Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries. Cardiovascular disease continues to claim the lives of more women in the U.S. than any other health problem, with coronary heart disease (CHD) as the number one cause of mortality . In earlEvidence from the literature suggests that GPER plays an important role in mediating cardiovascular actions of estrogen , howeverIn human coronary arterioles, estrogen potentiated angiotensin II-induced vasoconstriction via GPER and EGFR activation . The potAntibodies used in immunoblotting were: p-ERK1/2 from Cell Signaling , p-EGFR from Santa Cruz  and β-actin from Novus Biologicals . G-1 and G36 were purchased from Azano Pharmaceuticals Incorporation. Inhibitors used in the isometric tension studies were purchased from Tocris Bioscience. All other chemicals were purchased from Sigma Aldrich.2PO4, 2.5 mM MgCl2, 1.2 mM CaCl2, 2 g glucose and 2 g NaHCO3 in 1 L with pH 7.4 (The solution was previously oxygenated with 95% O2-5% CO2 for 30 minutes). Hearts were kept on ice during transport to the laboratory, where coronary arteries were dissected as described previously [Porcine hearts were obtained from an approved and regulated supplier; the geographic coordinates are latitude 30.372080° and longitude -96.070557°. The hearts were placed into ice-cold Krebs buffer of the following composition: 131.5 mM NaCl, 5 mM KCl, 1.2 mM NaHeviously , 13.2-5% CO2 and maintained at 37°C. Coronary arterial rings were equilibrated for 90 min under resting tension of 20 mN, and the bath solution was changed every 30 min. For relaxation response experiments, at the plateau of ET-1 (10 nM)-induced contraction, inhibitors were added 30 min prior to the measurement of a complete G-1 concentration-response relationship (1 to 3000 nM). Relaxation responses were calculated as the percent reduction in tension from ET-1 pre-contracted state. For vasoconstriction response, G-1 and inhibitors were also used to pretreat the arterial rings before adding endothelin 1  to induce a contractile response. Arterial rings were collected, oven-dry, and weighed after experiments. Contractile responses were calculated as force generated by arterial rings calibrated by artery dry weight with unit mN/mg.The left anterior descending (LAD) coronary arteries were dissected and cleaned of excess connective tissue and fat. To eliminate indirect effects of endothelium-derived vasoactive factors, the endothelium was removed physically by rubbing the intimal surface and tested by observing the absence of bradykinin-induced relaxation of PGF2α-induced contraction. The LAD was cut into ~3 mm rings and mounted on two parallel tissue supports in the tissue incubation chamber, with one support fixed to the stationary chamber and the other attached to a force transducer (AD Instruments). Isometric contractile force was recorded on a computer using LabChart software. The tissue bathing solution was Krebs buffer, oxygenated continuously with 95% O2, 0.16 mM CaCl2, 10 mM HEPES, 10 mM NaHCO3, 0.5 mM KH2PO4, 0.5 mM NaH2PO4, 0.49 mM EDTA, 10 mM Taurine 10 mM glucose and 15% fetal bovine serum (Lonza)) at 37°C for two hours. Then the tissue pieces were dispersed into single cell with complete SmBM basal medium (Lonza) and seeded into 60-mm gelatin-coated plastic dishes with density of 2x105 cells/ml. Primary cultured porcine coronary artery smooth muscle cells (PCASMCs) were maintained in complete SmBM basal medium. Cell cultures were kept at 37°C and under 5% CO2 in a humidified incubator. The purity of PCASMCs was verified by positive staining with smooth muscle-specific α-actin [We adopted the method of dispersing coronary artery cells by Chamley-Campbell et al. with some modification , 19, 20. α-actin . PrimaryCoronary artery rings were collected after various drug treatments in isometric tension studies and were snap-frozen in liquid nitrogen. The tissues were pulverized and then lysed in RIPA lysis buffer (Sigma) with protease and phosphatase inhibitors. PCASMCs were harvested and lysed in the same lysis buffer. Protein concentrations were determined by BCA protein assay (Pierce) and samples were separated on precast 4–12% Bis-Tris gels (Invitrogen) according to the manufacturer’s instructions. Then proteins were transferred to PVDF membrane (EMD Millipore) at 100 V for two hours. Membranes were blocked with 5% non-fat milk for one hour at room temperature and then incubated with primary antibodies in TBST, a mixture of tris-buffered saline (TBS) and Tween 20 (0.1%), containing 5% non-fat milk for overnight at 4°C. After washing and incubating with secondary antibodies, protein bands were detected with chemiluminescence. Then the membranes were stripped by washing in stripping buffer (Thermo Scientific), and then probed with β-actin antibody to be used as protein loading controls.The level of heparin binding epidermal growth factor (HB-EGF) in the PCASMCs samples was measured by enzyme-linked immunosorbent assay, according to manufacturer’s instructions (ThermoFisher Scientific). Briefly, PCASMCs, passage 4–5, were serum-deprived for 24 hours after cells reached approximately 50–60% confluence. After incubation with different drugs, cell culture media was collected in clean tubes and centrifuged at 3000 rpm for 20 min. The supernatants were kept for assay in duplicates. The optical density of each sample was measured by using a microplate reader at 450 nm and 550 nm. The average absorbance (450 nm minus 550 nm) of each sample was calculated from the standard curve, and the concentration of HB-EGF was expressed as pg/ml.Cyclic cAMP production was measured with Cayman’s cAMP assay, which is competitive enzyme-linked immunosorbent assay (ELISA) , as described previously (78). Briefly, PCASMCs (passages 4) were cultured in culture dishes (35-mm) in SmGM medium for three days. After reaching 85–90% confluence, cells were serum-deprived for 18 h in phenol red-free α-MEM buffer with 3-isobutyl-1-methylxanthine (100 μM) to inhibit phosphodiesterases. Then cells were treated with G-1 (1 μM), or ET-1 (10 nM), 0.1% DMSO was used as solvent control.All data were analyzed using GraphPad Prism . In isometric tension studies, the concentration-responses of G-1 and ET-1 with or without inhibitors were analyzed by two-way analysis of variance (ANOVA). For immunoblotting experiments and ELISA, one-way ANOVA was used. Bonferroni correction was used as post-hoc test to correct type 1 errors associated with ANOVA in all data sets. All data were expressed as means ± SEM. P value ≤0.05 indicated a significant difference.50 at 0.034 μM and maximum relaxation of 46.4 ± 3.4%; and the relaxation response was significantly inhibited by G36, without changing EC50 of G-1 (50 of ET-1 (8.6 nM)  . These f50 of G-1 increased from 0.034 μM in the absence of gallein to 0.017 μM in the presence of gallein  play an important role in GPCR-mediated HB-EGF shedding and EGFR transactivation. It has also been shown that MMP are involved in pressure-induced myogenic tone mediated by EGFR transactivation in mouse mesenteric resistance arteries . To inve50 values (0.034 μM). Furthermore, the inhibition of EGFR with AG1478 significantly reversed G-1 (1 μM) enhanced ET-1-induced contractile response  treatment of PCASMCs significantly increased EGFR tyrosine phosphorylation at 5 and 10 min, to a phosphorylation level similar to that observed with EGF (10 ng/ml), which served as a positive control. Pretreatment with AG1478 (5 μM) significantly attenuated G-1-stimulated tyrosine phosphorylation of EGFR . These dGPER activation can stimulate Src-related tyrosine kinase activity-dependent EGFR transactivation and then cause ERK1/2 phosphorylation in breast cancer cells . InhibitIn this study, we have found that GPER may mediate both the relaxation and contraction responses of coronary arteries. The contractile effect of a GPER agonist G-1 was induced through EGFR transactivation and subsequent ERK1/2 activation via Gβγ/Src/MMP signaling pathway.EGFR plays a key role in myogenic tone regulation as well as G protein-coupled receptor (GPCR)-mediated vasoconstriction response –35. MyogIn the current study, we determined that the inhibition of EGFR with AG1478 increased GPER-mediated coronary artery relaxation; but abolished GPER-mediated potentiation of ET-1-induced contraction without any effect on the contractile force induced by ET-1 alone, suggesting that there is a contractile component in the effect of GPER activation that involves EGFR. G-1 stimulation of heparin-binding EGF (HB-EGF) release and tyrosine phosphorylation of EGFR have further confirmed the notion that activation of GPER indeed causes transactivation of EGFR and thus induces enhanced vasoactive contraction of coronary artery. It appears that under condition pre-contraction, G-1-induced vasorelaxation dominates; however, when pre-treat the arterial rings with G-1 before applying vasoconstrictor ET-1, the vasoconstriction effect dominates. Apparently, in the pre-contracted arterial ring experiment, the constriction effect mediated by EGFR transactivation exerted opposing effect on the predominate response of relaxation mediated by cAMP in the action of GPER; when AG1478 was applied to block the EGFR pathway, the opposing vasoconstriction effect was inhibited, therefore, more vasorelaxation response of GPER activation being demonstrated. Vice versa, in the ET-1 concentration-response vasoconstriction experiment, pre-incubation of the arterial rings with G-1 induced more vasoconstriction response to ET-1 through transactivation of EGFR and AG1478 inhibited the vasoconstriction effect of G-1 pre-treatment. Together the evidence provided here suggests that GPER-mediated transactivation of EGFR is an important mechanism in the regulation of vascular tone.+ channel, voltage-gated Ca2+ channels and ERK1/2 [The GPER-mediated transactivation of EGFR occurs via Gβγ-subunit signaling in breast cancer cells , 41, 42,d ERK1/2 –46. Althd ERK1/2 , 46–48; d ERK1/2 , 41, 42.It is widely accepted that Src is coupled to nearly all GPCRs that lead to EGFR transactivation . This noIn conclusion, these studies demonstrate that the activation of GPER elicits both relaxation and contraction responses in isolated coronary arteries. Our previous work has shown that GPER also induces relaxation via cAMP , 14. How


Ice is one of the most extensively studied condensed matter systems. Yet, both experimentally and theoretically several new phases have been discovered over the last years. Here we report a large-scale density-functional-theory study of the configuration space of water ice. We geometry optimise 74,963 ice structures, which are selected and constructed from over five million tetrahedral networks listed in the databases of Treacy, Deem, and the International Zeolite Association. All prior knowledge of ice is set aside and we introduce “generalised convex hulls” to identify configurations stabilised by appropriate thermodynamic constraints. We thereby rediscover all known phases  except the metastable ice IV. Crucially, we also find promising candidates for ices XVIII through LI. Using the “sketch-map” dimensionality-reduction algorithm we construct an a priori, navigable map of configuration space, which reproduces similarity relations between structures and highlights the novel candidates. By relating the known phases to the tractably small, yet structurally diverse set of synthesisable candidate structures, we provide an excellent starting point for identifying formation pathways. Ice is one of the most well-studied condensed matter systems, yet new phases are still being discovered. Here the authors report a large-scale computational study of the configuration space of water ice, creating a navigable “sketch-map” including new predicted phases as well as relationships between different structures. Ice is a complex system of interest across much of science, ranging from astrophysics to biology. On the Earth’s surface and in its atmosphere, it plays a central role in determining climate and in countless natural processes and technological applications. Ice is also a key constituent of the Earth’s crust and mantle. Its phase diagram and properties have been investigated across a wide range of temperatures and pressures by experimentalists and theoreticians alike.1, 7 of which are metastable2. In addition, a number of hypothetical ice phases14 have been predicted and characterised using computer simulations. All of these phases are molecular crystals that fulfil the “Bernal–Fowler ice rules”15 and form four-connected networks. In most ice phases, the distinct ways of dressing the oxygen sublattice with hydrogen atoms within the ice rules  are quasi-energetically degenerate16. Theoretical studies have also suggested structures of water ice under ultrahigh pressures of up to many terapascals and its eventual decomposition18.A total of 18 crystalline ice phases have been formed under various conditions13 and the experimental description of ice XVII19 and of two-dimensional forms of ice21 have demonstrated that our understanding of ice is far from complete. Second, it has become apparent that the nucleation and melting of ice are complex processes in which metastable ice phases play a role23. In classical nucleation theory, an interfacial free energy advantage of a few percent will lead to preferential nucleation of metastable phases with free energies up to around 10 meV/H2O above the stable phase24.The phase diagram of ice has recently received renewed interest, first, because the theoretical discovery of the s-III clathrate hydrate25, to our knowledge no comprehensive study of (meta)stable ice phases and their formation has been published to date. The problem is two-fold: first, the enormous configuration space must be explored in a reasonably comprehensive manner. Second, in order to render the structure search relevant to experiment, the large number of theoretical (meta)stable structures generated in the process must be reduced to those that can be formed experimentally. This refinement must be a priori and quantitative. Finally, different stabilising factors—such as the absorption of guest molecules26—can be investigated further, and methods such as forward flux sampling30 and enhanced sampling metadynamics33 may be used to identify possible synthetic pathways.Despite valiant efforts using structure searching methods such as ab initio random structure searching (AIRSS)12 to explore the relevant parts of the configuration space of ice using databases of theoretically enumerated, four-connected networks. In the second section of the Results we rationalise the resultant structural data on the basis of purely energetic considerations and thereby identify structures that can be stabilised under pressure. By design, this approach cannot identify structures stabilised by thermodynamic and kinetic constraints other than pressure, such as temperature, electric fields, concentrations of guest molecules, etc. In the third section of the Results we overcome this limitation by developing a “generalised convex hull” (GCH) construction. Moreover, we use the sketch-map algorithm34 to construct a navigable map of the configuration space of ice. This primarily serves as an aide in developing an intuitive understanding of structural relationships. However, it also shows potential for helping to identify formation pathways for new candidate ice phases.This work aims for a comprehensive study of crystalline ice phases, focussing on the exploration of configuration and the reduction of the resulting intractably large amount of structure data to a small number of structures, which are likely to be accessible experimentally. In the first section of the Results we exploit the isomorphism between ice and silica networks12 and arises because both silica and water preferentially form four-connected networks composed of corner-sharing tetrahedral units. The basic building blocks of silica and water ice are so similar that it is even possible to form silica/water hetero-networks in which silicate oligomers form part of the hydrate lattice36.The strong isomorphism between ice and silica networks has previously been explored in ref. 37 and a number of very large databases of theoretically enumerated networks, such as the databases of Treacy38 and Deem39. Graph network enumeration has previously been applied to crystal structure prediction40 and, in particular, to sp2- and sp3-carbon42. The above databases have proven to be a valuable resource in searching for sp3 allotropes of carbon43 and constitute a comprehensive source of four-connected networks from which topologically distinct phases of ice can be constructed and geometry optimised to the respective associated local minimum energy structures using conjugate gradient methods. Recently, the search for computationally stable ultralow-density ices44 on the basis of the atlas of zeolites37 has hinted at the potential of this approach, despite its more limited scope and despite only considering stabilisation under (effective negative) pressure. The search for (meta)stable ice phases is facilitated by the strong correspondence between zeolite and ice structures. Figure 2 structures and their counterpart H2O polymorphs after geometry optimisation, indicating that structurally distinct SiO2 networks generally translate into structurally distinct H2O networks.There is a vast literature on four-connected structures, including an atlas describing the underlying networks of porous crystalline zeolites12 show that the energies and densities of low-density SiO2 networks and their counterpart H2O networks are correlated, but this correlation does not carry across to structures of densities comparable to and higher than that of ice Ih  have unit cells containing no more than 16 molecules, applying a cutoff to the unit cell size provides a reasonable method for preselection, which can be improved systematically by including structures with larger unit cells. In practice, we preselect only networks with unit cell volumes of no more than 800 Å3 and without 3-rings, which would normally induce excessive strain in an ice structure. Out of the 331,172 (Deem) and 5,389,408 (Treacy) zeolites, this leaves 74,731 structures. This selection contains duplicates since the databases are not mutually exclusive. Low-density structures with low SiO2 lattice energies are added back in by including the experimentally synthesised zeolite networks from the IZA database45  stable configurations that can be constructed increases exponentially with system size, but only those that can be observed experimentally are of interest. Their selection must take into consideration the uncertainty in the computational framework, the possibility of kinetic and/or surface effects promoting the formation of metastable phases and the (de)stabilisation of phases by different thermodynamic boundary conditions, such as pressure.Ech(ρ), is formed by structures that are stable against decomposition into two or more structures with lower average energy at the same average density and the so called “tie lines” that connect them. In the absence of kinetic effects, the only phases that can be observed by manipulating the density of the system (for example through pressure) are exactly those that constitute the vertices of the energy-density convex hull , Edr = E − Ech(ρ). Based on Edr, we refine the selection of ice structures as specified in Computational methods. Ultimately, only structures with Edr less than 10 meV are retained, for which kinetic, entropic and/or surface effects may plausibly lead to preferential formation during nucleation.To identify the polymorphs that are most likely to form at different pressures, we first consider a well-established approach based on a convex-hull construction. The convex hull of energy (as a proxy for free energy) as a function of density, see Fig. . In anal50. This clearly demonstrates the potential of our structure searching approach. However, not all known ice phases are classified as synthesisable, which highlights the limitations of the established convex-hull approach: it fails to identify synthesisable metastable structures (such as ice IV and XII/XIV) and structures that can be stabilised and made synthesisable by thermodynamic and kinetic constraints other than pressure . These limitations will be addressed in the following.Setting aside all prior knowledge of ice, this procedure identifies the theoretical i, 0 and quartz phases and the known Ih/XI, II, III/IX, V/XIII, VII/VIII and X phases of ice. Moreover, it identifies the structure that has since been identified experimentally as the porous ice XVII12. The IRR and IWV counterparts exhibit substantially lower densities than the known CS-I, CS-II and HS-III clathrate hydrates54, suggesting that they may only become stable at large negative pressures. Conversely, the counterparts of the PCOD8172143 and 11_2_15848 zeolites  must be accounted for, as highlighted by the stabilisation of ice Ih with respect to Ic by anharmonic quantum nuclear vibrations46, as well as by effects of similar magnitude observed in other H-bonded crystals55. Anharmonic QNE in particular stabilise ice XVII and the HS-III clathrate by a few meV/H2O with respect to Ih (see Table SIII). Their resultant zero pressure free energies exceed that of Ih by only 6.8 and 7.8 meV/H2O at the PBE-DFT level, respectively. The relative stability of the counterparts of the PCOD8172143 and 11_2_15848 zeolites with respect to Ih, on the other hand, is affected very little  obtaining a global picture of configuration space from which one can gather an intuitive understanding of the relations between different polymorphs; (b) assessing the effectiveness of the structure search, identifying more or less obvious “gaps”; and (c) selecting candidates stabilised by thermodynamic constraints other than pressure, such as absorption of guest molecules, electric fields, etc. All of these problems can be tackled effectively within a framework that borrows ideas from the machine-learning community. Points (a) and (b) are addressed by constructing an abstract, unbiased and general two-dimensional representation of configuration space in terms of the similarity relations between structures. Point (c) is addressed by generalising the conventional convex hull construction.56 combined with an entropy-regularised matching (REMatch) approach57. This captures the fundamental symmetries of the problem, such as invariance to alternative representations of the same periodic structure, particle labelling and rigid rotations and translations of the atomic coordinates. Based on the kernel-induced distance, we apply the sketch-map algorithm34 to obtain a two-dimensional representation that reproduces as accurately as possible the (non-linearly transformed) distance between each pair of structures. The construction and its parameters were designed to assess the oxygen lattice while being insensitive to proton-disorder and hydrogen-bonding defects.The first key ingredient of an intuitive representation of configuration space is a measure of the similarity of different configurations. We use the smooth overlap of atomic positions (SOAP) kernelThe resulting map is shown in Fig. 2 parent structures) are initialised with bond-centred protons.Several observations highlight the heuristic value of such a representation: (1) The positions on the map correlate well with both density and lattice energy  and very open structures  is sparse. At high density, this sparsity results from the increasing importance of geometric constraints, which limit structural diversity and prohibit the formation of energetically feasible “mixed phases” containing structural patterns from two or more low-energy configurations. At low-density, our preselection strategy leads to sparse sampling. Sketch map therefore provides indications of the quality of configuration-space sampling, which can be used to focus the structure search on the regions that need it most.Edr are projected onto the periphery of the map  by the application of appropriate thermodynamic constraints.Finally, structures with low E as a function of ρ, we consider E as a function of n variables measuring abstract structural features, E. The simplices of the GCH thus correspond to structures that are stable with respect to decomposition at a given set of these abstract structural features, rather than at a particular density. The conventional energy-density convex hull allowed the identification of structures, which can be stabilised by pressure, because pressure allows the manipulation of the density of an ice sample. Conversely, the GCH allows us to identify structures, which can be stabilised by imposing thermodynamic and/or kinetic constraints that couple to the abstract structural features. In analogy with Edr, one can then define a generalised dressed energy n structural features ϕ1 … ϕn.We define this GCH construction in analogy with the conventional energy-density convex hull in the second section of the Results, but instead of considering 59 is performed to extract the KPCA descriptors ϕi, which encode the key structural features and can be sorted in order of decreasing importance. Crucially, the KPCA components (unlike the collective variables defining the highly non-linear sketch-map projection) form a vector space in which the notion of convexity is well defined.In practice, a kernel principal-component analysis (KPCA)n of features considered, the screening becomes progressively more inclusive, since multiple axes of structural diversity are considered simultaneously. In practice, the selection was further refined by  eliminating structurally related configurations, as discussed in Supplementary Note The GCH construction provides a powerful tool for discovery, since one can select configurations that are both low in energy and “extremal” in the sense of structural features described by one or more KPCA descriptors. By increasing the number Including three KPCA descriptors in the GCH construction, we identify 50 structures within 20 meV of the GCH see Fig. , which i12, and two structures reminiscent of a high pressure structure with Pbcm symmetry proposed by Hermann et al.60. Notably, while the most promising candidates for experimental formation  energy calculations. As shown in Supplementary Note 48 instead of the PBE functional, despite significant differences with respect to the PBE lattice energies. The rPW86-vdW2 functional has been shown to be particularly accurate for the known phases of ice62. In contrast, the energy-density CH depends more sensitively on the choice of exchange-correlation functional.The 50 structures also include the theoretical i, 0, quartz and square phases, the CS-II clathrate hydrate  and the HS-III clathrate hydrate. Furthermore, we identify 34 new configurations that are excellent candidates for experimental formation and that we propose as candidates for ices XVIII through LI. Among them are, in particular, the ice counterparts of the DDR, SGT and NON zeolites, which were previously suggested as promising candidates for clathrate hydrates by Tribello et al.The success of the GCH construction in discovering the known ice phases and clathrate hydrates entirely a priori highlights that, although kinetic factors play an important role in determining which ice phases are formed in practice, structural and simple energetic considerations can provide a great deal of physical insight. More importantly, it demonstrates that the GCH approach does not simply discern structurally diverse configurations, but very effectively selects configurations that can be formed in experiment. It thereby provides strong support for the 34 proposed, new, structurally diverse candidates for ices XVIII–LI. This should spur experimental efforts to ratify our predictions.63, umbrella sampling64, forward flux sampling27 or enhanced sampling metadynamics approaches65, which have already proven successful in simulating the nucleation of ice32. The relation of the KPCA descriptors in the GCH construction to conventional quantities, such as density, vibrational spectra and concentrations of different types of guest molecules, promises to provide more direct guidance in identifying experimental formation pathways. However, this goes beyond the scope of this study.At this stage, the candidates are embedded in a human-readable sketch map of configuration space mainly as an aide in developing an intuitive understanding of the relation of the proposed candidates to the known ice phases. However, highlighting the phase transitions between the known ice phases suggests that proximity on the sketch map is a good indicator for the existence of a viable transition pathway. In conjunction with the GCH construction, the sketch map therefore provides a tractably small and yet structurally diverse set of synthesisable candidate structures and a means of identifying end points and suitable reaction coordinates for further investigation of formation pathways, for example, using transition state samplingIn addition, the approach demonstrated in this work sheds light on the energetics of proton-order/disorder and stacking-disorder, as well as H-bonding and planar defects, and also provides a glimpse of the preferred (quasi-) two-dimensional forms of ice.In its current state, the biggest limitation of our structure search is the preselection cutoff on system size. Relaxing this cutoff will drive the structure search towards completeness, which is the obvious next step. More generally, the connection between structural patterns and configurational energy exposed by the sketch-map dimensionality reduction suggests an expedient recipe for even more extensive database-driven searches. These need not be limited to crystalline water ice but could range from other tetrahedrally coordinated systems, such as silica or the carbon allotropes, to liquid, disordered and glassy systems.49 as parametrised by Raymand et al.66 and implemented in the Gulp package67 until the energies and forces were converged to within 10−4 eV and 10−3 eV/Å, respectively. This choice is motivated and justified in Supplementary Note Ice structures were initially geometry optimised using ReaxFF force fields47 as implemented in the Castep package68. The choice of density functional is discussed in detail in Supplementary Note k-point grids of maximum spacing of 2π × 0.07 Å−1, and on-the-fly generated ultrasoft pseudopotentials. The structures in the second section of the Results were further refined using PBE-DFT calculations with norm-conserving pseudo-potentials and, first, a plane-wave energy cutoff of 490 eV and Monkhorst-Pack k-point grids of maximum spacing of 2π × 0.07 Å−1, then a cutoff of 800 eV and Monkhorst-Pack k-point grids of maximum spacing of 2π × 0.04 Å−1, and finally a cutoff of 1200 eV and Monkhorst-Pack k-point grids of maximum spacing of 2π × 0.04 Å−1. The resulting energy differences between frozen-phonon configurations, atomic positions and residual forces were converged to within 10−4 eV/H2O, 10−5 Å and 10−4 eV/Å, respectively  was described by mapping one-dimensional (1D) subspaces along the harmonic normal mode axes up to large amplitudes of four times the harmonic root-mean-square displacements, where anharmonicity is important. The 3N-dimensional BO surface was then reconstructed from the 1D subspaces. The 1D energy surfaces were fitted using cubic splines The anharmonic vibrational Schrödinger equation was solved expanding the vibrational wave function in terms of simple harmonic oscillator eigenstates. The inclusion of 25 states for each vibrational degree of freedom was found sufficient to obtain converged results.Harmonic vibrational modes and frequencies were calculated using a finite displacement method. Anharmonic vibrations are calculated using the vibrational self-consistent field approach described in ref. 25 to the oxygen sublattices.Duplicates were identified by applying the “crysim” tool from the AIRSS methodhttp://cosmo-epfl.github.io), with the following choice of hyperparameters controlling the description of atomic environments:To assess the structural similarity between the configurations in the database under study, we used a REMatch-SOAP kernel, as implemented in the glosim.py package order in the definition of structural similarity. The choice of cutoff radius was tuned to achieve a clear separation between the known phases of ice in the database.http://sketchmap.org) was then applied to the SOAP kernel measure of similarity for 400 farthest-point-sampled landmark structures following the procedure described in ref. 70 and using the following parameters: σ = 0.12, A = 2, B = 4, a = 2, and b = 2.The non-linear sketch-map dimensionality reduction scheme (The data that support the findings of this study are available at the following 10.24435/materialscloud:2018.0010/v1.Supplementary InformationSupplementary Data 1Description of Additional Supplementary Files


Secondary phosphorylation develops in myocytes expressing phospho-mimetic cardiac troponin I (cTnI) but it is not known whether multiple substitutions (e.g. cTnISDTD and cTnIS4D) cause preferential phosphorylation of the remaining endogenous or the phospho-mimetic cTnI in intact myocytes. Western analysis was performed to determine whether the FLAG/total cTnI ratios are similar for phosphorylated versus total cTnI in myocytes expressing phospho-mimetic cTnI with Asp(D) substitutions at S43/45 plus S23/24 (cTnIS4D) or T144 (cTnISDTD). Representative Western analysis of phosphorylated S23/24 (p-S23/24) and S150 (p-S150) are presented along with re-probes using an antibody which detects all cTnI (MAB1691 Ab). The level of p-S150 also is compared to results obtained using single S43D and/or S45D phospho-mimetic substitutions. These results are discussed in more detail in Lang et al. [1] Specifications TableValue of the data•The distribution of p-S23/24 and p-S150 between FLAG and endogenous cTnI is compared to the overall cTnI distribution in myocytes expressing FLAG-tagged or non-tagged cTnI with phospho-mimetic substitutions  and thus, the distribution of p-S23/24 is not quantitatively analyzed for cTnIS4D.•+FLAG; panel C and Secondary p-S150 in myocytes expressing cTnIS4D is compared to myocytes expressing cTnI, cTnISDTD, S43D and/or S45D (1Western blots are presented which detect secondary phosphorylation at S23/24 and S150 in myocytes expressing cTnI with phospho-mimetic substitutions at PKC-targeted residues. The S23/24 phosphorylation is quantitated in the panel B of the 22.1GATGATGCCAACTACCGAGCCTATG-3′ (sense) and 5′-ATAGGCTCGGTAGTTGGCATCATCGCGACGTCGGACAGGAGCAG-3′ (anti-sense), and for cTnIT144D were 5′-GTGGCAAGTTTAAGCGGCCAGATCTCCGAAGAGTGAGAATC-3′ (sense) and 5′-GATTCTCACTCTTCGGAGATCTGGCCGCTTAAACTTGCCAC-3′ (anti-sense). Virus containing wild type cTnI (±FLAG), cTnI-S43D, -S45D (+FLAG), or -S43/45D (± FLAG) were prepared as described earlier The cTnISDTD and cTnIS4D were produced by sequentially replacing cTnI-S43/45 followed by -T144 or -S23/24, respectively with negatively charged D in full-length, wild type cTnI cDNA (rat) via site-directed mutagenesis , penicillin (50 U/ml) and streptomycin , and plated on laminin-coated coverslips at 37 °C for 2 h, as described earlier Animal work was carried out according to handling protocols and procedures developed by the University Committee for the Use and Care of Animals (UCUCA) at the University of Michigan. Isolated Ca2.3Prior to Western detection, cells were scraped into ice-cold sample buffer, and proteins were separated on 12% SDS polyacrylamide gels, electrophoretically transferred to PVDF membranes, and probed with primary and secondary antibody (Ab) pairs


Submarine glacial landforms in fjords are imprints of the dynamic behaviour of marine-terminating glaciers and are informative about their most recent retreat phase. Here we use detailed multibeam bathymetry to map glacial landforms in Petermann Fjord and Nares Strait, northwestern Greenland. A large grounding-zone wedge (GZW) demonstrates that Petermann Glacier stabilised at the fjord mouth for a considerable time, likely buttressed by an ice shelf. This stability was followed by successive backstepping of the ice margin down the GZW’s retrograde backslope forming small retreat ridges to 680 m current depth . Iceberg ploughmarks occurring somewhat deeper show that thick, grounded ice persisted to these water depths before final breakup occurred. The palaeodepth limit of the recessional moraines is consistent with final collapse driven by marine ice cliff instability (MICI) with retreat to the next stable position located underneath the present Petermann ice tongue, where the seafloor is unmapped. Submarine glacial landforms are used to reconstruct the Holocene retreat dynamics and stability of Petermann Glacier in northwest Greenland. Here, a large grounding-zone wedge at the mouth of Petermann fjord indicates a period of glacier stability, with final retreat likely driven by marine ice cliff instability. It drains the northwestern sector of the Greenland Ice Sheet into Nares Strait losing mass primarily through submarine melting3, but also via episodic calving events from a floating ice shelf, hereafter referred to as an ice tongue, extending in front of the grounding line. Two major calving events in 2010 and 2012 shortened the ice tongue by ~35 km, reducing its extent by nearly 40%6  and salty (S > 34.6) subsurface water of Atlantic origin has been observed to enter the Petermann Fjord from the north via Robeson Channel and Hall Basin after flowing through the Arctic Ocean5. This warmer water has contributed to thinning of the ice tongue from below making it more prone to calving5.Petermann Glacier is one of Greenland’s larger and faster flowing marine-terminating outlet glaciers Fig. 1. It dra8 show its margin close to the 2010 location prior to the major calving event  Oden, targeted Petermann Fjord to investigate the marine cryosphere, oceanography and geology in the fjord and the adjacent Nares Strait region . Here we analyse the submarine glacial landform record utilising the geophysical mapping data gathered during the Petermann 2015 Expedition. We identify a suite of glacial landforms providing information on the behaviour of Petermann Glacier that highlights the critical role of geology for the development of stability points in the seafloor landscape, where the ice retreat paused during deglaciation.During late summer 2015, the The mapping shows that Petermann Fjord has an overdeepened central thalweg with a maximum water depth of 1158 m ~10 km seaward from where the ice tongue margin was located in August 2015 Fig. . Peterma17, forming remarkably steep (some with slopes >70°) walls both above and below sea level. Seaward of the ice tongue margin, two outlet glaciers that drain an extension of the Greenland Ice Sheet debouch into the eastern side of Petermann Fjord.The eastern side of Petermann Fjord consists of exposed carbonate and other sedimentary rocks of Palaeozoic ageSkidbladner Glacier since we mapped its grounded margin and related glacial landforms with our survey vessel RV Skidbladner. Huge blocks, some >100 m wide and >20 m high, probably originating from mass wasting of the steep fjord walls , used as an indicator of fast-flowing, grounded ices20 Fig. . Among ts20 Fig. . We clasSets of streamlined landforms, dominantly crag-and-tails, also exist outside of the sill in Hall Basin Figs.  and 3a–dDirectional analysis of all mapped streamlined landforms shows that within Petermann Fjord, i.e., inside the sill, the ice stream of Petermann Glacier followed the shape of the fjord (towards ∼332°) and flowed through a 4-km-wide bottleneck before crossing the sill. In Hall Basin, the landforms show that ice flow progressively turned towards the northeast and eventually to a northerly direction from about 81°20′N Fig. . The ice22, from persistent sediment accumulation during halts in ice retreat23  is that, if present, any ice tongue that buttressed Petermann Glacier during its halt at the fjord mouth broke up prior to its retreat from the sill. The pattern of the ridges suggests a regular backstepping of the glacier margin, which makes us suggest that there was no ice tongue in place when they were formed.The surface of the GZW is characterised by small sinuous ridges with only a few metres of bathymetric expression Figs.  and 4a. Larger ridges with lengths 1–1.5 km and heights 5–10 m are mapped in Kennedy Channel transverse to the ice-flow direction spaced by about 500 to >2500 m Figs.  and 3c. The eastern part of Hall Basin displays prominent channels directly north of the GZW Fig. . Here, c27, an interpretation we adopt. Moats located on the ice-proximal side of MSGLs or crag-and-tails are commonly prescribed a formation from erosion of subglacial meltwater28, implying an abundance of subglacial meltwater in the eastern Hall Basin north of the GZW , though it is possible that some glacial landforms , may have been built over long time periods, even from multiple glaciations.The glacial landform record preserved on the seafloor will, under normal circumstances, be biased towards the final ice-retreat phase and breakup as earlier events are often erased by later events36. During the deglaciation, an ice stream confined to Nares Strait flowed north-eastward off Washington Land and Hall Land on the Greenland side36. Our mapping indicates that an ice stream debouched from Petermann Fjord and turned eastward to merge with an ice stream flowing towards the northeast in Nares Strait , except that we do not find morphological evidence of ice turning west out of Petermann Fjord’s southern side as suggested by England36  though enough englacial material could prevent the ice from floating.Near-circular sediment mounds with central depressions do exist in the heavily glaciated landscape of North Dakota, USA44. However, we suggest a mechanism where sediment accumulates on stagnant ice in depressions, much like that commonly proposed for kames. The accumulated sediment must eventually make its way through the ice by meltwater-aided transport, likely through systems of moulins or crevasses, to be deposited as a confined, high-relief mound on the seafloor once the stagnant ice breaks up and disintegrates. The horseshoe shape of some of the mounds remains an enigma. However, we speculate that it may originate from meltwater that breaches the edge of the deposited sediment mound once the central depression is formed, and flows towards the deeper side of the seafloor.Given the challenges of calling upon terrestrial analogues formed in stagnant ice for the formation of near-circular mounds in a marine environment, we find that we cannot readily adopt any previously published formation mechanism for similarly looking mounds, e.g., kames36 , West Antarctica49. We propose that Petermann Ice Stream was bounded by an ice shelf when it was pinned at the fjord mouth sill, with the ice shelf providing additional stability in the form of frontal buttressing50. Given evidence that Robeson Channel experienced reduced sea-ice cover from 9000 to 6000 cal. years BP46 it is likely that the ice shelf did not extend that far out of the fjord (>30 km). Relatively warm Atlantic water  was present in Nares Strait by 9000 cal. years BP46 and may have promoted ice shelf thinning to the point of ice shelf breakup, thus removing frontal buttressing and promoting retreat of Petermann Ice Stream into the fjord as a tidewater glacier margin.Current understanding of GZW formation implies the presence of a floating ice shelf beyond the grounding line, allowing the ice shelf to stabilise as the wedge grows vertically despite concurrent ice-sheet thinning36. The ice-proximal (landward) side of the GZW has a retrograding slope between about 2° and 4°, implying that the ice stream margin flowed upslope during this retreat phase when the transverse morainic ridges were formed  ice-stream margin Figs.  and 7 pemed Fig. , presumamed Fig. . Future 52 suggested that marine ice streams flowing on retrograde slopes are inherently unstable, specifically when lacking a buttressing ice tongue or shelf. If the vertical ice cliff at the margin rises more than about 100 m above sea level, the margin may begin fracturing and retreating rapidly, via massive calving, in a phenomena referred to as marine ice cliff instability (MICI)53. Assuming a density of ocean of water of 1026 kg m−3 and of ice 917 kg m−3, the draft of a floating ice mass is ∼0.89 × total ice thickness. This says that an ice cliff rising 100 m above sea level may form in the marine environment when the ice is grounded at water depths ∼809 m. Thus, grounding zones in more than 800 m water depth are considered to be particularly sensitive to MICI if there are no buttressing ice tongues or shelves in place53. Iceberg ploughmarks mapped in Pine Island Bay, West Antarctica, indicate that, during the last deglaciation, MICI was likely responsible for a rapid and sustained retreat of the Pine Island Glacier beginning around 12,300 cal. years BP14. The present depth of the Petermann Fjord (>1000 m), the retrograde landward slope of the GZW, and the location of iceberg plough ridges confirming calving all the way to the ice-stream base, lead us to suggest that MICI may have caused the Petermann Ice Stream to retreat rapidly from the fjord mouth sometime after 7600 cal. years BP. For ∼1100 years before the rapid retreat, the ice stream was stable at the fjord mouth facilitated by frontal buttressing by an ice shelf, lateral pinning points and a significant narrowing of the ice-stream trough. The palaeodepth of the GZW and Petermann Fjord can be derived for a given time if the isostatic depression of the area is known. At 7000 cal. years BP, the isostatic depression has been estimated to ~70 m56, which implies that the point where MICI would occur is merely 50 m deeper than the deepest mapped morainic ridge on the retrograde landward slope of the GZW.WeertmanTaken together, the subglacial landform record in Petermann Fjord and adjacent Hall Basin reveals the importance of geometric controls from the landscape, bedrock geology and of glaciological processes during the Holocene retreat of the Petermann Glacier to its present position, where the grounding line is located ~90 km from the major GZW at the fjord entrance. The landform record shows that the fjord geometry likely played a role in determining where the Petermann Glacier was stable for perhaps as long as 1100 years, while glaciological processes in the form of MICI may have put a drastic end to this stable period, but only after the buttressing ice tongue broke up. Crucially, it was the interplay of all these factors that governed the final retreat of Petermann Glacier. Therefore, this study highlights the need to improve our knowledge of the whole glacier system, in particular high-resolution bathymetry in front of marine-terminating glaciers or beneath their ice tongues, before they can be accurately portrayed in ice-sheet models.Petermann 2015 Expedition started and ended in Thule, Greenland on July 30 and September 3, respectively. Systematic multibeam bathymetric mapping and sub-bottom profiling was carried out with IB Oden in Petermann Fjord and the adjacent areas of Nares Strait, including Hall Basin, Kennedy Channel and Robeson Channel  multibeam echosounder and an SBP 120  chirp sonar sub-bottom profiler installed. Navigation was provided by a Seatex Seapath 330 GPS/GLONASS navigation system integrating heading and attitude, and an Seapath MRU5 inertial motion sensor providing pitch, roll, yaw and heave. The accuracy of the positions from the combined GPS/GLONASS solution varied with available satellites, but was commonly reported to be within a 5 m sphere, or better, both vertically and horizontally. Regular CTD  stations and XBTs (Expendable Bathy Thermograph) were used to provide the multibeam system on IB Oden with regular sound speed profiles of the water column.The nel Fig. . In tota2 of the seafloor was mapped from the 6.4-m-long RV Skidbladner using a Kongsberg EM 2040  multibeam system bow mounted on a pole that can be hoisted up for transit. A Valeport sound velocity probe was used to acquire sound speed profiles. The navigation system of RV Skidbladner consists of a Seatex Seapath 330 + with RTK  capability. The motion sensor unit measuring roll, pitch, yaw and heave was a Seatex MRU5 + mounted in a subsea bottle on the EM 2040 transducer casing. During mapping operations, a Hemisphere A325 Smart antenna connected to a SATELLINE-EASy Pro 35 W 403–473 MHz radio was stationed on nearby topographic high points to provide RTK corrections sent using the RTCM 3.0 protocol. The accuracy of the GPS/GLONASS positions after RTK corrections was generally reported by the Seapath 330 + system to be within a 10 cm sphere, or better, both vertically and horizontally.In some near-shore areas, ∼30 kmOden and 3 × 3 m for data from RV Skidbladner. Seafloor morphology was interpreted in the 3D environment of Fledermaus and maps were subsequently produced using the Open Source GIS software QGIS (http://www.qgis.org). The chirp sub-bottom profiler data were acquired using the Kongberg TOPAS software and the profile displayed in this work was post-processed using tools provided by the Geological Survey of Canada (courtesy Bob Courtney).All multibeam data were acquired using Kongsberg Seafloor Information System (SIS) and processed using a combination of Caris and QPS Fledermaus processing software. The processed data were gridded to a horizontal resolution of 15 × 15 m for data from IB https://bolin.su.se/data/contributions.php?d=1609&p=MjAxOC0wNS0xOCAxNTowNDozOS4yNzg4NjMgMzk3ODknfy8.The multibeam bathymetry shown in this work is available for download from Supplementary InformationPeer Review File