This highly unusual behaviour constitutes an interesting pathway from a glass to a crystal where translational periodicity is restored but the rotational degrees of freedom remaining completely random.It has been known for decades that certain aqueous salt solutions of LiCl and LiBr readily form glasses when cooled to below ≈160 K. This fact has recently been exploited to produce a « salty » high-pressure ice form: When the glass is compressed at low temperatures to pressures higher than 4 GPa and subsequently warmed, it crystallizes into ice VII with the ionic species trapped inside the ice lattice. Here we report the extreme limit of salt incorporation into ice VII, using high pressure neutron diffraction and molecular dynamics simulations. We show that high-pressure crystallisation of aqueous solutions of LiCl∙RH They present the limit of salt concentration where a homogeneous glass can easily form without applying extreme cooling speeds. We used deuterated samples to avoid the very large incoherent scattering of hydrogen, a standard method applied in powder neutron diffraction, including in almost all previous structural work on ice234For this purpose, deuterated aqueous solutions with R = 5.6 of LiCl and LiBr (LiCl∙5.6Dentirely due to hydrogen (deuterium), i.e. it has no contribution from oxygen. Therefore, its absence is a clear and unambiguous proof of hydrogen being strongly displaced from its normal position along <x,x,x>, see 2>1/2 ≈ 0.6 Å, which is indeed large. For comparison, the displacements in pure ice VII at 2.6 GPa and 295 K are <x2>1/2 ≈ 0.15–0.20 Å122>1/2 ≈ 0.30 Å. Fits based on this model correspond to the lines through the data shown in The most surprising feature of these patterns is, however, the total absence of the 111 reflection which should occur at ≈2.0 Å , the volume expansion was observed to be 43% smaller (i.e. only 8%)h, despite its considerably larger density. The destruction of the interpenetrating sublattices by the presence of ionic species must necessarily lead to an increase of volume which is expected to be highly sensitive to the ion concentration.The second remarkable feature is the strongly expanded lattice parameter: at the pressures measured by the lead marker the volume expansion relative to pure ice VII is 14% and 18% for the LiCl- and LiBr-system, respectively. These values scale The structural properties of these highly salt-loaded ice phases were then investigated by molecular dynamics simulations which give access to physical properties which cannot be extracted by diffraction measurements alone. For this purpose simulations were carried out with 15 × 15 × 15 ice VII supercells corresponding to 19227 atoms. Note that the structural results are insensitive to the choice of the isotope (hydrogen or deuterium) since the potentials used in the simulations are the same. Details of the calculations are described in the Methods section.2O compared to pure ice VII, viewed along the cubic [100] direction. Results for the LiBr-system are qualitatively similar and are not shown here. It is seen that inclusion of Li+ and Cl− leads to molecular displacements of several tenths of angstroms, but on average the H2O molecules are still at the bcc sites of ice VII. It is also seen that the Li+-ions prefer to locate themselves in the octahedral interstitials, whereas the much larger Cl-ions are on average closer to sites where H2O is expected. Radial distribution functions were calculated for the two systems and can be found as The most important detail in this context concerns however the spatial distribution of dipole moments. For this purpose the orientation of each water molecule was extracted from the last part of the 12 ns trajectory (1 ns) and employed to build the spherical probability maps shown in 2O and LiBr∙5.6H2O, however, the orientational probability is almost exactly isotropic. Close inspection of the dipole distribution shows a small residual preference for <100> directions in the case of LiCl∙5.6H2O which is hardly discernible in As shown in the The simulations therefore come to the same conclusion as the diffraction data: The ice VII-like structures emerging from crystallisation of highly concentrated LiCl and LiBr-solutions under pressure have completely disordered molecular orientations which means that a large fraction of hydrogen-bonds are broken and that the ice-rules are violated. This distinguishes it sharply from the orientational disorder present in most of the (pure) ice phases where the ice-rules are conserved. The crystallisation observed here could be classified as ‘fractional’ since it concerns mainly the three translational degrees of freedoms whereas the three rotational freedoms remain uniformly random. Breaking of hydrogen bond networks is a phenomenon which has been observed in liquid salt solutions at ambient pressure102O and LiBr∙5.6H2O are typically 4–5 times longer compared to pure ice VII at the same pressure. Therefore, although the ions severely perturb the H-bond network, their presence seems to hamper their orientational dynamics rather than to promote it.In this context the question arises if the inclusion of ionic species into ice VII promotes “plasticity”, i.e. the formation of an ice VII phase with freely rotating molecules, as predicted by several simulations1617not quenchable at 90 K, and probably even not at any temperature below, contrary to all forms of pure ice. 2O and LiBr∙5.6D2O return to an amorphous solid, a disordered system which is significantly different from the original glass. A comparison with published structure factors from ref. A further interesting aspect concerns the behaviour under de-compression at low temperatures: The two salty ice-forms are found to be 2 polymorphs incorporating small ions such as lithium are well known and can be formed at ambient pressureIn conclusion, we have presented neutron diffraction and molecular simulation results on highly concentrated LiCl- and LiBr aqueous solutions which were crystallized under high pressure. They indicate that the ionic species can be trapped inside a strongly expanded ice VII – like structure with random electric dipole orientation. These structures cannot be quenched (‘recovered’) to ambient pressure and low temperatures but transform back to a glass with a structure different to the initial one. The trapping of ionic species by crystallisation from a glass (devitrification) might be found in other network forming glasses, for example in silica. In fact, “stuffed” SiO2O) from Eurisotop, France. The solutions were filtered to remove particles larger than 3 μm to minimize the possibility of crystallisation during cooling, and loaded into TiZr gaskets together with a small piece of lead (10 mg). After sealing the gasket with a load of 10 kN onto the anvils the cell was cooled at a speed of 10 K/min to 80 K which transformed the liquids into pure glasses. The samples were then compressed at 80–110 K in steps up to 4–5 GPa and then heated at an average speed of typically 1 K/min up to the temperature where crystallisation occurred. After crystallisation, the samples were stable over at least the timescale of the diffraction experiments, i.e. 1–3 hours. Samples at lower pressures (below ca. 3.3 GPa for the LiCl compound and ca. 4.3 GPa for the LiBr system) crystallized into solids with unknown structure.Aqueous solutions of LiCl and LiBr with R = 5.6 were prepared from the salts purchased from Aldrich, and 99.8% enriched heavy water ∙5.63H2O. Each structure was initially relaxed at T = 10 K and constant volume for 30 ps (with a timestep of 0.1 fs). Then, the system was slowly heated up to T = 300 K employing a time constant of 50 ps for the thermostat (with a timestep of 1 fs) and equilibrated for 12 ns. The dipole moment orientation of each water molecule was extracted from the last part of the trajectory (1 ns) and employed to build the spherical probability maps. The orientational correlation time of water dipoles was estimated performing, for each system, additional 50 ns-long simulations using deuterium instead of hydrogen masses. In a second set of simulations a smaller 4 × 5 × 7 cell was used to generate 100 configurations which served to calculate neutron diffraction patterns and verify the consistency with experiment. Note that constraining the system at the experimental volume corresponds to a pressure of about 10 GPa: this is consistent with the domain of stability of ice VII with the interatomic potential employedSalty ices of stoichiometry Li(Cl/Br)∙5.6HHow to cite this article: Klotz, S. et al. Ice VII from aqueous salt solutions: From a glass to a crystal with broken H-bonds. Sci. Rep.6, 32040; doi: 10.1038/srep32040 (2016).


The guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII. However, because the monomers are thought to be arranged in parallel, dimerization may stabilize membrane anchoring via the paired myristoyl moieties. Plasma membrane anchoring has been shown to be essential for the activation of one of its key substrates in intestinal tissue, the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel -dependent protein kinase II (cGKII)1– channel .PRKG2) is abundantly expressed in intestinal epithelium and to a more limited extent in various other tissues of both epithelial and nonepithelial origin. In intestinal epithelium, cGKII relays signaling through a membrane-associated, cGMP-producing enzyme, guanylyl cyclase C (GCC). The catalytic activity of this receptor-enzyme is triggered by two locally produced ligands, the peptides guanylin and uroguanylin, but also by the heat-stable toxin (STa) produced by enterotoxigenic Escherichia coli (ETEC) strains. The (uro)guanylin/GCC/cGMP signaling axis stimulates intestinal salt and water secretion through coordinate activation of CFTR-dependent chloride and bicarbonate secretion and inhibition of sodium uptake through sodium-proton exchanger isotype 3 (NHE3) (–The gene encoding cGKII (3 (NHE3) . Dysregu3 (NHE3) –10. Inde (NHE3) –.In addition to its role in intestinal fluid homeostasis, one of the principal physiological roles of cGKII appears to be the regulation of the cell cycle and cellular differentiation in specific tissues. Thus, the most prominent phenotype of cGKII deficiency in rodents (and cattle) is dwarfism, which is caused by a defect in endochondral ossification, resulting from an impaired hypertrophic differentiation of chondrocytes –15.–Prkg2-null mice display subtle learning, emotional, and behavioral deficiencies -cGMP isomers (carrying a sulfur substitution on the phosphate group) and inhibitor peptides, respectively, is for the most part limited to in vitro studies , several phosphodiesterases (PDEs), and cyclic nucleotide–gated cation channels. Therefore, to establish the cGKI or cGKII dependence of cGMP-dependent signaling events, blockers of the cGMP-dependent protein kinases, which either antagonize binding of cGMP or peptide substrate, have been developed , 20. How studies . The com studies . Moreoveex vivo). It has been shown previously that the short-circuit current (Isc) response of mouse ileum to 8-pCPT-cGMP fully depends on cGKII and the CFTR anion channel . To evaluate the selectivity of these two compounds, we also assessed their activity toward cGKI and PKA, i.e. protein kinases that are phylogenetically and structurally closely related to cGKII. This showed that the potency of inhibition of cGKI and PKA was markedly lower than of cGKII (A).Compounds AP-C5 and AP-C6 concentration-dependently inhibited human cGKII activity r levels A. To evaof cGKII A.B). These results suggest that these compounds compete with substrate for binding to the catalytic domain of cGKII. Interestingly, the reported mK(ATP) values of cGKI and PKA are approximately 10-fold lower than that of cGKII -stimulated, PKA-mediated Ser-239 phosphorylation (The vasodilator-stimulated phosphoprotein (VASP), through its association with actin filaments, plays an important role in cytoskeletal dynamics . Like cGrylation .50 values for cGKII inhibition at cellular ATP levels (see inset). This may indicate that, at high levels, cGKII activity does not limit anion secretion because saturating levels of substrate phosphorylation are attained. Furthermore, the activity of these substrates may not limit the rate of anion secretion. This holds true for intestinal CFTR as it has been shown that ≤50% of the cellular pool is required for a full-anion secretory response . In fact, we found that AP-C5 potentiated the anion secretory response to forskolin of ileum (B). In view of this potentiation, we speculated that this compound, and its close analog AP-C6, might elevate cAMP levels, conceivably through inhibition of PDE activity. Consistent with this supposition, we found that in T84 cells these compounds moderately enhanced cAMP levels but only in the presence of forskolin . This strongly suggests that these compounds, at high concentrations, potentiate cAMP signaling through inhibition of PDE activity.In mouse ileum, we found that these compounds, at a concentration ≤50 μmol/liter, did not inhibit the anion secretory response elicited by the cAMP/PKA agonist forskolin A. In facof ileum B. In vieIn vitro, these imidazole-aminopyrimidines inhibited human cGKII at submicromolar levels. In contrast, these compounds showed little activity toward phylogenetically related ACG-type serine/threonine kinases (cGKI and PKA). To our knowledge, the presently developed compounds are the first to display selectivity in this respect and activity in native  tissue.We report the discovery of small-molecule inhibitors of cGKII that block protein phosphorylation and the ensuing ion secretory responses in the small intestinal epithelium elicited by cGMP-linked signal transduction pathways. reversed orientation has previously been described for blockers of PDK1 and PIM1  domain of cGKII. Aminopyrimidyl motifs are relatively common in this type of protein kinase inhibitors as these readily form hydrogen bonds with residues in the hinge region of the catalytic domain. However, molecular modeling indicated that the binding mode of the present set of compounds is more unusual as the aminopyrimidyl ring is projected to interact with residues in the catalytic loop. A similar, and PIM1 , 31. ThiIn mouse small intestine, these compounds effectively blocked 8-pCPT-cGMP–dependent VASP phosphorylation and anion secretion but only partially inhibited the STa-provoked anion secretory response. These data corroborate previous studies on cGKII-deficient mice showing that the response to STa is only approximately 80% dependent on cGKII and that, in addition, STa signals through cGKII-independent routes. Chiefly, cGMP is thought to also enhance PKA signaling through competitive inhibition of the cAMP-hydrolyzing PDE type 3 (PDE3), but direct cross-activation of PKA may also occur , 32. ImpApart from by a redundancy in prosecretory cGMP-dependent signaling pathways, the moderate efficacy of these cGKII inhibitors toward the STa-dependent Isc response of ileum may be compounded by their weak inhibition of PDEs, which may derive from their structural resemblance to previously identified PDE blockers . Thus, ai.e. an antiabsorptive action)  . Like cG action) . ConsequIn addition to its established role in controlling the intestinal fluid balance and the cell cycle, evidence accrued over the last decade points toward various other functions of cGKII in physiology. These often intriguing new prospects, such as cGKII's potential role in (feeding) behavior and psychosocial disorders, warrant further research , 37, 38.Mice (FVB) were maintained in an environmentally controlled facility at the Erasmus MC, Rotterdam, The Netherlands. All experiments were performed on animals 12–26 weeks of age and were approved of by the Ethical Committee for Animal Experiments of the Erasmus MC.2 (5 mmol/liter), BSA (25 mg/liter), and peptide substrate 2A3   or cGKII , enzyme (100 μg/liter) was added to assay solution containing Na-cGMP (50 μmol/liter), DTT (5 mmol/liter), Tris/HCl, pH 7.4 (20 mmol/liter), MgClg/liter) . For ass4WB5) as a structural template for modeling of ligand binding (aK ≤ 5.3). Induced fit docking of the ligand in the ATP-binding site, defined as all amino acids and those bonded to them within a 4.5-Å radius of the ligand, was performed using MOE.Because the X-ray structure of the cGKII catalytic domain is unresolved, we used a high-resolution (1.6-Å) X-ray structure of the PKA catalytic domain , supplemented with glucose  in 95% O2, 5% CO2, pH 7.3, at 37 °C. CFTR-dependent anion secretion was stimulated by addition of the adenylyl cyclase activator forskolin (Sigma-Aldrich), STa , or the membrane-permeable cGMP analog 8-pCPT-cGMP (Sigma-Aldrich). Data shown represent Isc measurements, obtained by clamping of the transepithelial potential difference at 0 mV.Collection of murine intestinal tissue and Ussing chamber assays were performed as described elsewhere . Tissue 2, pH 7.3, at 37 °C. After 30 min, medium was aspirated, and organoids were collected in ice-cold PBS supplemented with NaF (1 mmol/liter) and Na3VO4 (1 mmol/liter). After centrifugation , pelleted organoids were lysed in NaCl (150 mmol/liter), Tris/HCl, pH 7.6 (25 mmol/liter), Triton X-100 (1%), sodium deoxycholate (1%), SDS (0.1%), NaF (5 mmol/liter), Na3VO4 (3 mmol/liter) supplemented with a protease inhibitor mixture (Roche Applied Science). Lysates were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose membrane. Ser-239–phosphorylated VASP was detected by Western blot analysis . A fluorescent dye–labeled secondary antibody and the Odyssey IR imaging system  were used for quantitation. Detection of villin  served as a reference.Organoids derived from mouse ileum were cultured as described elsewhere . Organoi2 at 37 °C. Reactions were quenched by adding ice-cold ethanol (70%), and cAMP levels were assayed as described elsewhere . Derived ECP levels . VASP phM. J. B., H. R. d. J., and J. H. D. M. conceptualization; M. J. B. and G. T. data curation; M. J. B. and G. T. formal analysis; M. J. B., G. T., N. D. N., K. F. M., and H. R. d. J. investigation; M. J. B. and G. T. writing-original draft; A. H., J. A. S., and J. H. D. M. supervision; A. H., J. A. S., and J. H. D. M. funding acquisition; A. H. project administration; K. S., J.-P. B., and L. V. D. resources; K. S., J.-P. B., L. V. D., H. R. d. J., J. A. S., and J. H. D. M. writing-review and editing.


Animals often display a marked tendency to return to previously visited locations that contain important resources, such as water, food, or developing brood that must be provisioned. A considerable body of work has demonstrated that this tendency is strongly expressed in ants, which exhibit fidelity to particular sites both inside and outside the nest. However, thus far many studies of this phenomena have taken the approach of reducing an animal's trajectory to a summary statistic, such as the area it covers.Temnothorax albipennis, we demonstrate that this reductive approach suffers from an unacceptably high rate of false negatives.Using both simulations of biased random walks, and empirical trajectories from individual rock ants, To overcome this, we describe a site‐centric approach which, in combination with a spatially‐explicit null model, allows the identification of the important sites towards which individuals exhibit statistically significant biases.Using the ant trajectories, we illustrate how the site‐centric approach can be combined with social network analysis tools to detect groups of individuals whose members display similar space‐use patterns.We also address the mechanistic origin of individual site fidelity; by examining the sequence of visits to each site, we detect a statistical signature associated with a self‐attracting walk – a non‐Markovian movement model that has been suggested as a possible mechanism for generating individual site fidelity. The phenomenon of ‘recurrence’, in which the movement of an individual is biased towards a set of previously visited locations, is widespread in the animal kingdom  brains, rock ants exhibit impressive capacities for both internal , constructed by sandwiching a cardboard gasket between two glass microscope slides  the developmental stage and {ded Figs . For fur and the next, , that is, εt  between successive line‐segments  null model that uses constrained randomization to produce synthetic trajectories that exhibit no spatial biases but also preserve these distributions Munger . The RW , takes the synthetic ‘ant’ beyond the internal boundary of the nest, then the sample is discarded and another drawn. Third, because the trajectories contained some gaps, the exact temporal gap structure of each ant trajectory was imposed upon the corresponding synthetic versions. The end result of the RW procedure is a synthetic trajectory that exhibits realistic movement patterns, but which lacks any localized spatial biases present within the original −vkstop(t). However, as the first return times are defined by the intervals between successive visits, and as the observation period is finite, for each site s visited by ant i, the return time following the last visit, nis, is unknown, or more properly, it is ‘censored’. Censoring complicates the estimation of an average first return time, for example, ignoring the censored returns and instead taking the mean across the uncensored return times induces a downward bias in the mean. Therefore, to obtain an unbiased estimate of the typical first return time for a given site s, we calculate the restricted mean , Dis¯(z), and RTis¯(z). Finally, to identify the important sites for individual i, for each of the sites that it visits we test the null hypothesis that the observed number of visits nis, mean dwell time, Dis¯, or mean first return time, RTis¯, are statistically indistinguishable from the corresponding distributions of expected values produced by the RW null model. As there are no particular a priori reasons to predict whether ants should be biased towards or away from particular sites, for each site s visited by ant i we perform a two‐tailed permutation test using a significance threshold of α < 0·05. So in the case of the number of visits to a given site, nis, the null hypothesis is rejected if the observed nis is lower than the leftmost 0·025 quantile of the expected distribution, on the grounds that the ant made significantly fewer visits to s than expected. Similarly, the null hypothesis is also rejected if the observed nis is greater than the rightmost 0·975 quantile of the expected distribution, because the ant visited s significantly more than expected. However, as each ant i visits Ni different sites, there are Ni significance tests for each ant i, hence it is likely some sites may achieve a statistically significant P‐value just by chance. Therefore, to control for the effect of multiple comparisons, we apply the Benjamini–Hochberg false discovery rate procedure  to calculate a well‐established measure of spatial overlap, namely the volume of intersection  for R. We use this method because it allows the inclusion of censored time‐to‐event response variables, such as the unobserved return time that follows the last visit of individual i to site s, that is, RTvn→vn+1s, and also because the mixed‐effects extensions renders it robust to uncontrolled variation between individuals and colonies.We follow the general approach of Boyer, Crofoot & Walsh  and test & Walsh  pooled a & Walsh , with a  & Walsh . Survivavth visit, RTv→v+1s, and the predictor was the dwell time during the vth visit, Dvs. Furthermore, because the return time might have also been influenced by both the physiological or behavioural characteristics of the individual, or by other environmental features, we included three additional predictors in the model. These were, respectively, the reproductive caste of ant i (queen or worker), the community membership of i, as defined by the network partitioning, and the number of brood items  at site s during the vth visit of ant i.As in Boyer, Crofoot & Walsh , the resA, B, …, Y), (ii) each individual , and (iii) each site (198 levels). From these three random effects, we defined three candidate models. First, the maximal model that included all three random effects. The intermediate model retained colony and individual identity, but discarded the random effect with the greatest number of levels, namely, ‘site’. Finally, the minimal model retained the random effect with the fewest levels, namely colony identity. We used AIC to select among the three competing survival models; the model with the lowest AIC was the intermediate model, so we present and interpret the results from that model  each colony , whereas 152 ants visited a significantly smaller area than expected (site fidelity). All of the remaining 183 ants visited an area that was no larger or smaller than expected.Among 152 ants that the reductive approach classified as exhibiting fidelity, the site‐centric approach found 133 that visited at least one site significantly more than expected (site fidelity), whereas 18 visited at least one site less than expected (site avoidance). Therefore, by and large, the two approaches agreed over the identity of the ants that exhibit site fidelity. However, among the 183 ants that the reductive approach classified as exhibiting no spatial bias, the site‐centric approach found 57 that visited at least one site significantly more than expected (site fidelity), whereas 24 visited at least one site less than expected (site avoidance). Because 81 of the 183 ants that the reductive approach classed as exhibiting no spatial bias did in fact exhibit bias, we conclude that the site‐centric approach is considerably more sensitive.There is at least one additional reason to prefer the site‐centric approach over the reductive approach: because the reductive approach reduces the trajectory to a single statistic, such as the area covered, it can only assign an individual to one of three categories, namely, site fidelity , neutral , or ‘roaming’ (area larger than expectation). The site‐centric approach provides a richer classification scheme, as in addition to the above three categories, it can also identify individuals that are attracted to some sites whilst also avoiding other sites. Furthermore, by expressing the site visits statistics as standardized z‐scores, it can provide information about the magnitude and direction of the bias to particular sites Fig. .nis, Dis¯ and RTis¯, showed considerable variation in the extent to which ants exhibit fidelity to particular sites; the mean number of important sites per ant was 8·5 ± 0·30 for nis, 3·3 ± 0·17 for RTis¯, and 1·5 ± 0·1 for Dis¯. However, across the three metrics, there was an appreciable degree of consistency in the identity of the important sites . Therefore, the site‐centric approach revealed that despite outward appearances, queens were spatially isolated from workers.We now outline the results of the social network analysis of the spatial overlaps between ant pairs. One of the most obvious low‐level features of the spatial networks was the clear differences between the workers and the queens: even though queen movement was very biased towards the biological centre of the nest , made significantly more visits to each of the sites that they did visit , had a significantly longer site dwell time , and returned to previously‐visited sites significantly more rapidly  than ants in the ‘other’ community. So, nurse workers exhibited a much greater degree of spatial fidelity than the other workers.In the two community colonies, the movement patterns of the ants varied according to the identity of their community. In the 21 two‐community colonies, the ants in the ‘nurse’ community visited significantly fewer sites  candidate mechanisms, namely, chemical pheromones deposited onto the substrate, and internal place memory. As rock ants have evolved sophisticated strategies for chemical marking  contains information about the colony demographics, (ii) describes simulations that compare the reductive and site‐centric approaches to measuring site fidelity and (iii) shows the spatial networks for all 23 colonies.Click here for additional data file.


Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.Malaria is a life-threatening disease caused by different species of the protozoan parasite  Plasmodium parasites. Of these, P. vivax, P. malariae, P. ovale and P. falciparum cause malaria in humans with the latter being one of the most common and the most deadly. In recent years, P. knowlesi has also been reported to infect humans in certain forested areas of South-East AsiaPlasmodium parasites to most marketed antimalarial drugs34Malaria remains an important global health concern. According to the latest estimates, 214 million cases of malaria occurred globally in 2014 and approximately half million deaths, primarily children aged under 5 year, in AfricaP. falciparum membranes. Their content is unusually high representing 40–50% and 35–40% of the total phospholipids in P. falciparum, respectively68910P. falciparum by the Kennedy pathways from choline (Cho) or ethanolamine (Etn)via a common choline/ethanolamine phosphotransferase (CEPT). In addition, P. falciparum possesses an alternative route for the synthesis of PC from Etn that involves the methylation of PEtn by a phosphoethanolamine N-methyltransferase (PMT) to form PCho which subsequently form PC, thus connecting both Kennedy pathways6Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major and essential phospholipids in We have previously focused on the Cho phospholipid metabolism in cancer cells and tumors as a therapeutic target by inhibiting the human CK activity. In humans, CK is encoded by two genes, which express three isoforms CKα1, CKα2 and CKβ. An overexpression of the α-isoform of CK has been identified as an oncogene that mediates human cell transformation. As a consequence, CKα has been proposed and validated as a molecular target for the development of novel cancer therapeutic agents1416171819202121HsCKα1) and the high identity between the human CKα1 and the P. falciparum CK (PfCK) at the binding site  with ECceptible , in agrePfCK or PfEK might explain the resulting phenotype induced by these compounds. While we selected PfCK due to the previous demonstration that these compounds inhibited the human enzymePfEK was also taken into account because of the PE reduction-associated phenotype found in P. falciparum. At the ATP and Cho-binding sites, the similarities in sequence between both enzymes were high . Note that HsCKα1 was also kinetically evaluated to compare its kinetic parameters with the P. falciparum enzymes. As expected and in agreement with the literature1828HsCKα1 showed a high specific activity and affinity for both Cho and ATP, which were reflected by a high kcat and catalytic efficiency , PfCK is likely to be responsible for the phosphorylation of all the Cho and the vast majority of the Etn in the parasites Kennedy pathway.On the contrary, the ficiency . In factHsCKα1 with IC50s of 940 and 520 nM, respectively  and inhibition tends towards being competitive. For PfCK, BR23 and BR25 also showed mixed inhibition against Cho with Kis ~35 uM, but in this case alpha factors were lower than for the human enzyme implying less impact on substrate binding. In contrast, both inhibitors were found to be competitive inhibitors of PfCK with respect to Etn with Kis of ~4 uM . These values were also comparable to the obtained for the HsCKα1 , in agreement with the high similarities inferred at the protein sequence level for both enzymes pyridinium fragment to access the adjacent binding site  and S11.ith Y354 .As expected, the benzyl-4-(dimethylamino)pyridinium fragment of compounds BR23 and BR31 superposes fairly well with an atomic shift of <0.30 Å, supporting the high similarities between both compounds . In partPfCK showing very similar features as found for the human enzyme  for Cho in accordance with previous studiesPfCK, despite its lower specific activity, is also able to phosphorylate both Cho and Etn with similar Kms to the human enzyme. The PfEK enzyme has a very low catalytic efficiency (~60 times less that PfCK) to phosphorylate Etn and showed no ability to phosphorylate Cho at all. This clearly shows that PfCK is potentially responsible for the phosphorylation of all the Cho and likely the major source of PE in the parasites for the Kennedy pathway leading to PC and PE, respectively. The overall pool of PC and PE would be also dependant upon the enzymes’ expression levels and possibly cellular location. However, both are expressed at similar levels according to PlasmoDB, and are located in the cytosol, suggesting that these two latter factors would not be the rate-limiting step for the synthesis of PC and PE, the later being essential for GPI biosynthesis and maintaining the correct membrane composition, fluidity for numerous cellular processes and organelle function. Furthermore, it is obvious that the phosphoethanolamine N-methyltransferase acting upon PE is not the major source of PC as these levels remain relatively unchanged. Also the relative available concentration of Cho and Etn is crucial for the final effects of the inhibitors. In particular, intracellular Etn has been shown to be lowerThis study set out to chemically validate either or both BR23 and BR25 as lead compounds that can effectively kill  pathway . VariousHsCKα1 with respect to ATP and Cho, with Kis~4 uM, and Kis~100 nM respectively. The latter suggests that both compounds are competitive with respect to preventing the binding of the substrate (Cho/Etn). From the high sequence similarity between HsCKα1 and PfCK especially within the active site, it is not surprising that BR23 and BR25 also showed mixed inhibition against PfCK for Cho (Kis ~35 uM), but compared to the human enzyme having less impact on Cho binding. However, both inhibitors were found to be competitive inhibitors of PfCK with respect to Etn with Kis of ~4 uM. This implies that the absolute binding positions of the two alternative substrates (Cho and Etn) are different within the active site of PfCK. This is supported by the crystal structure of HsCKα1 with BR25, where the compound adopts an unusual binding mode that induces the aperture of an adjacent binding site located at the back of the Cho-binding site. However, BR23 and related analogues with a short and rigid linker consisting of a biphenyl group were computationally predicted and experimentally proven to adopt a typical binding mode within the Cho-binding site of HsCKα121The potential inhibitory effect of BR23 and BR25 on both kinases was examined in detail by enzyme kinetics. Both BR23 and BR25 showed mixed inhibition of PfCK, their respective binding modes were very similar to those found for the human enzyme where BR23 adopts the typical binding orientation and BR25 adopted the unusual conformation mentioned above. Collectively, this data clearly shows that both compounds behave differently depending upon the enzyme and substrate identity, thus causing a more potent inhibition of PfCK when Etn is a substrate. A better understanding and exploitation of this adjacent binding mode near the Cho binding site, may lead to greater potency and selectivity. While there is a Tyr (Y256) in the human enzyme, a Trp (W238) is present in the parasite enzyme  the compounds may accumulate within the parasite, hence reaching a higher local concentration; and (b) the accumulative effect of inhibiting PE biosynthesis even at only 10–20% over a period of 24 hours might cause a significant decrease in the pool of PE that is lethal to the parasite. The latter may be especially true during periods of rapid cell growth/division when the demand on PE is high as will be the flux of PE biosynthesis.Despite the fact that neither BR23 nor BR25 showed any inhibition towards PfCK in the presence of either Etn or Cho as substrate. This also highlights that both PfEK, which only phosphorylates Etn, and the possible decarboxylation of PS . Inhibition of GPI synthesis disrupts the anchoring of essential cell-surface proteins to the membrane (i.e. MSP-1), as PE is the PEtn donor that links the proteins via their C-terminus to the third mannose of the GPIPfCK is responsible for producing the vast majority of the PEtn used by the parasite to make PE; (2) the type of inhibition by BR23 and BR25 on PfCK are influenced differentially by the presence of the alternative substrates, Etn or Cho; (3) the direct action of BR23 and BR25 on the parasites is to inhibit the formation of PEtn directly reducing the de novo biosynthesis of PE via the Kennedy pathway, which is ultimately lethal to the parasite; and (4) BR23 and BR25 are lead compounds with potent and selective nanomolar antiplasmodium activity that have been chemically validated against PfCK.In summary we have shown the following conclusions: (1) PfCK have been previously reported20Details about cloning and purification of human CKα1 and PfEK, defined as pfek, was made synthetically and codon optimized by GenScript to be expressed in E. coli. pfek was amplified using the forward primer,The DNA sequence encoding amino acid residues 2–420 of CATATGGAATACCAACTGCGTGAAATTG-3′ containing a NdeI site , and the reverse primer5′-CGGAATTCGTCGACTCACAGTTTGCTGCGGAATTTCAC-3′ containing a SalI site  and a stop codon (underlined). Subsequently the PCR product was digested with NdeI and SalI and cloned into a modified pET15b vector containing a histidine tag and a PreScission protease (PP) cleavage site, resulting in the expression plasmid pET15bPP-pfek. The plasmid was verified by sequencing.5′-CGGAATTCpfek was transformed into BL21 DE3 (star) cells grown in 2XYT medium with 100 μg/ml of ampicillin. Cells were grown at 37 °C until reaching an OD of 0.6 at 600 nm, after which the expression of the protein was induced overnight by 1 mM IPTG at 18 °C and 180 rpm.pET15bPP-The cells were harvested by centrifugation at 3480 g for 30 min and suspended in buffer A , containing lysozyme (1 mg/ml)), DNAse (0.1 mg/ml) and a cocktail of protease inhibitors . The cells were disrupted by sonication and centrifuged at 19500 g at 4 °C for 30 minutes.PfEK was assessed by SDS-PAGE. PfEK concentrations were determined spectrophotometrically using the extinction coefficient at 280 nm of 1.129 mg ml−1 cm−1. Finally the protein was stored in buffer B at −80 °C.The supernatants were applied onto a 1 × 5 ml cobalt HP column  previously equilibrated with buffer A. The column was washed with buffer A and the fusion protein was eluted with buffer A containing 500 mM imidazole. The fusion protein was dialysed with buffer B  and subsequently was cleaved overnight with PP  at 4 °C. The PP was removed by a GSTrap column and the unbound untagged protein was applied onto a 1 × 5 ml cobalt HP column  previously equilibrated with buffer B. The unbound protein was further purified by size exclusion chromatography using a Superdex 75 XK26/60 column previously equilibrated with two volumes of buffer B. The eluted peaks were concentrated and used for biophysical experiments. The purity of the recombinant P. falciparum 3D7 was cultured according to standard methods90s) after 6 additional hours of incubation (i.e. 6–12 hours post-invasion). Thin blood smears were Giemsa stained every 6 hours until an entire intraerythrocytic cycle was completed.P. falciparum 3D7 were cultured with BR23 or BR25 inhibitors at their EC90s for 3h after sorbitol synchronization. After washing, P. falciparum trophozoites were cultured for 18h without inhibitors, collected, treated with saponin to release the parasites from the red blood cells, and washed twice with PBS prior to Bligh & Dyer lipid extraction8 parasite cells were dissolved in 375 μl 1:2 (v/v) chloroform:methanol and vigorously vortexed for 15 min. 125 μl of chloroform were added and 500 pmols of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine were included in each sample as internal controls. 125 μl of water were added to the sample, vortexed and centrifuged at ×1000 g (RT) for 5 min to make the sample biphasic. Lower organic phase was collected and upper (aqueous) phase was re-extracted twice with 125 μls of chloroform. Lipid extracts were pooled, dried under nitrogen and stored at 4 °C for ESI-MS/MS analysis.2, 1 mM PEP, 1 mM NADH, 6 U/mL pyruvate kinase, 9 U/mL lactate dehydrogenase, 4 mM Cho or Etn, 2 mM ATP (for PfEK) or 4 mM ATP (for CKs) and 0.1–50 μg enzyme. The reaction was initiated by addition of enzyme or substrate, as appropriate, and the rate of reaction was monitored by following the decrease in absorbance at 341 nm for 10 min at room temperature using a Thermo Multiska spectrophotometer. Experiments were carried out to ensure that coupling enzymes were not rate-limiting under any of the conditions used and control experiments lacking substrates and/or enzyme were carried out to correct for any background rate.A continuous spectrophotometric 96-well plate assay was established for CK and EK activity based on an assay describedPfEK only). Curves were plotted and Km and Vmax values estimated using Grafit 5 (Erithacus Software Ltd).One substrate was held constant at the concentration specified above while the second substrate was varied. Any ATPase activity was controlled for and subtracted from the rates .The enzyme was pre-incubated for 5 min with varying concentrations of inhibitor prior to reaction being started by addition of 0.5 mM each substrate. The ICis were estimated from the best-fit model and results were reinforced by linear re-plots of the data Activity was measured at 3–4 different concentrations of inhibitor and a fixed concentration of one substrate (as specified in the standard assay) while the second substrate was varied. GraphPad Prism 6 (GraphPad Software Inc.) was used to fit data to different inhibition models with the best fit being determined by Aike’s Informative Criteria. Kwww.lipidmaps.org).Lipid extracts, were dissolved in 15 μl of choloroform:methanol (1:2) and 15 μl of acetonitrile:iso-propanol:water (6:7:2) and analysed with a Absceix 4000 QTrap, a triple quadrupole mass spectrometer equipped with a nanoelectrospray source. Samples were delivered using a Nanomate interface in direct infusion mode ~125 nl/min). The lipid extracts were analysed in both positive and negative ion modes using a capillary voltage of 1.25 kV. MS/MS scanning  were performed using nitrogen as the collision gas with collision energies between 35–90 V. Each spectrum encompasses at least 50 repetitive scans. Tandem mass spectra (MS/MS) were obtained with collision energies between 35–90 V. Assignment of phospholipid and neutral lipid species is based upon a combination of survey, daughter, precursor and neutral loss scans, as well previous assignments nl/min. HsCKα1 and PfCK. The PDB entry 4CG9 has been used for predicting the docking pose of compound BR23 in HsCKα1 because it contains compound BR31Whereas Maestro 9.3 softwareHsCKα1) and 3FI8 (PfCK) were generated using the Shrödinger Glide modulePfCK Cho-binding site as a reference for the Grid generation, being the Grid box centered in these molecules and defined with a high volume (25 Å) to allow the insertion of compounds BR23 and BR25. Once the 3D structures were prepared for each compound , each molecule was optimized by means of the MacroModel module using the OPLS 2500 force field and a convergence gradient of 0.05 Å. After minimization, the LigPrep moduleIn a first step, all the proteins were aligned using the Protein Structure Alignment tool included into Maestro 9.3. Then, Protein Preparation Wizard module43PfCK binding pocket (3FI8) in the same conditions.Finally, Glide was used to flexibly dock compounds BR23 and BR25 into the active site of both proteins (4CG9 and 3FI8 pdb codes) with extra precision, and BR25 into ds for compounds BR23 and BR25 against HsCKα1 and PfCK were measured by monitoring the quenching of tryptophan fluorescence. All experiments were carried out in a Cary Eclipse spectrofluorometer at 25 °C with the enzymes at 1 μM, and concentrations of compounds varying from 0.05 to 8 μM for HsCKα1, and from 0.05 to 16 μM for PfCK in 25 mM Tris, 150 mM NaCl, pH 7.5. Fluorescence emission spectra were recorded in the 300–400 nm range with an excitation wavelength of 280 nm, with slit width of 5 nm. Controls were determined by incubating the enzymes with equivalent amounts of DMSO. As indicated previously, data analysis was performed in Prism (GraphPad software) considering a model with a single binding site . The sitting-drop vapour diffusion method3212) grew within 2–4 days and then were soaked for one day with 20 mM compound BR25 . The crystals used in this study were cryoprotected in the previously stated mother liquor solution containing an additional 20% ethylenglycol and frozen in a nitrogen gas stream cooled to 100 K.Diffraction data of the binary complex was collected at XALOC . The data was processed and scaled using the XDS packageHow to cite this article: Serrán-Aguilera, L. et al. Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine, Causing Parasite Death. Sci. Rep.6, 33189; doi: 10.1038/srep33189 (2016).


Glycogen Synthase Kinase-3 (GSK3) was originally reported as a key enzyme of glucose homeostasis through regulation of the rate of glycogen synthesis. It has subsequently been found to influence most cellular processes, including growth, differentiation and death, as part of its role in modulating response to hormonal, nutritional and cellular stress stimuli. More than 100 protein targets for GSK3 have been proposed although only a small fraction of these have been convincingly validated in physiological cell systems. The effects of GSK3 phosphorylation on substrates include alteration of enzyme activity, protein localisation, protein:protein interaction and protein stability. This latter form of regulation of GSK3 substrates is the focus of this review. There is an ever-growing list of GSK3 substrates that upon phosphorylation are targeted to the beta-transducin repeat containing protein (β-TrCP), thereby allowing ubiquitination of bound protein by cullin-1 and so initiating destruction at the proteasome. We propose the existence of a GSK3-β-TrCP ‘destruction hit-list’ that allows co-ordinated removal (or stabilisation) of a set of proteins with a common physiological purpose, through control of GSK3. We identify 29 proteins where there is relatively strong evidence for regulation by a GSK3-β-TrCP axis and note common features of regulation and pathophysiology. Furthermore, we assess the potential of pre-phosphorylation (priming) of these targets  to provide a second layer of regulation delineated by the priming kinase that allows GSK3 to mark them for destruction. Finally, we discuss whether this knowledge improves options for therapeutic intervention. GSK3 β deletion results in postnatal lethality, with multiple developmental defects and loss of hepatic function GSK3 is central to multiple intracellular pathways including those activated by Wnt/β-catenin, Sonic Hedgehog, Notch, growth factor/RTK, and G protein-coupled receptor signals N-terminal phosphorylation . Meanwhile the canonical Wnt signaling pathway disrupts the interaction between GSK3 and Wnt-specific targets N-terminal phosphorylation of GSK3 There are several regulatory mechanisms which control GSK3 activity; a) phosphorylation of Ser21 in GSK3 α and the equivalent residue in GSK3 β, Ser9 2.23or4S(P), with the C-terminal serine phosphorylated) The majority of GSK3 targets require prior phosphorylation (by a distinct kinase) to generate a GSK3 consensus sequence   E3 ubiquitin ligase β-TrCP. Interestingly, β-TrCP binding of proteins is enhanced by dual phosphorylation of the substrate, with a similar spacing of the two phosphorylated residues to that generated by GSK3 phosphorylation of primed substrates .Fig. 1Sc3The ubiquitin proteasome system (UPS) is the predominant route by which eukaryotic proteins are turned over in the cell, and defects in the UPS have been implicated in a range of diseases such as immune disorders, neurological disorders and cancer 3.1Ubiquitin is a small (8.5 kDa) ubiquitously expressed protein, encoded by four human genes . The covalent attachment of ubiquitin to a target protein (ubiquitination) most commonly results in its degradation by the proteasome.C-terminal carboxylate group to a particular lysine residue within the target protein, and the chain can then be elongated by linking ubiquitin molecules to one of the seven lysine residues  of the previous ubiquitin molecule. It is the polyubiquitination on specific lysines, mostly on Lys48 and Lys29 of ubiquitin that leads to degradation of the target protein by the proteasome Ubiquitination involves three basic steps: 1) ATP-dependent activation of ubiquitin, 2) conjugation of ubiquitin to an intermediary protein, and 3) ligation of the ubiquitin to the target protein. These steps are performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s), respectively. Importantly ubiquitination can involve attachment of a single ubiquitin molecule (mono-ubiquitination) or the generation of a chain of linked ubiquitin molecules (poly-ubiquitination) Some specificity to, and regulation of, this process is provided by the existence of a large family of E3 ubiquitin ligases (>600 members), all with the ability to transfer ubiquitin to the protein target 3.2One of the best examples of the large family of RING E3 ligases is provided by SCF containing complexes, of which there are 69 known in humans β-Transducin repeat containing proteins  are the best studied of the WD40 subfamily of F-box proteins. β-TrCP proteins are responsible for the ubiquitination of a wide range of critical proteins including; IκK phosphorylated IκB, GSK3 phosphorylated β-catenin, cell cycle regulatory proteins CDC25 and APC, and many more Misregulation of β-TrCP proteins, has been implicated in cancer development and progression, mainly because it controls the degradation of several tumour suppressor proteins 3.33or4S(P), with the C-terminal serine phosphorylated) it is perhaps not surprising that so many GSK3 substrates are now reported to be β-TrCP binding proteins . In addition, the stoichiometry of priming ; secondly, those with a minimal S/TXXXS/T sequence ; thirdly, those lacking the dual phosphorylation motif .4.3.13or4S-phos) it seems reasonable to propose that this phosphodegron is central to allowing degradation by a GSK3 and β-TrCP dependent mechanism. One could therefore predict that the presence of the DSGXXS motif within a protein, where phosphorylation occurs at the C-terminal Ser, would enhance subsequent phosphorylation at the N-terminal Ser by GSK3, binding of β-TrCP, poly-ubiquitination and degradation. Gumbiner and colleagues performed an in silico search for protein sequences containing “D/ESGXXS/TXXXS/TXXXS/T”, and identified 38 proteins with this sequence (and with evolutionary conservation) As mentioned above, the proposed motif for β-TrCP binding is a doubly phosphorylated DSGXXS sequence where X is any amino acid, and as this closely resembles the GSK3 consensus sequence  regulates epigenetic modifications of chromatin. Ectopic GSK3 expression resulted in β-TrCP-mediated poly-ubiquitination and degradation of CHD1, while GSK3 inhibition reduced poly-ubiquitination and stabilized CHD1. In normal cells PTEN reduces Akt signaling which reduces the Akt-mediated inhibition of GSK3. This stimulates the GSK3-mediated phosphorylation of two CHD1 phosphodegrons, resulting in CHD1 degradation via the β-TrCP-mediated ubiquitination-proteasome pathway. PTEN deficiency (often found in neoplastic disease) results in hyperactivity of Akt, reduction in GSK3 activity, and stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network in vivoSnail (a zinc-finger transcription factor) triggers the epithelial-mesenchymal transition (EMT) during embryonic development through its inhibition of E-cadherin expression FGD1 gene disruption results in Faciogenital Dysplasia , a skeletal dysplasia and multiple congenital anomaly syndrome, implying a crucial role in aspects of mammalian development. The gene product contains domains that indicate it is a guanine nucleotide exchange factor (GEF) for the Rho family GTPase Cdc42. FGD1 contains a DSGXXS motif and is recognized by β-TrCP when the two serine residues in this sequence are phosphorylated. This promotes ubiquitination and subsequent degradation of GEF by the proteasome in vitroin vivo, providing priming for GSK3 phosphorylation of Ser728. If so, this would mean there is a requirement for three different kinases to phosphorylate Sp1 before it was shuttled to the proteasome.Sp1 is a ubiquitous transcription factor linked to the regulation of dozens of genes. Glucose starvation or treatment with the thiazolidinedione derivative (OSU-CG12) both induce specific binding of Sp1 to β-TrCP followed by Sp1 degradation in LNCaP cells in vitro, and this phosphorylation is required for the recognition of PRLr by β-TrCP, as well as for PRLr ubiquitination and degradation 349SGRGS) is a perfect DSGXXS motif there is no report as yet of phosphorylation of Ser353 being required for GSK3 to phosphorylate PRLr or for binding to β-TrCP. One might predict the existence of a priming kinase that initiates the pathway by phosphorylation of Ser353.The prolactin receptor (PRLr) is a cytokine receptor best known for its role in mediating prolactin control of mammary growth. However, it is expressed in many cell types besides mammary cells, including pancreatic beta cells and adipose tissue, and so it contributes to reproduction, islet differentiation, fat storage and immunomodulation. GSK3 phosphorylates PRLr on Ser349 C-terminal sequence in TAZ to promote its degradation. However, there is also an N-terminal DSGXXS sequence within TAZ that is regulated by PI3K signaling TAZ  is a transcriptional co-activator mainly controlled by the Hippo pathway. The Hippo pathway is a key regulator of organ homeostasis through modulation of cell proliferation and apoptosis. TAZ is inhibited by large tumour suppressor (LATS)-dependent phosphorylation, leading to cytoplasmic retention and ubiquitin-dependent degradation. The LATS kinase, a core component of the Hippo pathway, phosphorylates a NFE2L2), a cap‘n’collar (CNC) basic-region leucine zipper (bZIP) transcription factor that mediates intracellular redox homeostasis. It is best known for its activation during oxidative stress, but it is becoming more appreciated for its contribution to other cellular stress responses One protein with a DSGXXS-like motif is Nuclear factor-erythroid 2 p45-related factor 2 -driven gene expression. Under non-stressed conditions the ubiquitination of NRF2 is predominantly performed by cullin-3 (CUL3) using Kelch-like ECH-associated protein-1 (KEAP1) as a substrate adaptor protein for the CUL3 RING-box 1 (RBX1) E3 ligase complex. KEAP1 functions as a stress sensor, under oxidative stress conditions several key cysteine residues on the surface of KEAP1 are modified. The modified KEAP1 loses its ability to induce cullin-3 ubiquitination of NRF2. As a consequence, the half-life of newly synthesised NRF2 protein is extended and the expression of ARE-driven NRF2-target genes is induced.378 or DSGIS338 (n.b. amino acid numbering based on mouse Nrf2) were able to interact with β-TrCP, with the latter exhibiting much higher affinity if it were doubly phosphorylated Besides KEAP1, NRF2 is also targeted for proteasomal degradation through ubiquitination by cullin-1, using β-TrCP as a substrate adaptor. However, in this instance, ubiquitination of NRF2 by cullin-1 requires the transcription factor to be first phosphorylated by GSK3 in order to generate the phosphodegron to which β-TrCP binds . In NRF2N-terminal Ser (Ser335) within the β-TrCP binding motif is still lacking. Indeed, the spacing between the Ser residues in the DS335GXS338 is less than three amino acids, indicating it is highly unlikely that GSK3 phosphorylates Ser335 using Ser338 as the ‘priming site’. However, there is strong evidence that GSK3 phosphorylates Ser338 using Ser342 as a priming site There are two key differences between NRF2 and the GSK3-β-TrCP targets listed above: firstly, it is a DS335GXS338 motif; secondly, robust evidence that GSK3 directly phosphorylates the Interestingly, the related CNC-bZIP transcription factor NRF1 is also degraded by the proteasome in a β-TrCP-dependent manner through a DSGLS motif 4.3.2N-terminal residue within the SXXXS sequence (by GSK3). Thus, amongst the 27 proteins in The β-TrCP binding motif is clearly more degenerate than the ‘classic’ DSGXXS motif. For example, our work on NRF2 found DSGXS to be a high affinity interaction sequence after dual phosphorylation However there doesn’t appear to be any consistent pattern in the primary sequence of the ‘phosphodegron’  to explain recognition of this series of proteins by β-TrCP . These pThe simplest explanation is the existence of a scaffold protein(s) that enhances interactions between GSK3, β-TrCP and the substrates for degradation, in an analogous fashion to the canonical Wnt signaling complex 4.3.2.1Mcl-1 is an anti-apoptotic member of the Bcl2 family, that is lost from cells exposed to UV irradiation bcl-3 gene. Interestingly phosphorylation of BCL-3 modulates its association with HDAC1, -3, and -6 and attenuates its oncogenicity by selectively controlling the expression of a subset of target genes such as SLPI and Cxcl1 BCL-3 is a proto-oncogene that regulates immune cell growth through its interaction with NF-kB fbw7 leading to polyubiquitination and degradation of Myc, primarily during the G1 phase of the cell cycle fbw7Myc is a transcription factor that enhances expression of a broad spectrum of genes involved in the biogenesis of nucleotides and ribosomes, thereby stimulating protein translation. In addition, Myc induces the expression of several cyclins and suppresses the transcription of cyclin-dependent kinase inhibitors, thereby promoting exit from quiescence and stimulating progression through G1 phase. GSK3 phosphorylates Thr58 of Myc and this is enhanced by priming at Thr62, probably by ERKs. Initially phosphorylation of Thr58 of Myc by GSK3 was reported to enhance binding of the E3 ligase SCFS65) it has not yet been confirmed that β-TrCP is the E3 ligase responsible for targeting to the proteasome. GSK-3-mediated Maf phosphorylation also modulates extracellular matrix remodeling which could modify cancer progression The Maf oncoproteins  are transcriptional regulators of the AP-1 superfamily. They regulate developmental, metabolic, and tumorigenic pathways in multiple tissues and are overexpressed in about 50% of human multiple myelomas Foxp3-expressing Treg cells limit anti-tumour responses and allow the persistence and growth of cancer. Phosphorylation of Foxp3 by GSK3 promotes β-TrCP binding and polyubiquitination leading to degradation by the proteasome. EGF-like growth factor Amphiregulin (AREG), which is frequently upregulated in human cancers, reduces GSK3 activity and stabilizes Foxp3 S390A phosphorylation deficient mutant in human breast MCF-10A epithelial cells enhances proliferation and formation of aberrant acini structures in 3D culture. Reduced phosphorylation of Ser390 of PR-A and relatively low GSK3 β activity is found in the Brca1-deficient mammary gland Progesterone receptors (PR-A) are over expressed in Brca1-deficient mammary epithelial cells. BRCA1 germ line mutations increase the risk of breast and ovarian cancer, whereas treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 knock-out mice. All of these data indicate that progesterone has a critical role in initiation and/or progression of breast carcinogenesis. GSK3-mediated phosphorylation of Ser390 in PR-A promotes its ubiquitination and degradation C-terminal to the proposed GSK3 target site. δ-Catenin can be degraded by the proteasome and also by lysosomes. Numerous downstream processes are regulated by δ-catenin, including control of cognitive function, E-cadherin signaling and angiogenesis. Enhanced δ-catenin stability is associated with several neoplastic diseases including those of prostate, lung, ovary, brain and colon.δ-Catenin polyubiquitination occurs following phosphorylation at Thr1078 by GSK3 and generation of the phosphodegron which recruits β-TrCP1 4.3.2.2Xenopus embryos, these Smad4 phosphorylations regulate germ-layer specification and Spemann organizer formation. Human Smad4 mutations that enhance phosphorylation at Thr273/Thr269 and Thr265 are less stable, inactivate TGF-β signaling and are associated with tumourigenesis Smad4/DPC4 is an essential transcription factor in the TGF-β pathway and is frequently mutated or deleted in prostate, colorectal, and pancreatic carcinomas. Smad4 activity is directly regulated by the Wnt and fibroblast growth factor (FGF) pathways through hierarchical phosphorylation by GSK3 and Erk. FGF activates ERK, which primes three sequential GSK3 phosphorylations on Smad4 that generate a Wnt-regulated phosphodegron bound by β-TrCP PHLPP1 is a member of a family of Ser/Thr protein phosphatases (PPases) that serve as tumour suppressors by negatively regulating Akt signaling and its expression is reduced in colorectal cancer 4.3.2.32 tension (in particular hypoxia). Under normal O2 tension it is rapidly degraded following polyubiquitination by the E3 ligase, VHL. However, phosphorylation of Hif1a by GSK3 induces ubiquitination and proteasomal degradation, independent of VHL, and this pathway is proposed to underlie regulation of Hif1a action by growth factors, nitric oxide and hormones Hif1a is a critical factor that initiates an adaptive and protective transcriptional programme to protect cells against changes in OREDD1 (regulated in development and DNA damage responses 1) is a hypoxia-inducible factor-1 target gene, with a protein half-life of <15 min. It has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling during hypoxic stress through regulation of TSC proteins. Other environmental stresses such as energy stress, glucocorticoid treatment and reactive oxygen species can enhance REDD1 gene transcription. REDD1 is subject to ubiquitin-mediated degradation mediated by β-TrCP, dependent on phosphorylation by GSK3 RASSF1 gene. RASSF1A is implicated in the regulation of apoptosis, microtubule stability and cell cycle arrest. RASSF1C is a very unstable protein that is polyubiquitinated and degraded via the proteasome. RASSF1C degradation is enhanced when cells are exposed to stress signals, such as UV irradiation RASSF1A [Ras association  domain family member 1A] and RASSF1C are two ubiquitously expressed isoforms of the Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) is a lipid synthesizing enzyme key in the production of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho) and hence plays a vital role in lung physiology. It is a member of the 1-acyl-sn-glycerol-3-phosphate acyltransferase family converting lysophosphatidylcholine to phosphatidylcholine in the presence of acyl-CoA. This process is also important in the synthesis of platelet-activating factor (PAF). Lipopolysaccharide (LPS) treatment of cells causes LPCAT1 degradation, altering the surfactant properties of the lung. Phosphorylation at Ser178 by GSK3 following priming at Ser182 generates the β-TrCP binding site and enhances polyubiquitination and degradation of the protein in response to LPS 4.3.2.4in vitroCryptochrome 1 (CRY1) and 2 (CRY2) are flavoproteins that act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals In Drosophila, Cubitus interruptus (Ci) is a key transcriptional regulator of limb development. The long form (Ci-155) is an activator of gene transcription while a shorter proteolytic fragment is a repressor Gli3 is the human homologue of Ci-155, and is the only Gli isoform that can be processed to a repressor. It also regulates limb development under the control of Shh signaling. In a similar fashion to the Drosophila Hh Ci-155 system, PKA phosphorylation of Gli3 on several sites primes it for subsequent phosphorylation by GSK3 and CK1 Paraxial protocadherin (PAPC) is involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. PAPC cellular location, ubiquitination and stability were all affected by treatment with a GSK3 inhibitor (BIO) or expression of a dnGSK3. These deficits were reversed by introduction of a Ser816Asp/Ser820Asp ‘double phosphorylation mimic’ mutant PAPC 4.3.3Cip1 contain a Glu or Asp three or four residues C-terminal to the phosphorylated serine and thus it is conceivable that the acidic amino acid side chain can partially mimic a phosphorylated serine and enhance phosphorylation by GSK3. This remains to be confirmed and in our experience an acidic residue does not prime peptide substrates to enhance phosphorylation by GSK3 (Sutherland unpublished data). In addition, the evidence that the degradation reported is mediated by β-TrCP, or even by ubiquitination is still lacking.There are two proteins in Cip1 by the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the proper control of endothelial cell (EC) proliferation and survival Cip1 is a short-lived CDK binding protein with a high proteasomal degradation rate. The PI3K inhibitors LY294002 and wortmannin reduced p21Cip1 protein abundance in human umbilical vein EC. GSK3 phosphorylated p21Cip1 at Thr57 within the CDK binding domain Cip1 protein levels in EC, whereas LiCl interfered with p21Cip1 degradation and increased p21Cip1 protein about 10-fold in EC and cardiac myocytes. This suggests that GSK3 triggers p21Cip1 degradation, while stimulation of Akt increases p21Cip1 protein levels via inhibitory phosphorylation of GSK3.Regulation of the DNA repair protein p21C. elegans. Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a rapidly dividing embryo. The zinc finger protein, OMA-1 is expressed only in oocytes and 1-cell embryos, and is degraded rapidly after the first mitotic division allowing embryogenesis to proceed normally. The OMA-1 protein is directly phosphorylated at Thr239 by the DYRK kinase MBK-2, and this is required both for OMA-1 function in the 1-cell embryo and its degradation after the first mitosis in vitro. Phosphorylation at both Thr239 and Thr339 are essential for correctly-timed OMA-1 degradation in vivo. Therefore, GSK3 regulation of Oma1 is required for correctly-timed OMA-1 degradation in one cell embryos Oma1 has mostly been studied in 4.3.4There are two proteins in Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key determinant of the angiogenic response and is decreased in diabetic hyperglycemic mice exposed to oxidative stress. A phosphorylated form of VEGFR-2 was reduced in the mice by hyperglycemia and VEGFR-2 protein was reduced in cells directly exposed to oxidative stress. This coincided with co-location of β-TrCP and VEGFR-2, and increases in ubiquitination of VEGFR-2 in vivo and is the ligase responsible for securin degradation after UV irradiation. While GSK3 may mediate this degradation of securin, the site(s) phosphorylated by GSK3 is not known Securin is a chaperone protein which binds to separase to inhibit premature sister chromatid separation until the onset of anaphase, and which also modulates cell-cycle arrest after UV irradiation. At metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome (APC/C), allowing activation of separase. UV radiation induces a marked reduction of securin protein, while GSK3 inhibitors prevent this securin degradation 5The prior phosphorylation of a substrate at a residue 4 or 5 amino acids C-terminal to the GSK3 target site greatly enhances the rate of phosphorylation by GSK3. This priming occurs on the majority of validated substrates of GSK3 and introduces an opportunity for substrate selective control of GSK3 action, especially where priming is an essential condition for the substrate to be recognized by GSK3 However, of the 27 proteins regulated by the GSK3-β-TrCP axis where the GSK3 site is known there are 25 substrates where priming could exist . Yet a pClearly most of these proposed priming kinases are part of dynamically regulated pathways, and thus it would seem likely that the priming of the GSK3 substrates will also be dynamically regulated. One obvious example of this is Mcl-1 which becomes a GSK3 target in cells exposed to UV irradiation due to activation of a priming event mediated by JNK 6GSK3 has been touted as a therapeutic target ever since it was discovered as a regulator of glucose homeostasis. Indeed several major pharmaceutical companies developed potent and selective small molecule chemical inhibitors which entered preclinical trials for diabetes and dementia KEAP1, NFE2L2, or CUL3, and associates with increased cell proliferation and resistance to anticancer drugs. In tumour cells in which the KEAP1-CUL3 ubiquitin ligase is unable to repress NRF2, stimulation of GSK3 activity has been found to suppress NRF2, presumably by increasing ubiquitination of the CNC-bZIP transcription factor by β-TrCP-cullin-1. In this circumstance, and in −/−Keap1 mouse embryonic fibroblasts, inhibition of GSK3 by CT99021 increases NRF2 activity activators rather than inhibitors as possible anti-cancer therapeutics.The wide range of GSK3 inhibitors available for pre-clinical studies has been comprehensively reviewed previously inhibition has been proposed as a potential therapeutic option in specific forms of cancer That said, GSK3 and GSK3 substrate phosphorylation status of the diseases being targeted. For example, is there enhanced GSK3 activity , is there only a subset of GSK3 targets dysregulated, and is any GSK3 defect related to enhanced basal GSK3 or loss of GSK3 regulation? This information is key in order to develop patient-based therapeutic options to interfere with GSK3 function in a manner specific to the pathological issue.What is very clear is that if one is to develop safe GSK3-based therapeutic approaches for the clinic then it will be important to understand more about GSK3 activity in vitro expression cloning strategy in cytoplasmic extracts from Xenopus identified 35 novel polypeptides whose expression was modified by LiCl, and a subset of these were also regulated by interfering with Wnt regulation of the axin complex To this end, the development of a panel of GSK3 substrate biomarkers would permit more detailed classification of the many ‘GSK3-associated diseases’. This would normally involve the development of robust phosphospecific antibodies to the residues on the GSK3 targets, ideally with a companion non-phospho antibody to provide a ratio of phosphorylated to non-phosphorylated sequence in each case. The subject of this review (the GSK3 mediated destruction of the target) may provide the opportunity to develop tools to classify these diseases by measuring substrate protein abundance, since in the case of the destruction hit-list this would be directly related to the rate of phosphorylation. In this way a more detailed, extended, and quantitative investigation of the ‘GSK3 destruction proteome’ has the potential to provide biomarker panels for the classification of GSK3 associated disease, which could be assessed in an antibody-based or MS-based assay. An elegant In summary, the emergence of the growing list of GSK3 substrates which are labelled by ubiquitination for destruction via the proteasome may provide some mechanistic understanding of why GSK3 activity is linked to so many diseases. At the same time, there may be opportunities to develop disease specific diagnostic and prognostic biomarker arrays, and hence disease specific or even patient specific therapeutic interventions, based on the GSK3 substrate profile of the disease. It remains to be seen just how large the GSK3-β-TrCP hit-list turns out to be, but it should continue to provide important insights into how cells respond to various stimuli to modify their protein compliment.


The relative abundance of various OTUs and functional genes were significantly correlated with multiple environmental parameters, highlighting many potential environmental drivers and ecological strategies.Climate change has resulted in an accelerated decline of Arctic sea ice since 2001 resulting in primary production increases and prolongation of the ice-free season within the Northwest Passage. The taxonomic and functional microbial community composition of the seawater and sea ice of the Canadian Arctic is not very well known. Bacterial communities from the bottom layer of sea ice cores and surface water from 23 locations around Cornwallis Island, NU, Canada, were extensively screened. The bacterial 16S rRNA gene was sequenced for all samples while shotgun metagenomics was performed on selected samples. Bacterial community composition showed large variation throughout the sampling area both for sea ice and seawater. Seawater and sea ice samples harbored significantly distinct microbial communities, both at different taxonomic levels and at the functional level. A key difference between the two sample types was the dominance of algae in sea ice samples, as visualized by the higher relative abundance of algae and photosynthesis-related genes in the metagenomic datasets and the higher chl  The Canadian Arctic Archipelago constitutes an extensive Arctic continental shelf area, of socio-economic and ecological importance due to its highly productive ice-associated ecosystem, its role as a refuge for multiyear ice, and as a northern navigation route via the Northwest Passage12157Gammaproteobacteria, Alphaproteobacteria and Flavobacteria were observed to be common to both sea ice and seawater11a maximum, obtained from SSU rRNA gene amplicon sequencing, was also found to be influenced by bio-physical forcings, in particular complex organic matter contentHowever, the microbial community composition and genomic content of seawater and sea ice around the Northwest Passage, or in the Arctic in general, is poorly understood. Metagenomic studies conducted in the marine environment in the Canadian high Arctic are still limitedThe objectives of the present survey were to 1) characterize bacterial community structures in seawater and the immediate bottom layer of sea ice cores (referred to as sea ice) recovered near Cornwallis Island in the Northwest Passage, and 2) relate the relative abundance of key taxa and functional genes to environmental parameters. In order to attain these goals, the 16S rRNA gene was amplified and sequenced from sea ice and seawater samples taken at 23 locations around Cornwallis Island in the Canadian high Arctic, while 16 selected samples (6 sea ice and 10 seawater samples) were also subjected to shotgun metagenomic sequencing.Samples of surface water and ice bottom samples were collected at 23 stations surrounding Cornwallis Island, in Lancaster Sound, Wellington Channel and adjacent channels, between 74.10′ and 75.93′N and 92.64′ and 101.34′W . The sama (chl a), bacterial abundance, particulate organic carbon (POC), dissolved organic carbon (DOC), dissolved nitrogen (DN), macro-nutrients  and salinity. Chl a fluorescence was read on a 10AU Turner Design fluorometer calibrated using pure chl a extract  and concentrations were calculated according to Parsons et al.et al.Sea ice and seawater samples were analyzed for chlorophyll et al.DNA extractions were performed on duplicate samples of the melted sea ice (50–100 ml) or seawater samples (1000 ml) filtered on 0.22 μm nitrocellulose filters . The filters were immediately frozen and stored at −80 °C until DNA and RNA extraction. The Powerwater RNA Isolation Kit  was used for seawater nucleic acid extraction of the filters according to the manufacturer’s protocol, modified for the presence of lysis-resistant organisms and the omission of DNAse treatment for total nucleic acid extraction. The protocol for DNA extraction of the sea ice filters was based on a modified CTAB (cetyltrimethylammonium bromide) method of Ausubel Libraries for sequencing were prepared according to Illumina’s “16S Metagenomic Sequencing Library Preparation” guide (Part # 15044223 Rev. B), with the exception of using Qiagen HotStar MasterMix for the first PCR (“amplicon PCR”) and halving reagent volumes for the second PCR (“index PCR”). The template specific primers were (without the overhang adapter sequence): 515f (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The first PCR (“amplicon PCR”) was carried out for 25 cycles with annealing temperatures of 55 °C. Diluted pooled samples were loaded on an Illumina MiSeq and sequenced using a 500-cycle MiSeq Reagent Kit v3.7 molecules were used in an emulsion PCR using the Ion Xpress 200 Template Kit (Life Technologies) as described in Sanschagrin and YergeauFollowing UPGMA cluster analysis of the samples based on the 16S rRNA gene dataset (not shown), representative samples of major clusters were selected for metagenomic sequencing. Each DNA library was prepared for sequencing using the Ion Xpress Plus Fragment Library Kit (Life Technologies) with the Ion Xpress Barcode Adapters 1–16 (Life Technologies), using the Ion Shear Plus Reagents. Size selection was performed using a Pippin Prep instrument  set to “tight” collection with a base pair target setting of 315 to collect fragments with a median of 200–300 bp as recommended in the “Ion Express Plus gDNA Fragment Library Preparation” manual . Barcoded libraries were pooled in an equimolar ratio three by three. A total of 3.50 × 10Sequences were analyzed through our internal rRNA short amplicon analysis pipeline as previously described19et al.et al.−5. MG-RAST classifies sequences using the “subsystems technology”, in which each sequence is assigned to a manually curated subsystemShotgun metagenomic sequences were submitted to MG-RAST 3.0s) using the “cor.test” function, dissimilarity calculation using the vegdist function of the vegan library, principal coordinate analyses (PCoA) using the “cmdscale” function and Permanova using the adonis function of the vegan library. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was carried out based on the UniFrac distance matrices using the “agnes” function of the vegan library.All statistical analyses were carried out in R . Paired and non-paired t-tests were performed using the t.test function (or the non-parametric wilcox.test if needed), Spearman rank-order correlations  concentrations in the sea ice compared to seawater . In contrast, Si(OH)4 and salinity were significantly higher in the seawater as compared to the sea ice .A summary of the biochemical characteristics of sea ice and seawater is presented in Not surprisingly, when plotting principal coordinates calculated from UniFrac distances (based on 16S rRNA gene sequences) a significant difference  was observed between the microbial communities in seawater and sea ice samples . The micSimilar differences between the sea ice and seawater samples were visible in the metagenomic dataset, both for the species-level taxonomic table  and the Thaumarcheota, Euryarchaeota, Actinobacteria, Cyanobacteria, Fimicutes, Planctomycetes, Betaproteobacteria, Deltaproteobacteria, and Verrucomicrobia and significantly depleted in Bacteroidetes, Alphaproteobacteria and Gammaproteobacteria when compared to sea ice samples. Some of these trends were also visible in the metagenomic datasets , Actinobacteria , Alphaproteobacteria , Betaproteobacteria , Deltaproteobacteria , Planctomycetes  and Verrucomicrobia  in the seawater as compared to sea ice and a significantly lower relative abundance of diatoms  and Gammaproteobacteria  in the seawater as compared to sea ice. As expected in view of the biases associated with PCR amplification, the relative abundance of some phyla varied considerably between 16S rRNA gene and metagenomic datasets  . Seawatedatasets  with sigdatasets .Alteromonadaceae, Polaribacter, and Colwelliaceae . There were 160 “Level 2” categories and 988 “Level 3” categories, therefore only categories having a direct link to our data are presented in 4 were selected. For the MG-RAST “Function” dataset, NO2+NO3 was selected for the sea ice, while no variable was significant for the seawater.Correlation and canonical correspondence analyses (CCA) were used to identify linkages between OTUs, functional genes and measured environmental variables/constituents (described in 4 was the factor having the highest number of positive (23) and negative (37) correlations. For the sea ice MG-RAST “Function” dataset, 499 correlations were significant (94 positive and 405 negative), with total bacteria showing the highest number of positive correlations with functions (26) and NO2+NO3 showing the highest number of negative correlations (68). For the seawater MG-RAST “Function” dataset, 1627 correlations were significant (659 positive and 968 negative), with DOC showing the highest number of positive correlations (149) with functions and POC showing the highest number of negative correlations (235). The complete list of significant (P < 0.05) correlations is presented in Many OTUs showed significant correlations with environmental variables. In the sea ice 16S rRNA gene datasets, 461 Spearman correlations were significant (P < 0.05), with 387 being negative and 74 being positive. Salinity was the factor most often positively correlated with OTUs (15 times), while POC was the factor most often negatively correlated with OTUs (71 times). In the seawater 16S rRNA gene dataset, 392 Spearman correlations were significant . Si(OH)a concentrations. Metagenomic datasets from this study showed that these algae were mainly diatoms, confirming visual observations during sampling and corresponding with ice algal taxonomic studies near the study areaGammaproteobacteria, and this taxon was significantly more abundant in sea ice as compared to seawater. A symbiotic relationship was proposed to exist between algae and bacteria by production of vitamin B12303233In the present study, we compared seawater and the immediately overlying sea ice at 23 locations around Cornwallis Island in the Canadian high Arctic. It is one of the first studies to simultaneously look at linked seawater and sea ice samples using shotgun metagenomics and 16S rRNA gene sequencing and to relate this genomic data to potential environmental drivers. As expected, seawater and sea ice exhibited large differences in terms of chemistry, community composition and functional gene content. A key difference between the two sample types was the dominance of algae in sea ice samples, as visualized by the higher relative abundance of algae and photosynthesis-related genes in the shotgun metagenomic datasets and the high sea ice chl The mean values and range for the physico-chemical indicators are similar to values previously reported for surface water in the Northwest PassageMost results showed higher variability in microbial communities in sea ice samples as compared to seawater. Sea ice is highly variable in terms of physiochemical properties, especially within its microenvironments37Gammaproteobacteria and Bacteroidetes were abundant in sea ice samples, similar to multi-year ice sampled near the geographic North PoleActinobacteria, Alpha- and Betaproteobacteria, which are also common to other first-year and multi-year sea ice samplesProteobacteria and Bacteroidetes were relatively abundant in seawater samples, similar to previous reports from Antarctic and Arctic seawater81235Although previous studies used different PCR primers7123341In conclusion, our study has shown that the microbial communities and their associated functional genes present around Cornwallis Island in the Canadian high Arctic are very different between sea ice and seawater, even though they were quite variable between sampling sites. The functional differences observed could be at the root of the different capacities of sea ice and seawater to degrade hydrocarbon, as recently shown using samples from the same areaHow to cite this article: Yergeau, E. et al. Metagenomic survey of the taxonomic and functional microbial communities of seawater and sea ice from the Canadian Arctic. Sci. Rep.7, 42242; doi: 10.1038/srep42242 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP‐dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to  CytOxcytochrome c oxidase (E.C. 1.9.3.1.)HHill slope coefficient (according to GraphPad Prism software)PEPphosphoenolpyruvic acidPKpyruvate kinase (E.C. 1.7.1.40)ROSreactive oxygen speciesTNturn over numberMitochondrial respiration is the most common mechanism for ATP production and energy supply. Energy demand varies and depends on different forms of activities in cells, tissues and organisms. Early in earth's history, there were Prokaryotes that were dependent on their own metabolism and on nutrients that were provided by their environment. Approximately 250 million years ago, after dramatic changes occurred in earth's atmosphere, its composition changed to 21% oxygen Box 1G°’) of −50 kJ/mol (NADH to the Fe‐S centre of NADH‐Q Reductase), −42 kJ/mol (Cytochrome b to Cyt c1 in Cytochrome – Reductase) and −100 kJ/mol (Cytochrome a to oxygen in CytOx) Cellular respiration involves oxygen consumption to produce ATP. The mitochondrial electron transport chain (ETC) is composed of five elements: Four enzymatic respiratory complexes and ATP synthase. Electrons that are delivered from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. Electron transmission between respiratory chain complexes liberates free enthalpies . This potential drives the ATP synthase (complex V) toward production of ATP (Mitchell‐theory). In a second feedback mechanism, the binding of ATP to mitochondrial complex IV results in an allosteric inhibition of Cytochrome c Oxidase (CytOx) activity (Kadenbach theory), thus inhibiting excessive rates of ROS production, which could render the mechanism a pivotal element understanding degenerative diseases, and in turn, for developing modern therapeutic concepts Box 2As is generally known, electrons are transferred through the ETC (from complex I to IV). Additionally, complexes I, III and IV act as proton pumps, translocating hydrogen ions (protons) across the inner mitochondrial membrane and creating a mitochondrial membrane potential. This ΔΨm drives complex V (ATP‐synthase) toward production of ATP. As reported by Kaim and Dimroth, an optimal range between 80 mV <ΔΨm <120 mV guarantees sufficient synthesis of ATP, resulting in the transfer of 4 electrons to dioxygen (reduction) and oxidation of hydrogen to water without excessive production of ROS. At ΔΨm >>120 mV, large amounts of ROS are produced Under physiological conditions, the cell contains extremely high quantities of ATP. In the so called ‘relaxed’ state, ATP binds to subunit IV of the phosphorylated CytOx and induces an allosteric conformational change that results in sigmoidal enzyme kinetics. Therefore, CytOx is ‘ATP‐dependent allosterically inhibited’ and an increased production of ROS during respiration is avoided. Although the membrane potential is sustained by the ATP demand, in this state (within a stable range of ΔΨm), the enzymatic activity of CytOx after ATP binding is regulated exclusively by the mitochondrial ATP/ADP ratio.In the case of cellular stress, allosteric inhibition of the enzyme by ATP is switched off. Although the maximum rate of ATP synthase is known to be beyond 100–120 mV Kadenbach's Hypothesis states that the regulation of the membrane potential and ROS formation in mitochondria are determined by the ATP‐induced allosteric inhibition of CytOx, and represents a second mechanism for respiratory control. Under relaxed conditions, feedback inhibition of CytOx by ATP maintains the membrane potential at low values. Stress factors increase the cytosolic and/or mitochondrial [Ca2+], which activates calcium‐dependent protein phosphatases and dephosphorylates CytOx. Without allosteric inhibition of CytOx by ATP, the membrane potential increases with a consequent increase of ROS formation. Question regarding initial mechanisms have yet to be answered. There is a competition between ADP and ATP for the binding sites on the enzyme The molecular mechanisms are currently not fully understood. With regard to the present concepts see Fig. 2A–D.selective electron transfer by an electron donor (ascorbic acid 18 mM) and an electron transmitter (cytochrome c in increasing concentrations) to the mitochondrial respiratory chain complex IV (CytOx). Subsequently, Kadenbach and co‐workers used this system to perform measurements either in the presence of 5 mM ADP or 5 mM ATP. For measurements in the presence of ATP, an ATP regenerating system  was also used to maintain the ATP concentrations high enough and to demonstrate the effect of inhibited CytOx.Respiratory steady states have already been defined by Chance and Williams HK from 1.09 to 1.97) at increasing cytochrome c concentrations (from 0.25 to 60 µM). However, at high concentrations of cytochrome c, the enzyme kinetics became hyperbolic. In these experiments, CytOx kinetics was measured as ‘Turn over number’ (TN [S−1]) 3 content inside the proteoliposomes was constant, but addition of increasing amounts of cytochrome c resulted in a steady increase of electron transmissions to aa3 and in higher TN's at the end. According to our hypothesis, the shift from sigmoidal to hyperbolic kinetics described in this report is attributed exclusively to the electron transmission rate to CytOx, whereas the enzymatic consumption of oxygen itself depends on the uptake of electrons by aa3 when increasing amounts of cytochrome c are transferred to dioxygen for reduction Studies by Arnold and Kadenbach Acceptance of this type of CytOx activity measurements has always been controversial. First, criticisms were based on the fact that original measurements from Arnold and Kadenbach 3 content at the same protein ratio of 1–2.25 between homogenate and mitochondria. During measurements of CytOx kinetics, we employed experimental conditions that were similar to those reported by Arnold and Kadenbach 3 inside the CytOx molecule created a pivotal point for understanding the inhibitory effect of ATP. Hill‐slope (H) values were calculated using an allosteric sigmoidal programme (GraphPad Prism software) as a parameter of allostery, where H = 1 indicates that the equation is identical to standard Michalis‐Menton kinetics. Hyperbolic curves were obtained in all ADP‐measurements. When H is greater than 1, the curve is sigmoidal due to positive cooperativity, as in the presence of ATP and an ATP‐regenerating system. We observed sigmoidal curves during tissue homogenate measurements of both rat heart and bovine heart. . The theory of Kadenbach describes a mechanism that maintains the ΔΨm at low values under normal conditions, thus preventing excessive formation of ROS. This mechanism is switched off under conditions of stress and excessive work to maximise the rate of ATP synthesis and is accompanied by a decreased efficiency Box 3Small amounts act as ‘secondary messengers’ and are probably formed by cells after binding of agonists  to receptors of signaling cascades through NADPH oxidases at the plasma membrane Measurements of ROS formation at high Ψm values were performed in isolated mitochondria and were additionally confirmed in cultured cells The basis of Kadenbach's theory is the proposal of a ‘second mechanism of respiratory control’ Recently, we observed that the ATP‐dependent inhibition of CytOx is also associated with ‘ischaemic preconditioning’ (IPC) KATP channels openers, i.e. dioxazid, pinacidil or nicorandil KATP channels ATP channels Preconditioning was observed after a brief ischaemia/reperfusion event, and may be generated experimentally by hypoxia, oxidative stress, heat shock or by activation of α1 receptors Iksoo Lee was the first to demonstrate in her thesis an obvious correlation between the phosphorylation of cytochrome c oxidase by a cAMP‐dependent protein kinase A and ATP‐dependent allosteric enzyme inhibition 32P] ATP, but did not identify the phosphorylated amino acid. The signaling pathways leading to phosphorylation and modification of CytOx activity are still largely unknown. Hüttemann and co‐workers 2‐HCO3−‐sAC‐cAMP‐PKA regulatory pathway for oxidative phosphorylation. The latest findings of Hess et al. indicate a ‘CO2‐HCO3−‐sAC‐cAMP‐ signalosome’ that is responsible for PKA activation and phosphorylation of subunit Va at positions T65 and S 43 of CytOx in Saccharomyces cerevisiae under normoxic conditions Hüttemann and co‐workers Yaniv et al. Regarding the data from the Manfredi group, Lee and co‐workers studied signaling pathways targeting mitochondria and examined phosphorylation of CytOx subunits by the cAMP‐dependent pathway. Using phospho‐antibodies against phospho‐tyrosine, they detected phosphorylated cow liver CytOx subunit I in the presence of theophylline, a phosphodiesterase inhibitor (PDE inhibitor) that induces high levels of cAMP. This type of phosphorylation of Tyr304 in CytOx decreased V(max) and increased K(m) for cytochrome c. It shifted the reaction kinetics from hyperbolic to sigmoidal as CytOx is fully or strongly inhibited up to 10 μM concentrations of cytochrome c sigmoidal kinetics of the enzyme at high intramitochondrial ATP/ADP ratios with ADP bound to the CytOx subunit IV and replaced by ATP remains to be clarified 91‐containing binding site. Cytochrome c bound to Cardiolipin and ATP has a high level of Peroxidase activity that favours protein structures with an open haeme pocket 3 acts as part of the binuclear centre and site of oxygen reduction (Fig. The inhibition of CytOx by ATP presents a ‘second mechanism of respiratory control’ ion Fig. A–D. KadeThe authors declare no conflict of interests regarding the publication of this paper.