer to the Mg2+ concentration in the water layer under ice) was used to characterize the migration ability of Mg2+. The results showed that during the freezing process, the concentration distribution of Mg2+ in the ice and water two-phase system was as follows: ice layer < water before freezing < water layer under ice; in other words, it migrated from ice layer to the water layer under ice. “K” decreased with increasing ice thickness, freezing temperature, initial concentration, and initial pH; the higher the ice thickness, freezing temperature, initial concentration, and initial pH were, the higher the migration efficiency of Mg2+ into the water layer under ice was. During the melting process, Mg2+ was released in large amounts (50–60%) at the initial stage (0–25%) and in small amounts (25–100%) uniformly in the middle and later periods. According to the change of Mg2+ concentration in ice melt water, an exponential model was established to predict Mg2+ concentration in ice melt period. The migration law of Mg2+during the freezing and melting process was explained by using first principles.To explore the migration law of magnesium ions (Mg Icebound is an important hydrological feature of surface water at high latitudes. More than 50 million lakes regularly freeze every year in the world, and the annual icebound time is more than 150 days  To study the effect of ice thickness on the migration law of Mg2+ in the freezing process, a standard solution of Mg2+ with a concentration of 500 mg·L−1 was prepared and placed in five freezing simulators. Then, an 8-L water sample with pH 7 was put into each simulator, the height of water was 28 cm, and devices were placed in a low temperature box at – 5 ℃,—10 ℃,—15 ℃,—20 ℃, and—25℃. When the ice thickness reached 12 cm, the reactors were removed.(2) To study the effects of freezing temperature on the migration law of Mg2+ in the freezing process, referring to Guidelines for Drinking-water Quality set by the WHO (3) of drinking water shall not exceed 500 mg·L−1. The standard solutions of Mg2+ with concentrations of 300 mg·L−1, 400 mg·L−1, 500 mg·L−1, 600 mg·L−1, and 700 mg·L−1 were placed in five freezing simulators. Then, an 8-L water sample with pH 7 was placed into each simulator, the height of water was 28 cm, and devices were placed in a low-temperature box at – 15 ℃. When the ice thickness reached 12 cm; the reactors were removed.(3) To study the effects of initial concentration on the migration law of Mg the WHO , the max2+ in the freezing process, a standard solution of Mg2+ with a concentration of 500 mg·L−1 was prepared and placed in five freezing simulators. Then, an 8-L water sample was put into each simulator; the height of water was 28 cm; and the pH of the water samples were adjusted to 5.5, 6.5, 7.5, 8.5, and 9.5 by dropping HCl and NaOH solution and placing devices in a low temperature box at – 15 °C. When the ice thickness reached 12 cm, the reactors were removed.(4) To study the effects of initial pH on the migration law of MgTo explore the migration law of MgThe ice sample obtained from the above freezing experiment was put into a beaker, melted in a constant temperature box (25 °C), and the water under the ice was evenly mixed and put into a beaker for detecting.2+ were detected respectively.The process of ice samples acquisition in the melting experiment was the same as that in the simulated freezing experiment. The ice samples with different ice thickness, freezing temperature, initial concentration, and initial pH are weighed, placed in the ice-melt device, and melted in the incubator (25 °C). Take the ice melt water four times on average according to the ice sample weight, and the weight of each melt water accounts for 25% of the total weight of the ice sample. The melt water weight reaches 25% of the total weight of ice sample for the first time as melting 1, 25% of the total weight of ice sample for the second time as melting 2, 25% of the total weight of ice sample for the third time as melting 3, and 25% of the total weight of ice sample for the fourth time as melting 4. The concentrations of Mg2+ concentrations in ice melt water and the water layer under ice were measured in accordance with the Water and Wastewater Monitoring Analysis Method (4th ed.) (The Mg4th ed.) , and the2+ in the ice layer and the average concentration of Mg2+ in the water layer under ice, which reflects the migration ability and discharge effect of Mg2+ in the process of water freezing:The distribution coefficient “K” is defined as the ratio between the average concentration of Mg2+ in the ice layer and 2+ in the water layer under ice (mg·L−1).where 2+ were measured , the concentrations of Mg2+ were 46.838 mg·L−1, 68.734 mg·L−1, 97.745 mg·L−1, 108.335 mg·L−1, and 136.476 mg·L−1, respectively, which were 1.74–2.14 times of the average concentration of Mg2+ in the ice layer. The ratios of Mg2+ concentration in melting 1 to Mg2+ concentration in corresponding ice layer were 43.57%, 48.06%, 52.64%, 50.67%, and 53.41%, respectively. For meltings 2–4 of ice samples with different initial concentration, the average concentrations of Mg2+ were 20.222 mg·L−1, 24.772 mg·L−1, 29.310 mg·L−1, 35.162 mg·L−1, and 39.680 mg·L−1, respectively, which were 0.62–0.75 times of the average concentration of Mg2+ in the ice layer. The ratios of the average Mg2+ concentration in meltings 2–4 to the Mg2+ concentration in the corresponding ice layer were 56.43%, 51.94%, 47.36%, 49.33%, and 46.59%, respectively.As shown in Fig. 2+ were 113.596 mg·L−1, 97.745 mg·L−1, 90.125 mg·L−1, 80.29 mg·L−1, and 65.523 mg·L−1, respectively, which were 1.77–2.31 times of the average concentration of Mg2+ in the ice layer. The ratios of Mg2+ concentration in melting 1 to Mg2+ concentration in corresponding ice layer were 59.01%, 54.69%, 52.64%, 48.79%, and 44.59%, respectively. For meltings 2–4 of ice samples with different initial pH, the average concentrations of Mg2+ were 27.696 mg·L−1, 29.310 mg·L−1, 31.422 mg·L−1, 32.171 mg·L−1, and 27.497 mg·L−1, respectively, which were 0.55–0.74 times of the average concentration of Mg2+ in the ice layer. The ratios of the average Mg2+ concentration in meltings 2–4 to the Mg2+ concentration in the corresponding ice layer were 40.99%, 45.31%, 47.36%, 51.21%, and 55.41%, respectively.As shown in Fig. Guidelines for Drinking-water Quality set by the WHO (3) of drinking water shall not exceed 500 mg·L−1. In the simulated freezing experiment, the detection range of Mg2+ in ice layers was 26.077–66.477 mg·L−1 when the concentration of Mg2+ was 500 mg·L−1, which is lower than the limit value of hardness in the Guidelines for Drinking-water Quality, while the detection range of Mg2+ in the water layers under ice was 573.08–1468.248 mg·L−1, which is significantly higher than the initial concentration of 500 mg·L−1 and far beyond the limit value of hardness in the Guidelines for Drinking-water Quality. Under different conditions, the concentration relationship of Mg2+ in the ice and water two-phase system is as follows: ice layer < water layer before freezing < water layer under ice; in other words, Mg2+ migrate from the ice layer to the water layer under ice during the freezing process. The migration process can be explained by first principles.According to  the WHO , the max2+ in water are tightly surrounded by water molecules, a large amount of Mg2+ were rapidly released in a short time. The concentration of Mg2+ in the initial melting stage was much higher than the average concentration of Mg2+ in the ice layer, and then the release rate of Mg2+ rapidly decreased, showing a small amount of uniform release phenomenon. The concentration of Mg2+ in the middle and late stages was significantly lower than the average concentration of Mg2+ in the ice layer.The results of ice melting experiments showed that the release law of Mg2+ can be attached to the growing droplets, which is defined as “solute elution” .where A, t, and y0 are appropriately selected and substituted into the above equation to calculate the Mg2+ concentration of ice melt water in the melting process under specific conditions.By fitting the curve, the parameter values under various conditions can be obtained, as shown in Tables 2+ is discharged, which leads to Mg2+ migrating from the ice layer into the water layer under ice, and the concentration of Mg2+ in the water layer under ice increases. The migration effect has a more significant impact on shallow lakes at high latitudes, so more attention should be paid to the detection of Mg2+ pollution in the water under ice in winter.(1) In the process of water freezing, Mg2+ to the water layer under ice. Other factors (such as light and radiation) may also influence Mg2+ migration during the freezing process, which must be further explored.(2) In the water freezing process, distribution coefficient “K” decreases with increasing ice thickness, freezing temperature, initial concentration, and initial pH; in other words, higher freezing temperature, ice thickness, initial concentration, and initial pH are conducive to the migration of Mg2+ are rapidly released in a short time, and the release rate of Mg2+decreases rapidly in the middle and later periods (25–100%), showing a small and uniform release phenomenon. Therefore, in the early melting stage of lake ice, the preferential centralized release of pollutants may have a great impact on the ice water environment, which needs the attention of limnologists.(3) In the early period of ice melting (0–25%), a large number of Mg