Psychrophiles thrive permanently in the various cold environments on Earth. Their unsuspected ability to remain metabolically active in the most extreme low temperature conditions provides insights into a possible cold step in the origin of life. More specifically, metabolically active psychrophilic bacteria have been observed at −20 °C in the ice eutectic phase . In the context of the RNA world hypothesis, this ice eutectic phase would have provided stability to the RNA molecules and confinement of the molecules in order to react and replicate. This aspect has been convincingly tested by laboratory experiments. Theronments . As far ronments . These hronments , and theronments .Although attractive and well documented , the hypIt is frequently overlooked that the majority (>80%) of the Earth’s biosphere is cold and permanently exposed to temperatures below 5 °C . Such loPlanococcus halocryophilus, isolated from high Arctic permafrost, was found to divide at −15 °C and to remain metabolically active at −25 °C [Psychrophiles thrive in permanently cold environments in thermal equilibrium with the medium and even at sub-zero temperatures in supercooled liquid water. Such extremely cold conditions are encountered, for instance, in salty cryopegs at −10 °C in the Arctic permafrost , in the t −25 °C , which pt −25 °C . Cryocont −25 °C . Antarctt −25 °C , while pt −25 °C . Glaciert −25 °C . These et −25 °C ,40,41,42There is no formal lower temperature limit for life under natural conditions, as most microbial species can be maintained for extended periods of time in low temperature freezers at −80 °C, then revived under appropriate conditions. Bacterial survival has been reported in frozen permafrost samples up to half a million years in age, and such viability was correlated with the capacity to slowly repair DNA, therefore preserving the cell genetic program .The concept of ice as a habitat for cold-adapted organisms has been initially described by Priscu and Christner . CurrentIn the context of a possible cold origin of life, some parameters are worth mentioning here, as they are relevant for both extant psychrophiles and prebiotic macromolecules.(i)Liquid veins between ice crystals: the eutectic phase . In glac(ii)Unfrozen water film in contact with minerals. Ice in contact with rocks, mineral particles, or clay grains harbor a nanometer-thick layer of unfrozen water. Cells attached to mineral particles have been observed in both glacier ice and permafrost. As a result of the small size of the water film, these cells are immobilized in this microenvironment and are not able to move freely.(iii)Inside ice crystals. Microorganisms can be trapped inside individual crystals in polycrystalline ice . As withThree habitats for psychrophiles in ice have been described.It follows that ice, but also frozen minerals or clay and permafrost, all display liquid microenvironments, ranging from thin water molecule layers to large veins, which are compatible with the water-based chemistry of prebiotic molecules.2 and CH4 in deep Greenland glacier ice have been related to ongoing in situ production by microorganisms trapped in ice 400,000 years ago [Cells can remain metabolically active in the above-mentioned microenvironments, as they can obtain a limited amount of gases and nutrients by diffusion, including through individual ice crystals, whereas waste products diffuse away from them. For instance, huge excess concentrations of COears ago ,57.Exponential growth with doubling time in the range of 5 h at 0 °C recorded for psychrophilic bacteria cultivated in the laboratory in rich medium  are unliIn the context of prebiotic chemistry, the main message here is that extremely low environmental temperatures do not totally abolish the metabolism of a complex microorganism like a bacterium, which relies on a huge number of biochemical reactions. Accordingly, simple prebiotic chemical reactions might have been slowed down but not cancelled in ice at low temperature.Vitrification of the intracellular content—which does not result in cell death (in contrast to freezing)—has been proposed for survival at very low subzero temperature . HoweverThe long term survival of microorganisms in ice suggests that diffusion of nutrients and waste products is still effective in frozen environments. It follows that prebiotic reactions might have been confined in an icy medium, but with sufficient diffusion potential to avoid poisoning of the niche. Furthermore, ice appears as a better environment to escape natural radiation as compared with minerals and permafrost, although none of these environments could protect from cosmic radiation until buried several meters in ice.Several studies have shown that the polymerization of prebiotic molecules is favored in ice veins and at the surface of clay grains at subfreezing temperatures because precursors are concentrated from initially dilute solutions and are organized, for instance, on a mineral surface that provides a scaffolding, while low temperature reduces the tendency to hydrolyze (see ,62,63 foIn this respect, an outstanding contribution in this field from the Holliger’s lab has convincingly shown that ice can be a protocellular medium for RNA replication . The autIt was also shown that the eutectic phase is required for polymerase activity, as further freezing to −25 °C abolished ribozyme activity . FurtherHowever, the ribozyme used in this study was optimally active at ambient temperature, and only synthesized short nucleotides. Therefore, the same group has subjected the ribozyme to in-ice directed evolution and engineering in order to produce a variant adapted to subzero temperatures able to synthesize RNA in ice at temperature as low as −19 °C . This reIt is currently elusive to draw a precise scenario for the emergence of life, which should obviously include a wide range of diverse factors such as geothermal chemistry, meteoric input of organic precursors, clay geochemistry, or vesicles formation, amongst others ,25. NeveIce is certainly not the unique environment for the origin of life, but as elegantly stressed by Price , “It is


H,1H,2H,2H-perfluorooctyltriethoxysilane (POTS) using spin coating and chemical vapor deposition methods. Scanning electron microscope images reveal the porous structure of the silica nanoparticles, which can trap small-scale air pockets. An average water contact angle of 163° and bouncing off of incoming water droplets suggest that a super-hydrophobic surface has been obtained based on the silica nanoparticles and POTS coating. The monitored water droplet icing test results show that icing is significantly delayed by silica-based nano-coatings compared with bare substrates and commercial icephobic products. Ice adhesion test results show that the ice adhesion strength is reduced remarkably by silica-based nano-coatings. The bouncing phenomenon of water droplets, the icing delay performance and the lower ice adhesion strength suggest that the super-hydrophobic coatings based on a combination of silica and POTS also show icephobicity. An erosion test rig based on pressurized pneumatic water impinging impact was used to evaluate the durability of the super-hydrophobic/icephobic coatings. The results show that durable coatings have been obtained, although improvement will be needed in future work aiming for applications in aerospace.A super-hydrophobic surface has been obtained from nanocomposite materials based on silica nanoparticles and self-assembled monolayers of 1 Ice formation and accretion may hinder the economic and environmentally friendly operation of aircraft  and poseA surface that exhibits a water contact angle of 150° or greater with very little flow resistance is considered to be super-hydrophobic . Super-hCoatings incorporating silica nanoparticles have been attracting significant interest due to high thermal and mechanical stability and high surface roughness . Among lH,1H,2H,2H-perfluorooctyltriethoxysilane (POTS), were grafted onto the silica nanoparticle surface by the chemical vapor deposition method to obtain a low surface energy. The hydrophobicity, icephobicity and durability of the coatings were investigated.In the current work, silica nanoparticles were deposited by the spin-coating method to form a nanostructured rough surface to trap small-scale air pockets. Self-assembled monolayers (SAMs) of fluoroalkyl silane, 1Ra) of 2.64 nm in area of 5 µm × 5 µm are alloy (2024-T4). For all other tests, the substrates are glasses with Ra of 1.66 nm in area of 5 µm × 5 µm.Silica nanoparticles, polystyrene and POTS (98%) were purchased from Sigma-Aldrich Company . Chloroform was purchased from Fisher Scientific Company . All chemicals were used as received. 0.5 g silica nanoparticles and 0.019 g polystyrene were dissolved into 30 mL chloroform by continuous stirring for about 1 h. The mixture was deposited onto substrates at a speed of 1500 rad/min for 1 min using a spin coater . For ice adhesion test, the Al substrates with roughness average  with pumping out rate of 1 µL/s.The surface morphology was investigated by a scanning electron microscope  under an acceleration voltage of 20 kV after Pt was deposited on the samples to prevent charging by electron beam. The composition was measured by energy dispersive X-ray spectroscopy  with an electron accelerating voltage of 20 kV by accumulating the counts for 60 s. The binding energies of elements were characterized by an X-ray photoelectron spectroscopy  using Al Kα X-ray as the radiation source with wavelength of 1486.6 eV. The Fourier transform infrared (FTIR) spectra were recorded by a spectrometer  using attenuated total reflection mode in the range between 650 cmF is the centrifugal force (N), m is the mass of ice block (kg), r is the radius of the beam (m) and ɷ is the speed of rotation (rad/s). From the centrifugal force, the shear stress is determined:A is the Area iced (m2), τ is the shear stress (Pa). Six silica-based coating samples were measured for better accuracy.Ice adhesion tests were performed using a centrifuge method with a glaze ice block (mass of 1.3 g) in a low temperature chamber with temperature of −5 °C. Using the rotation speed at the detachment of the glaze ice block, the ice adhesion strength is calculated using the ice block mass and beam length :(1)F=mrThe water droplet icing tests were performed by monitoring the water droplets on three spots of coated samples and uncoated samples on a cold plate setting at −10 °C. By observing the video of the water droplets, icing duration can be obtained.To evaluate the durability, erosion test rig  will form low-surface-energy surfaces which will contribute to the super-hydrophobicity. The self-assembled monolayer of 1A is the apparent contact angle measured on the substrate surface; θ is the water contact angle on the fluoridated smooth surface and it was 100° [f1 and f2 are the fractions of the solid surface and air in contact with water droplets; and f1 + f2 = 1 [f1 calculated using the average water contact angle of 163° is 5.3% and it indicates that 94.7% of the surface is occupied by air, which indicates that a combination of silica nanoparticles and POTS allows air to be trapped easily, resulting in a super-hydrophobic surface.The Wenzel model and the Cassie-Baxter model are generally used to explain the hydrophobicity of coatings with high roughness. In the Wenzel model, water droplets follow the profile of a rough surface and are pinned to the surface, which results in them being unable to slide on the surface . Howeverwas 100° ; f1 and + f2 = 1 . The f1 According to classical nucleation theory and observation, it was reported that the nucleation rate and macroscopical growth velocity of ice can be greatly reduced by a super-hydrophobic surface owing to an extremely low, actual solid-liquid contact area caused by the trapped air pockets . As preve, with θe standing for the estimated equilibrium contact angle which implies that a low ice adhesion strength can be obtained from super-hydrophobic surfaces [Besides the icing delay performance, the ice adhesion strength is another important parameter for icephobicity. With low ice adhesion strength, the ice can be removed easily which is desirable for de-icing. It was revealed that the average ice adhesion strength is linearly correlated with 1 + cosθsurfaces . From Fisurfaces . It is wA strict definition of icephobicity remains unclear. It was suggested that a surface should be called icephobic if it delays ice formation at temperatures below the freezing point of water and/or if it has a weak adhesion strength to ice of less than 100 kPa . The bouWhen an aircraft flies through the atmosphere, its surfaces may undergo impact from hydrometeors such as rain, which can adversely affect the structure of the aircraft and reduce the lifecycle of the components . TherefoH,1H,2H,2H-perfluorooctyltriethoxysilane were grafted onto the silica nanoparticle surface by the chemical vapor deposition method to reduce the surface energy. The morphology, composition, and functional groups were characterized to reveal the relationship between the characteristics of the nanocomposite material and the hydrophobicity. An average water contact angle of 163° suggests a super-hydrophobic surface was obtained on silica nanoparticles with surface modification by SAMs of POTS. The water droplet icing test results show that the icing formation of silica-based nano-coatings was significantly delayed compared to bare substrates and commercial icephobic products due to the existence of the low surface energy and air pockets on the surface. The ice adhesion strength test results show that the shear stresses between the treated surface/ice block are much lower than those between the bare substrate/ice block. The icing delay and low ice adhesion strength suggest icephobic surfaces have been obtained from the super-hydrophobic silica-based coatings. To evaluate durability, a test rig of erosion from pneumatic water impinging was designed and set up. The erosion test results show that super-hydrophobicity remained after testing for 60 min. Further optimization aiming for aircraft applications is in progress.Silica nanoparticles were deposited onto glass substrates to form a nanostructured rough surface with the function of trapping small-scale air pockets. Self-assembled monolayers (SAMs) of 1


We report on the discovery and characterization of ice β-XV, which represents the second hydrogen ordered polymorph related to ice VI. –1, which is much larger than for the known hydrogen ordered proxy of ice VI, ice XV. Raman spectroscopy indicates the 1.80 GPa sample to be more ordered than ice XV. It is further distinct from ice XV in that it experiences hydrogen disordering above ≈103 K which is 26 K below the ice XV to ice VI disordering transition. Consequently, below 103 K it is thermodynamically more stable than ice XV, adding a stability region to the phase diagram of water. For the time being we suggest to call this new phase ice β-XV and to relabel it ice XVIII once its crystal structure is known.In the last twelve years five new ice phases were experimentally prepared. Two of them are empty clathrate hydrates and three of them represent hydrogen ordered counterparts of previously known disordered ice phases. Here, we report on hydrogen ordering in ice VI samples produced by cooling at pressures up to 2.00 GPa. Based on results from calorimetry, dielectric relaxation spectroscopy, Raman spectroscopy, and powder X-ray diffraction the existence of a second hydrogen ordered polymorph related to ice VI is suggested. Powder X-ray data show the oxygen network to be the one of ice VI. For the 1.80 GPa sample the activation energy from dielectric spectroscopy is 45 kJ mol Oxygen atoms are arranged in two interpenetrating but not interconnecting networks of hexamer cages. This “self-clathrate” is one of the most complicated of all known crystalline ice phases.et al. reported the structure of ice XV, the hydrogen ordered form of ice VI.Cc space group symmetry for ice XV,P1.Cc nanocrystallites was proposed, which are destabilized as soon as the dielectric constant of the transforming ice VI is reduced.One of water's anomalies is its rich polymorphism. Currently 17 crystalline phases are known.The complicated nature of ice VI involving two unconnected networks implies that point defects migrating in one of them are unable to switch to the other network. This is a source for complex thermal signatures associated with the disordering in ice XV. For other ordered ices, such as ice XI,2O to 77 K, compressing to 1.0 GPa using our ZWICK BZ100 material testing machine with a piston-cylinder setup (8 mm diameter bore), heating to 255 K and quenching with liquid nitrogen at ≈–45 K min–1. The ice VI reference was prepared the same way except that no dopant was introduced, i.e., pure H2O instead of 0.01 M HCl was used. The new polymorph identified in this work was prepared by varying this protocol. After cooling 0.01 M HCl to 77 K at 1 bar, pressures of 1.0–2.0 GPa were applied before heating to 255 K. To induce hydrogen ordering the subsequent high-pressure cooling rate was reduced to ≈3 K min–1. At 77 K the pressure is released to recover the hydrogen ordered ice at ambient pressure.The ice XV reference was prepared following the protocol by Shephard and Salzmann,–1. Therefore, we will refer to those samples based on their preparation pressure. That is, in the following the term “1.80 GPa sample” refers to a sample prepared by cooling ice VI from 255 to 77 K at 1.80 GPa and ≈3 K min–1. In addition to studying the quench-recovered ices directly, we also studied these latter ice samples after heating and recooling them at ambient pressure. This procedure was described by Whale et al. in the literature.e.g., in the following the term ice XVrec(130 K) will refer to a recooled ice XV reference sample that is studied at ambient pressure after heating to 130 K and recooling to 77 K. A 1.80 GParec(114 K) sample refers to a sample of the new polymorph that was heated to 114 K and then recooled at ambient pressure.Except for the ice VI and ice XV references, all samples studied in this work are cooled at ≈3 K min–1 from 93 to 253 K – which first results in the disordering of the H-atom network and subsequently to a rearrangement of the O-atom network to produce ice I.–1.–1, independent from the baseline choice and including all random and systematic errors. Additionally recooling experiments–1.All ices were scrutinized calorimetrically using a Perkin Elmer DSC 8000 and compared with earlier work of Shephard and Salzmann.–1 the 1.80 GPa sample was heated directly to the temperature range above 130 K, in which ice VI is stable. In addition we studied sample 1.80 GParec(114 K) and heated it from 102 to above 130 K. Finally, an ice XV reference sample prepared at 1.00 GPa was also measured. In the Arrhenius plots that will be used to determine activation energies, closed symbols represent relaxation times, which were evaluated by fitting the dielectric loss peak using a Havriliak–Negami function;To measure the dielectric response of the ice crystals, the samples were transferred from Innsbruck to Dortmund under liquid nitrogen conditions. A Novocontrol Alpha-A impedance analyzer was used to investigate the dielectric response as described in earlier works.nd-order Savitzky–Golay filter (7 points). Samples containing 5 wt% D2O were studied in order to decouple the OD from the OH vibrations as in many studies before.2O samples containing 5 wt% D2O by comparing these samples with pure H2O samples. The agreement of DSC scans and OH and translational regions in Raman spectra shows that the ordering to the new polymorph is not affected by adding small amounts of D2O.All Raman spectra were recorded with a WiTec WMT50 Spectrometer  inside an Oxford N Microstat controlled by a Lakeshore 331S temperature controller. Samples were powdered and loaded into the microstat at 84 K. Crystals were selected under the microscope for Raman measurements in confocal geometry. Raman spectra were taken using accumulation times of about 30 minutes. The data are smoothed with a 2For powder X-ray diffraction measurements samples were powdered under liquid nitrogen and transferred to the sample chamber. X-ray patterns were recorded in flat geometry on a Cu sample holder with an XPERT III PANalytical Xpert Pro MPD diffractometer  at 129 K. The second interpretation involves a homogeneously ordered sample that converts to ice VI in two distinct steps, where both steps involve hydrogen disordering. In that latter interpretation the previously inaccessible phase transforms in the first step at 103 K to a less hydrogen ordered phase, which is presumably ice XV, which in the second step at 129 K disorders to produce ice VI.This naturally raises the question what the two phases before and after the first endotherm are. Our temperature dependent X-ray studies (see below) indicate that the network of oxygen atoms is the same as the one in ice VI. At ambient pressure this network is retained up to about 150 K, where the oxygen network rearranges to form ice I. An amorphous nature and all other known ice polymorphs, rec(114 K) sample. For comparison, we have also studied an ice XV reference sample prepared at 1.00 GPa. Based on the previous section for this reference sample an additional phase transition (related to the first endotherm at 103 K) is not expected.In order to learn more about the nature and the relaxation dynamics of the 1.80 GPa sample we have studied quench-recovered crystals using dielectric spectroscopy. Runs on three different 1.80 GPa samples obtained from the same batch were performed. During the first two runs dielectric loss spectra were recorded by heating the sample to temperatures at which ice VI is stable. As part of the third run the 1.80 GPa sample was recooled after heating it up to 114 K resulting in the 1.80 GPaT = 3 K. via τ = 1/(2πνpeak) signals a reduction of the dipolar relaxation times τ. An interesting feature is observed in T step changes significantly near 105 K. This behavior was detected in all runs carried out for the three different 1.8 GPa samples (see below). The dielectric data presented in this work thus hint at the occurrence of a phase transition near 105 K, in accord with the conclusion drawn from the first, low-temperature endotherm observed in DSC scans.Dielectric loss spectra were obtained upon heating in temperature steps of typically ΔThe ice XV reference sample was studied in the same temperature range and the corresponding dielectric loss spectra are shown in T < 129 K followed by an increase in amplitude  sample, cf.To further investigate the nature of the previously inaccessible low-temperature state, rec(114 K) sample is narrower, cf.A comparison of the spectra of the 1.80 GPa samples recorded at 102 K with and without prior recooling reveals that the spectrum of the 1.80 GPaνpeak at which the dielectric loss peaks appear, the temperature dependent relaxation times τ can be obtained. activation energies Ea are calculated for each linear regime. For the 1.80 GPa sample this results in an activation energy of 45 kJ mol–1 below 108 K, an activation energy of 18 kJ mol–1 from 108 to 124 K, and 34 kJ mol–1 above this temperature. Dotted lines in From the frequencies rec(114 K) sample displays only one kink near 124 K, see via a separate analysis of the dipolar relaxation times above and below the phase transition temperatures observed in the 1.80 GPa sample, a slight change of the activation energy of the ice XV to ice VI transition becomes visible. In the intermediate temperature regime the activation energy of the 1.80 GPa sample differs from that of sample 1.80 GParec(114 K) and from that of the ice XV reference sample. This behavior cannot be explained with our current knowledge, but might be due to different degrees of relaxation in the H-network for samples that were or were not previously recooled at ambient pressure.The relaxation map of the recooled 1.80 GPaTaken together, the dielectric data hint at the existence of a low-temperature phase transition in the 1.80 GPa sample that occurs in addition to that from ice XV to ice VI. The new low-temperature phase (i) appears to be inhomogeneous, probably due to the presence of a mixture with ice XV or ice VI and (ii) undergoes a phase transition for temperatures near 108 K which under ambient pressure is non-reversible.rec(130 K) reference (panel b) and ice VI reference (panel c). Please note that the ice XV in panel b was recooled from 130 K at ambient pressure. The comparison between the spectra for these three samples  is quite revealing. In general, the 1.80 GPa sample shows the most narrow and most structured bands, see e.g., the bands at 3206 cm–1 and 2449 cm–1 are blueshifted by 16 and 10 cm–1 compared to ice XV, respectively. Similar effects, including band shifts to higher and lower wavenumbers upon ordering, were observed for ice XII and XIV.e.g., the band at 131 cm–1 is absent for ice XV (panel b) but of medium intensity for the 1.80 GPa sample (panel a). The three librational bands near 450 cm–1, 500 cm–1 and 540 cm–1 are strong, weak and strong for ice XV, but absent, strong and absent for the 1.80 GPa sample. These observations indicate that the 1.80 GPa sample is more ordered than ice XV.The nature of the hydrogen ordering was additionally scrutinized using Raman spectroscopy  and 9 be24rec(130 K) (rec(120 K) (–1 is absent for ice XVrec(120 K) and the intensity ratio for the two OD-stretching band differs significantly. This implies that there is a difference in terms of the extent of hydrogen ordering in ice XV prepared by recooling at 1 bar from different temperatures. This issue will be studied and discussed further in a forthcoming publication. Upon heating ice XVrec(120 K) from 85 to 120 K, i.e., just prior to the transition to ice VI, there are barely any spectral changes  remains stable up to 120 K. The completed transition to ice VI is evident for the spectra recorded at 133 K. The OD-band shape has changed and adopts the intensity ratios also seen for the reference spectrum of ice VI in –1. By contrast, upon heating the 1.80 GPa sample the spectra change significantly prior to reaching 120 K. While four maxima in the OD-stretching part are resolved at 82 K, only two maxima are resolved at 108, 112, 116 and 128 K. At 96 K and 104 K an intermediate situation is encountered. In the librational regime the most intense band is the 510 cm–1 band up to 97 K, whereas the 450 cm–1 band is the most intense one at 110 K. At 102 and 104 K the 450 cm–1 band is seen to grow, whereas the band at 510 cm–1 redshifts to 533 cm–1. This indicates that the librational bands are particularly sensitive to hydrogen ordering and useful to investigate phase changes in the H-atom network. Clearly, the changes near 104 K are associated with the first endotherm in –1 suggests ice XV formation from the 1.80 GPa sample, whereas the growth of the feature at 533 cm–1 suggests ice VI formation. The band at 533 cm–1 shows a decreased intensity again at 110 and 115 K in c(130 K) , middle c(120 K)  are diffnges see , i.e., i–1, 510 cm–1 and 533 cm–1 are overlapping, we have analyzed peak heights rather than the peak area. In particular we have analyzed the heights at 450 cm–1 and 510 cm–1 and defined the relative intensity of the band at 450 cm–1 RI450 as follows: RI450 = I450/(I450 + I510), where I450 and I510 denote the intensities at 450 cm–1 and 510 cm–1, respectively. 450 as deduced from the data in rec(120 K) (blue circles) and ice VI . For ice VI the intensity ratio RI450 slowly increases from 0.4 at 100 K to 0.5 at 132 K. For XVrec(120 K) the RI450 ratio is 0.90 ± 0.05 below 100 K and decreases to 0.7 at 120 K and then to 0.5 at 132 K as the sample transforms to ice VI. For the 1.80 GPa sample RI450 is 0.28 at 84 K and gradually increases to 0.70 at 110 K. The gradual change is consistent with the complex nature of the hydrogen disordering transition. Near 104 K the Raman spectra resemble more the ones of ice VI, but at 110 K and above a closer similarity with the spectra of ice XV is noted. However, the spectra are neither identical to the ice VI nor to the ice XV reference spectra. This suggests that the 1.80 GPa sample has indeed to be viewed as inhomogeneous, as also indicated by dielectric spectroscopy. Taken together it has become clear that also Raman spectroscopy provides evidence for a distinct ice polymorph more hydrogen ordered than ice XV present in an inhomogeneous sample recovered from 1.80 GPa.Since the librational peaks at 450 cmi.e., a pronounced isotope effect precludes such experiments.2O sample cooled at 1.4 GPa at 0.5 K min–1 does not show the second endotherm. This is consistent with the neutron diffraction results obtained by Salzmann et al.et al. did not report any hints for the existence of a phase other than ice VI and ice XV in deuterated samples, even though the pressure range up to 1.60 GPa was investigated.In order to learn more about the nature of the hydrogen order in the new polymorph, in particular whether or not it displays ferroelectricity and what its space group is, neutron diffraction experiments on deuterated samples would be desirable. However, in the fully deuterated samples studied for this work a second endotherm, analogous to that observed in 2O samples. Hydrogen atoms barely scatter X-rays. Therefore, different types of hydrogen ordering can be inferred merely from indirect effects such as slight changes in the oxygen network, as for instance exploited by Kamb et al. for the example of ice II.29For a crystallographic comparison of the new polymorph prepared in this work with conventionally prepared ice XV we used powder X-ray diffraction on Hd = 0.265 nm  sample than for the 1.80 GPa sample containing domains of the new hydrogen ordered polymorph. The steeply increasing part that is observed for the 1.80 GPa sample up to 104 K is missing for the 1.80 GParec(118 K) sample. Instead, the data of the 1.80 GParec(118 K) sample show overlap with those of the 1.80 GPa sample above 124 K, when both finally transform to disordered ice VI. Above 124 K, all Bragg peaks converge to the position of the ice VI Bragg peaks. However, below 124 K there is a clear discrepancy in the Bragg peak position between ice XV and the new polymorph. This rules out that the new polymorph is identical to ice XV and again demonstrates the presence of three distinct phases upon heating in the sample prepared at 1.80 GPa. All of the three distinct phases share a highly similar oxygen network. Since with ice VI and ice XV only two such phases are known, our findings imply the discovery of a novel variant of ice XV/VI.The temperature dependence of the lattice spacing  for the cf. the ice VIII ticks in It is suggested that our experimental data imply the presence of a new hydrogen ordered phase of ice VI. Here, we discuss arguments indicating that alternative interpretations can be ruled out. An important question we consider is, whether or not the new polymorph is the same as a known ice phase or a mixture of different known phases. The X-ray data clearly exclude all ice polymorphs except for ice VI and its hydrogen ordered proxies.rec(114 K) only displays one kink upon heating, consistent with the results for the ice XV reference sample.The dielectric loss data are consistent with this conclusion. For the 1.80 GPa sample the appearance of three linear regimes in the relaxation map suggests the presence of three phases. The location of the two kinks separating the three phases is close to the location of the two endotherms in 2O ice nor for systems such as spin ices.It is not entirely clear from our data whether or not the phase identified in this work transforms first to ice XV near 100 K and then to ice VI above 120 K. Such a two-step disordering separated by more than 20 K would be unique. We are not aware of a related two-step disordering neither for Hvia ice XV or whether the transformation takes place via states of hydrogen ordering distinct from ice VI as well as from ice XV.Raman spectroscopy indicates the new polymorph to be more ordered than ice XV based on more narrow and structured bands in the former than the latter sample. Analysis of the transformation behavior upon heating suggests that the new polymorph transforms neither to ice VI nor to ice XV upon heating to 103 K, but adopts a hydrogen ordering pattern different from these two phases. It needs to be settled first what the Raman spectra of ice XV at 115 K really imply before Raman spectroscopy allows one to clarify whether the new polymorph develops towards ice XV or not. The difference between the spectra of ice XV recooled from 130 K , middle,Cc phase originally envisaged for ice XV which is now considered to be antiferroelectric.et al.In summary, we found strong indications for the existence of a second hydrogen ordered phase of ice VI. The new phase revealed by our 1.80 GPa sample was carefully compared with ice XV, both in its “pristine” form obtained from cooling at 1.00 GPa as well as in its recooled form obtained at 1 bar. The phase behavior is markedly different for the various samples as consistently observed in X-ray, dielectric, calorimetric and Raman experiments. The observation of a discontinuous transition involving latent heat in  the phase boundaries between the ice β-XV/ice II and the ice β-XV/ice VIII neighbors have to be almost vertical. This is because of the small change in entropy occurring at the phase transition between two ordered phases of differing densities. Furthermore, the densities of ice XV and ice β-XV are similar, as inferred from the X-ray experiments. Therefore, the phase boundary between these two phases needs to be almost horizontal, resulting in an almost rectangular stability domain. Thus, two triple points, each associated to three hydrogen ordered phases, are identified at T/pII–XV–βXV = 103 K/0.8 GPa and T/pVIII–XV–βXV = 103 K/1.5 GPa. The phase boundaries of the ices II–XV and XV–VIII are based on work of Salzmann et al.Based on the calorimetry and Raman results the new polymorph seems to be more ordered and hence thermodynamically favored over ice XV at temperatures below 103 K. This means that ice β-XV represents a new stable phase. Its stability range in the phase diagram is shown as the colored rectangle in 2O samples, an investigation of the effect of quantum tunneling on the formation of ice β-XV and neutron diffraction to possibly unravel the crystal structure. Also the states of hydrogen ordering involved in the phase transitions merit further investigation.Future experiments to further increase our understanding of hydrogen ordering will involve DT. G. prepared all samples and conducted X-ray diffraction and calorimetry experiments, A. T. conducted Raman experiments. L. P. and K. K. conducted dielectric experiments, T. G. and T. L. analyzed Raman, X-ray and calorimetric data, A. T. analyzed Raman data and L. P., K. K. and R. B. analyzed the dielectric data, M. E. analyzed the X-ray data, T. G. and T. L. designed the research, T. G., T. L., L. P. and R. B. wrote the manuscript.thomas.loerting@uibk.ac.at).The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to T. L. (Email:


The discovery of an exclusive local production of IgE antibodies dates back to the 1970s, but only recently the pathophysiology of such phenomenon was deeply investigated, leading to the concept of local allergic rhinitis (LAR). Currently, LAR is defined by the occurrence, in patients with symptoms clearly suggesting allergic rhinitis but with negative results to common allergy testing, of allergen specific IgE in the nasal mucosa. Most studies investigating LAR were based on the development of rhinitis symptoms following nasal provocation test (NPT) with the suspected allergens, but such test may be performed by a number of options, none of them being as yet acknowledged and recommended in consensus document. On the other hand, also the mere detection of IgE in the nasal mucosa indicates, as for IgE measurement in blood or other tissues, allergic sensitization but cannot give the certainty of clinical allergy. Therefore, the combination of IgE detection in nasal mucosa and a positive result of NPT should be used to diagnose LAR. Recent data on the use for in vitro testing of molecular allergy diagnostics in place of whole allergen extracts suggest that this method could improve the sensitivity and specificity of laboratory tests, and an appraisal of the basophil activation test as a third level technique, to be implemented when the results of local IgE testing and NPT are uncertain, is currently ongoing. Dermatophagoides could be found in nasal secretions [The discovery of the exclusively local production of IgE antibodies in the nasal mucosa was made thanks to Huggings and Brostoff, who in 1975 reported that in patients with symptoms clearly suggesting allergic rhinitis (AR), but with negative results of allergy testing, IgE antibodies to cretions  and in tcretions . Indeed,cretions . In the Dermatophagoides pteronyssinus was positive in 54% of patients with a diagnosis of nonallergic rhinitis (NAR) based on the negative results to common allergy testing [p = 0.623). This defines LAR as a well-differentiated clinical entity with a low rate of progress to AR and a natural history towards worsening [Dermatophagoides in 54% of patients, but only 22% of patients had specific IgE in the nose. Fuiano et al. [Alternaria spores were present in the air, a positive result of nasal IgE and NPT was observed in 69.6% of patients, while positive SPT and NPT occurred in 26.8% of patients (p < 0.0001); this suggests that the association of NPT and nasal IgE has higher diagnostic capacity [Alternaria allergy was confirmed also in adults, in whom both positive allergen-specific NPT and specific IgE in nasal secretions were found [The definition “local allergic rhinitis” (LAR) was first proposed by Rondón et al.  in a rev testing . The samorsening . Indeed,orsening . While corsening . Therefoo et al.  reportedcapacity . Such dire found .D. pteronyssinus. Similar ratios between LAR and AR were reported in a study on 219 elderly patients (mean age 65.8 years): 21% of patients had LAR and 40.2% had AR, D. pteronyssinus being the major culprit [The actual prevalence of LAR is uncertain. LAR was long considered a rare disorder, until Rondón et al.  in 2012  culprit . Such daThe pathomechanisms of AR are triggered by an inflammatory response in the nasal mucosa including an immediate IgE-mediated mast cell response and a late-phase response with recruitment of eosinophils, basophils and T cells expressing a Th2 cytokine profile, comprising interleukin (IL)-4 and IL-5. Recently, cytokines regulating the Th2 response, such as thymic stromal lymphopoietin, IL-25, and IL-33, were added as important factors . The patAs discussed above, NPT alone cannot achieve a certain diagnosis of LAR. On the other hand, it was recently hypothesized that the nasal IgE production may represent a form of spontaneous immune response, based on a study on three small groups of subjects, one with AR, one with NAR, and healthy controls undergoing measurement of specific IgE to various allergens in nasal scrapings. Allergen-specific nasal IgE were detected in all groups , leading the authors to conclude that the presence of nasal IgE to allergens seems to be a non-specific phenomenon . Indeed,


The curling motion of the curling stone on ice is well-known: if a small clockwise rotational velocity is imposed to the stone when it is released, in addition to the linear propagation velocity, the stone will curl to the right. A similar curl to the left is obtained by counter-clockwise rotation. This effect is widely used in the game to reach spots behind the already thrown stones, and the rotation also causes the stone to propagate in a more predictable fashion. Here, we report on novel experimental results which support one of the proposed theories to account for the curling motion of the stone, known as the “scratch-guiding theory”. By directly scanning the ice surface with a white light interferometer before and after each slide, we observed cross-scratches caused by the leading and trailing parts of the circular contact band of the linearly moving and rotating stone. By analyzing these scratches and a typical curling stone trajectory, we show that during most of the slide, the transverse force responsible for the sideways displacement of the stone is linearly proportional to the angle between these cross-scratches. The tendency of rotating and moving objects to travel along a curved trajectory is essential in many sports, and understanding the underlying physical mechanisms is a fascinating problem. Indeed, depending on the system, counter-clockwise rotation may lead to a curved trajectory towards left or right, or to no turn at all, see Fig. In the Olympic winter sport of curling, two teams slide granite stones across an ice sheet towards a target area. In each of the predetermined amount of game-parts (ends), points are awarded only to the team with a stone closest to the center of the target area: one point is scored for each stone beating the opposing team’s best stone. The essential characteristic of the game is a curled trajectory of the rotating curling stone and is well-known among the players: if a small clockwise rotational velocity is imposed to the stone when it is released, in addition to the linear propagation velocity, the stone will curl to the right. A similar curl to the left is obtained by counter-clockwise rotation. This effect is widely used in the game to reach spots behind the already thrown stones and rotation makes stone travel more predictable. What makes this phenomenon intriguing is that even if its existence is well-known, the underlying tribological mechanism remains to be explained properly. In fact, the mechanism that puts this curl in the curling stone is under scientific debate and novel experimental evidence is required.The system of interest consists of the granite stone, weighing a little less than 20 kilograms, moving on the ice surface. The geometry of the stone is such that the apparent contact area between the stone and the ice surface is given by a circular band with a width of approx. 6 mm and a diameter of 120 mm. Both the bottom surface of the stone and the ice surface are rough: the bottom of the stone is intentionally roughened to control the magnitude of the ice-stone friction, as well as the curvature of the trajectory of the stone. The ice surface consists of many small protrusions known as pebbles, produced by spraying water droplets onto an initially flat ice surface. These pebbles, rough stone bottom surface, stone weight and small real contact area clearly form a combination favorable for a curled trajectory. As an example, the ice hockey puck does not have these properties and consequently it does not curl. and The stone is put to motion by releasing it before the release (hog) line, approx. 28 meters from the center of the target area. The player throwing the stone aims at adjusting the initial linear propagation velocity as well as the rotational velocity of the stone in order to get it to a desired final position within the target area. In addition, team mates may attempt to affect the motion of the stone by sweeping the ice in front of the sliding stone with special brooms. Under typical conditions of the game, with 3–4 revolutions during the propagation of the stone, the sideways displacement of the stone tends to be of the order of 1–1.5 meter.1. Indeed, if the leading half of the stone experiences a smaller friction force than the trailing half, the stone will be subject to a net lateral force towards right (left) in the case of clockwise (counter-clockwise) rotation, as illustrated in Fig. 2. Mechanisms that have been proposed to lead to a front-rear asymmetry in friction include frictional melting of ice1, pickup of ice debris along the leading half of the contact band3, and temperature reduction originating from evaporation of a liquid film formed by the leading half4. However, the explanatory power of any such ideas is limited by the maximum lateral displacement they may account for: even in the extreme case of assigning all the friction to the trailing half, the resulting lateral displacement has been estimated to be approx. half of that empirically observed5.With the aim of explaining the empirically observed curved trajectory of the curling stone, several mechanisms have been proposed in the literature. One often-presented argument is related to a proposed asymmetry of the friction force between the leading and trailing parts of the contact band6. There, the idea is that the front half of the rough contact band scratches the ice, with the scratches tilted to the right or left from the propagation direction depending on the rotation direction of the stone (clockwise or counter-clockwise). The asperities of the trailing part of the rough contact band have to cross these scratches, generating another set of scratches, with the opposite tilt as compared to those due to the leading part , we observe cross-scratches caused by the leading and trailing edges of the linearly moving and rotating stone. Moreover, by analyzing the angle at which these scratches are formed for different combinations of translational and rotational velocity of the stone, we establish a strong correlation between the angular difference between the two sets of parallel scratches angle and the sideways displacement of the stone during a typical trajectory of the stone. Our observations and analysis thus supports the scratch guiding theory as the dominating mechanism explaining the curved trajectory of the curling stone.In this paper, we focus on observing the details of the topographical changes that occur on the surface of the ice sheet during the sliding of the curling stone. By directly scanning the ice surface in an almost in situ manner, rather than studying a replica of the ice sheet as done in previous studies6.To study the topographical features formed in the ice surface due to the sliding curling stone, we built a custom test track in a cold room (temperature around −5 °C). The track consists of an ice sheet with a removable part. This part of the ice sheet can be removed quickly from the track and placed in the white light interferometer for measurement immediately after sliding the curling stone over it, see Methods for details. This procedure allows us to directly scan the ice surface in an almost Figure Moreover, Fig. 4. As shown above, the angle between the velocity vectors of the leading and trailing edges equals the angle between the two sets of scratches observed in white light interferometry measurements; in what follows, we’ll refer to this angle difference as the scratch angle difference (SAD). Initially, as the longitudinal velocity dominates, SAD is small, see Fig. 8, where the sliding and spinning degrees of freedom were found to be coupled. In the middle panel of Fig. Finally, we consider the typical time-evolution of the longitudinal and rotational velocities, and hence of the angle between the velocity vectors of the leading and trailing edges of the contact annulus during the typical dynamics of the curling stone; the data analyzed is obtained from ref.Ftrans should be some function Ftrans(θ) of SAD, denoted here by θ. For small θ, one may linearize Ftrans(θ), and thus arrive at an equation of motion describing the transversal motion of the stone for small θ,M is the mass of the stone, and C a constant. Integrating Eq. (vtrans(t) is proportional to the time integral of θ. In the bottom panel of Fig. Ftrans(θ) employed above breaks down. The linearization seems to work well for SAD-values smaller than θ ≈ 25°, corresponding to the dark blue region in the top panel of Fig. The general SAD dependence of the transverse force responsible for the curved trajectory of the stone originating from the scratch-guiding mechanism is difficult to establish or derive, due to the complexity of the underlying microscopic processes. However, one may argue that the transverse force in situ observation of cross-scratches created by the leading and trailing halves of the contact annulus of the curling stone. The orientation of these scratches is given by the local velocity vectors along the contact annulus of the propagating and rotating stone. The presence of these two sets of parallel scratches allows one to define the quantity we refer to as the scratch angle difference (SAD), which depends on the relative magnitudes of the longitudinal and rotational velocities of the stone. We found that the transverse force responsible for the curved trajectory is linearly proportional to SAD (denoted by θ) for small θ (θ < 25° or so), and hence the time-dependence of the transverse velocity is linearly proportional to the time integral of θ. Together, these two observations provide strong evidence that at least up to a few seconds before the stone comes to a halt, the dominating contribution responsible for the curl of the curling stone is given by the scratch-guiding mechanism6.In this paper we have presented the first experimental, nearly Ftrans(θ), may become non-linear, so that the dominating contribution to Ftrans would still be due to the scratch-guiding mechanism, or (ii) some other mechanism may start operating. For instance, one could argue that the scratch-guiding mechanism may become stronger for small longitudinal velocities because of different fracture behaviors operating for different time scales (“more time to follow scratches”), thus giving rise to the strongly curved trajectory at the very end of the slide. On the other hand, one could imagine other mechanisms that might play a role, including for instance possible local sticking of the “small sliding velocity” quarter of the contact annulus. Obviously, several different mechanisms may be operating simultaneously. In any case, the final stages of the sliding process appear to be governed by complex, non-linear mechanisms originating from microscale contact mechanics. Thus, understanding the related details poses an important challenge to be tackled in future work.For large SAD, i.e., typically during the last few seconds of the slide of the stone, the above-discussed linearization breaks down. This could be due to two main reasons: (i) the dependence of the transverse force on SAD, The experimental setup was built in a cold room (temperature around −5 °C and relative humidity 80–85%). A sketch of the setup is shown in Fig. Three sheets of plywood were put on a base with metallic edges to construct the basis of the ice sheet. The smallest plywood sheet (length: 20 cm) was removable and positioned as indicated in Fig. 9 and ice skate friction10. A plate was installed in the interferometer in order to attach the removable piece of ice exactly in the same position in the different experiments. The spatial resolution of the interferometer is 55 nm, but accuracy is restricted by the scanning light wavelength. Images of a width of 6 mm and length of 4,5 mm were scanned before and after each slide. After each slide the sheet was repaired by spraying water on it.For scanning the surface of the ice sheet a white light interferometer (Bruker Contour GT-K Automated System), which was also placed in the cold room, was used. The same experimental methodology has been previously used to study rubber-ice frictionDifferent sliding configurations were used to mimic diverse scenarios: We considered rotating the stone without propagation, rotating clockwise and counterclockwise, slides with different combinations of translational and rotational speeds, as well as slides in which either only the front edge or the whole stone crossed the pebble.The directionality of surface topography was shown by two-dimensional PSD plots in Fig. The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.


Effects of CTS in vitro on cell proliferation, cycle, migration and invasion were evaluated. The activation of JAK/STATs signaling was detected by western blot and immunofluorescenc staining. SHP-2 inhibitor or SiRNA were used to determine the involvement of SHP-2. The in vivo anti-MGs activity of CTS was studied with nude mice bearing intracerebral U87 xenografts. Our results revealed that CTS significantly inhibited the proliferation of MGs in vitro via inhibiting STAT3 signal pathway. The cell cycle was arrested at G0/G1 phase. Although CTS did not change the expression of total SHP-2 protein, the tyrosine phosphatase activity of SHP-2 protein was increased by CTS treatment in a dose-dependent manner in vivo and in vitro. SHP-2 inhibitor or SiRNA could reverse the inhibitory effect of CTS on phosphorylation of STAT3 Tyr705. In vivo study also showed that CTS inhibited the intracranial tumor growth and extended survival of nude mice bearing intracerebral U87 xenografts, confirming an inhibitory effect of CTS on MGs. Our results indicated CTS may be a potential therapeutic agent for MGs. The inhibitory action of CTS is largely attributed to the inhibition of STAT3 Tyr705 phosphorylation with a novel mechanism of upregulating the tyrosine phosphatase activity of SHP-2 protein.Malignant gliomas (MGs) are one of the most common primary brain cancers in adults with a high mortality rate and relapse rate. Thus, finding better effective approaches to treat MGs has become very urgent. Here, we studied the effects of cryptotanshinone (CTS) on MGs  In 2010, a literature published in Nature reported that STAT3 has an important role in mesenchyme differentiation and predicts poor clinical outcome in human glioma.3 Thus, STAT3 signaling is considered a potential target for therapeutic intervention of MGs.5 Many STAT3 modulators have been demonstrated, such as metal complex,7 peptide,8 small interference of specific RNA (SiRNA),9 natural products.10 STAT3 activation is negatively regulated by protein tyrosine phosphatase (PTPs), protein inhibitor of activated STAT family proteins (PIAS), suppressor of cytokine signaling (SOCS) and ubiquitin–proteasome pathway.11 For instance, some PTPs, such as SHP-1, SHP-2 and TC-PTP, can regulate STAT3 activation through tyrosine dephosphorylation.14 Activating SHP-1 by Morin was reported to have an inhibitory effect on STAT3 activation.15Signal transducer and activator of transcription 3 (STAT3) belongs to the STATs family, and may be the most intimately linked to tumorigenesis.17 Cryptotanshinone (CTS), one of the major representative components isolated from the root of Salvia miltiorrhiza Bunge has been reported to have inhibitory effects on STAT3 activation. It was also reported to have other pharmacological effects including anticardiovascular diseases,18 anti-inflammation,20 neuron protection21 and antiproliferation of cancer cells.22 We recently observed that CTS suppressed the proliferation of human MG cells T98G and U87 through inhibition of STAT3 signaling pathway.23 However, little is known about its in vivo effect on tumor, particularly MGs. Because CTS can pass through the blood–brain barrier,24 we inferred that CTS may have a potential therapeutic effect for MGs. In the present study, we explored the role and mechanism of CTS by both in vitro and in vivo MG models. Our results disclose that CTS may be a potential drug candidate for the treatment of MG via upregulating the PTP activity of SHP-2.Using structure-based virtual screening method, a lot of STAT3 inhibitors from nature products have been identified.μM) for different time periods . The results showed that the proliferation of C6, U251, U87 and T98G cells was all inhibited by CTS in a time- and dose-dependent manner  of CTS for 48 h followed by BrdU cell proliferation ELISA kit measurement  for 16 h. Cell migration into the wound was measured according to the distance between the wound edges before and after CTS treatment. We observed that CTS significantly impaired cell migration into the wound . However, no changes were detected in U251 cells  for 24 h, we detected the expression of these proteins. The results showed that the expression of cyclin D1 and survivin was decreased in a dose-dependent manner  were injected intracerebrally. The nude mice were treated with CTS (30 mg/kg/day or 60 mg/kg/day) from day 8 to 28. On day 28, the animals were killed and their brains were harvested, fixed and sectioned. As shown in in vivo effect of CTS on SHP-2, we measured its tyrosine phosphatase activity in harvested tumor tissues. We found that after CTS treatment, the tyrosine phosphatase ativity of SHP-2 was increased significantly , an indicator for reactive gliosis in the brain tissue adjacent to the tumor by immunohistochemistry in paraffin-embedded glioma sections. The results revealed that CTS treatment reduced the intensity of p-STAT3 (Tyr705), Ki67 and GFAP staining . The numSalvia miltiorrhiza Bunge, has been reported to possess a great antiproliferation effect on different cancer cells.31 In the present study, we confirmed that CTS could significantly inhibit the proliferation of MGs cells in vitro and the growth of intracranial tumor in vivo. The survival period of nude mice bearing intracranial U87 xenografts was obviously extended. These results suggest that CTS may be a potential therapeutic agent for MGs.Classical therapeutic approaches for MGs include surgical resection, postoperative radiotherapy and chemotherapy . However, since the invasive growth, incomplete surgical resection, losing insensitivity to radiotherapy and chemotherapy, it remains a high mortality and relapse rate. Therefore, more effective therapeutic approaches are urgently needed to improve the outcome of glioma treatment. CTS, as a main active component isolated from the root of 32 In breast cancer cells, CTS inhibited breast cancer cell growth via suppressing estrogen receptor signaling.29 In HepG2 and MCF7 cells, CTS induced ER stress-mediated apoptosis.33 In colorectal cancer cells, CTS inhibited proliferation and growth via STAT3 signaling as well.30 Shin reported that CTS inhibited phosphorylation of STAT3 Tyr705, which was independent on the suppression of JAK2 phosphorylation.17 STAT3 has two phosphorylation sites, Tyr705 and Ser727. The Tyr705 site is the main phosphorylation site of STAT3. It has been reported that more than 50% p-STAT3 (Try705)-positive immunohistochemical staining in WHO grade III or IV glioma.5 Ser727 phosphorylation is considered a secondary event after Tyr705 phosphorylation.34 In our present study, we also found that the inhibitory effect of CTS on phosphorylation of STAT3 Tyr705 in MGs cells was independent on activation of JAKs. Also, our results showed that CTS had no significant effect at Ser727-phosphorylated site (35 survivin36) expression, and has a very important role in malignant growth and proliferation of MGs.37 In the present study, we found that CTS inhibited both cyclin D and survivin which control cell proliferation, but not cyclin E. During the regulation of cell cycle process, cyclin D1 controls the checkpoint from G1 phase to S phase;38 survivin controls the checkpoint from S phase to G2/M phase.39 Our results showed that CTS treatment increased significantly the cell percentage in G1 phase, while decreased cell percentage in G2/M phase. This result is similar as previous report,23 indicating that the cell cycle arrest caused by CTS treatment in MG is a whole stage process but mainly in G1 arrest.Different mechanisms underlying antitumor effect of CTS have been reported in different types of tumor cells. In lung cancer cells, CTS induced autophagy via JNK signaling.ted site . Persistin vitro antitumor effect of CTS has been reported in different types of tumor cells, there have been few papers reporting in vivo antitumor effect of CTS,40 and so far, its in vivo effect on MGs have not been reported. Compared with subcutaneous models, orthotropic xenograft model of U87 cells transplantation has more similar glioma-like growth characteristic and better stereotactic localization.41 We successfully established the orthotopic implantation model of human glioma cells in nude mice, which provided the tool for evaluating the therapeutic effects of CTS on MGs in vivo. Our results showed that CTS markedly inhibited the tumor growth with prolonged lifetime of tumor xenograft mice. CTS could also decrease the phosphorylation level of STAT3. These results were accordant with the data from cultured cells.Although the 15 We detected the biological effect of CTS on the expression of some other upstream of STAT3 signaling, which is also MGs growth and proliferation-related, including Akt, PTEN, GSK3β, Erk and Chk-1.45 However, no change was found in the phosphorylation of these protein kinases.It has been reported that CTS has antitumor effect independent on p-JAKs, Tyk2, Src, which are the upstream kinases of STAT3 signaling.PTPN11 gene, which is also known as PTPN11 or SH-PTP2. SHP-2 was earlier reported as proto-oncogene.48 Over the past few years, a number of disease-associated SHP-2 mutations have been identified in Noonan syndrome, leukemias and other malignancies.49 Bard-Chapeau et al.50 reported that SHP-2 has a tumor-suppressor function in liver. Deletion of SHP-2 dramatically enhanced diethylnitrosamine-induced hepatocellular carcinoma development through the STAT3 pathway. The direct connection between activating mutates of PTPN11 and the disease makes SHP-2 provide an attractive target for mechanism-based therapeutics. As a PTP, SHP-2 can reduce the phosphorylation of STAT3 specifically, and is a STAT3 negative regulator.51 Although SHP-2 has two-way biological functions, the precise mechanisms for the pivotal role of its catalytic activity in cell signaling pathways remain to be further illustrated. Our results showed that SHP-2, but not SHP-1 and TC-PTP knockdown reversed the inhibitory effect of CTS on STAT3 phosphorylation. It indicates that SHP-2 is involved in the CTS-mediated inhibition of STAT3 signaling. It was confirmed by the result from SHP-1/2 inhibitor NSC-87877. Furthermore, we detected the tyrosine phosphatase activity of SHP-2 directly. We found that although the expression of SHP-2 protein did not change after CTS treatment, the tyrosine phosphatase activity of SHP-2 was dose-dependently increased. It is reported that SHP-2 phosphorylation at Tyr542 and Tyr580 relieves basal inhibition and stimulates SHP-2 tyrosine phosphatase activity.52 However, CTS did not alter either Tyr542 or Tyr580 site. So how CTS increase SHP-2 activity remains to be elucidated in our future study.PTP SHP-2 is encoded by the in vitro and in vivo anti-MGs activity of CTS. Here, we may have a schematic summary for the mechanisms of inhibitory effect of CTS on STAT3 signaling pathway in mind, which is shown in In our study, we show the Four- to six-week-old female BALB/c athymic nude mice were purchased from the Model Animal Research Center of Sun Yat-sen University . The mice were housed and maintained in laminar air-flow cabinets under specific pathogen-free (SPF) conditions. All animal procedures were performed following the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (NIH) and were approved by the ethics committee of Experimental Research, Zhongshan Medical College, Sun Yat-sen University. All efforts were made to reduce the number of animals used and to minimize their suffering.2 atmosphere.C6 cells were purchased from American Type Culture Collection , and other three human MGs cell lines, U251, T98G and U87, were kindly provided by Professor Panasci . The cells were cultured in high-glucose Dulbecco’s Modified Eagles Medium  supplemented with 10% fetal bovine serum  at 37 °C under 5% (v/v) CO3VO4; Sigma-Aldrich) and CTS (C5624) (Sigma-Aldrich) used in vitro. CTS used in vivo were provided by Professor Pei-Qing Liu in Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, and was suspended in 0.5% carboxymethylcellulose sodium for intragastric administration. NSC-87877  was used as SHP-2 inhibitor.MTT  , dimethyl sulfoxide , Sodium orthovanadat  for 72 h as specified by the manufacturer. The sequence of negative control (NC), SHP-1, SHP-2 and TC-PTP SiRNA duplexes  were as shown in the Cell growth curve was assessed using a colorimetric MTT assay. Furthermore, cell proliferation was tested using BrdU ELISA kit according to the manufacturer's instructions .in vitro ‘scratch’ wounds were created by scraping cell monolayers with a sterile disposable rubber as described by Tamura et al.53 and U251 cells invasion experiment was evaluated using the xCELLigence System  according to the manufacturer’s protocol.For detection of U251 cell migration, 23 The percentage of cells in each phase of the cell cycle was detected by FACS Calibur Flow Cytometry  with ModFIT using Cell cycle analysis kit .U251 cells were treated prepared for flow cytometry detection as described previously.6) suspended in 5 μl of PBS were stereotactically implanted into the corpus striatum in the right cerebral hemisphere  of the nude mice, as previously described with slightly modification.25The anesthetized mice were placed in a Kopf small animal stereotaxic instrument. The syringe needle was adjusted to the bregma, then U87 cells , followed by analysis of tumor size at the largest brain section using a light microscope as previously described with slight modification.54 Immunohistochemical staining was performed according to Guo's instructions.55 Primary antibodies were Ki67 , GFAP , STAT3  and p-STAT3 .At day 28, mice were killed and the brains were removed, fixed with formaldehyde solution and sectioned routinely into 4–5 Cell or tissue lysates were subjected to immunoprecipitation (IP) with antibody against SHP-2 (BD Transduction Laboratories). The immunocomplex was pooled down by protein A/G agarose  beads. SHP-2 PTP activity was measured using Tyrosine Phosphatase Assay Kit  as specified by its manufacturer.56 Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 ; STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 ; antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 ; GAPDH (Beyotime).Nuclear protein was extracted according to the manufacturer's instruction of the nuclear and cytoplasmic protein extraction kit (Beyotime). The protocol for western blot has been described in detail in our previous publication.t-test, and differences among the groups were assessed by one-way ANOVA and LSD post hoc test. Results were analyzed using SPSS 13.0 software . Statistical significance was accepted for P<0.05.All of the described experiments were performed more than three times, and the data were expressed as means±S.E.M. The statistical difference between the two experimental groups was determined using the independent-samples


Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on α/β and β/α interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit ε, work in synergy to fine-tune the enzyme during adverse conditions.ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the  FO is composed of the membrane ring (III14) and subunit IV. The catalytic centre of the cATPase is represented by the α/β-‘head’ with catalytic (β/α) and regulatory (α/β) nucleotide binding sites at the interfaces. The rotor of the enzyme comprises the membrane ring (III14) together with γ and ε subunits. The “head” and the rotor are linked by a peripheral stator stalk containing subunits δ, I, II and IVMuch less well studied than the eukaryotic and bacterial enzymes is the cATPase, located in the thylakoid membrane of plant chloroplasts. cATPase utilises a proton gradient established during photosynthesis for ATP production. The FTo date there are relatively few reports of PTMs and their functional role in ATP synthases. However PTMs are involved in a variety of cellular functions and have emerged as being essential for eukaryotic lifeTurning to the ATP synthases the most prominent PTM reported to date is trimethylation of Lysine-43 in the membrane ring subunit, which was found to be complete and conserved across several vertebrate species17cATPase has been the target of proteomic investigations which have focussed primarily on PTM regulation in the absence and presence of light. For example subunit β was identified as a target of Casein Kinase II in the dark period21Here we extend our knowledge of the role of PTMs in ATP synthases. We identify novel phosphorylation and acetylation sites in the chloroplastic enzyme and determine their occupancy levels. Comparing mass spectra of the naturally modified and deacetylated cATPase exposes both similarities and differences between the two modifications. Specifically, we found that the deacetylated cATPase is less stable and that nucleotide occupancy is decreased after partial deacetylation. Importantly the location of the highest abundance modification sites in the structure of the cATPase suggests that acetylation and phosphorylation control different interfaces in the α/β catalytic core as well as differentially modulate interactions with the δ and ε subunits.We purified chloroplasts from spinach leaves and extracted cATPase using a protocol described previously24Arabidopsis thaliana) chloroplasts we observed a high level of conservation. We found co-location of modifications in conserved regions together with similar sequence stretches without modification  before and after deacetylation reveals peak splitting which corresponds to acetylated and deacetylated forms of the ε subunit . After iTo investigate the importance of acetylation for stable protein interactions we employed a comparative cross-linking strategy described previouslyMany of these cross-linked peptides were found to be acetylated. Since acetylated amino groups will not be available for cross-linking we anticipate an increase in cross-linking following deacetylation if no conformational changes take place. A significant decrease in intensity was observed however for two intra-protein interactions within the ε subunit itself following deacetylation. The cross-link located in the hinge region of the anti-parallel double-α-helix decreased by a factor of >50 (K105:K112) while the N-terminus and the adjacent β-domain (N-term:K20) reduced by a factor of seven . This de293132331 head and subcomplexes reveals populations that differ by the mass of a single nucleotide  with cATPase subjected to incubation in the presence of SIRT3 and CIP. Mass spectra, recorded under the same conditions, are complicated since the stability of the deacetylated/dephosphorylated complex was compromised .To quantify this combined effect we applied comparative cross-linking to investigate changes in protein interactions after simultaneous deacetylation and dephosphorylation. As above, naturally modified ATP synthase and the unmodified ATP synthase were cross-linked with BS3-d0 and BS3-d4, respectively. Most of the cross-linked peptides contain one, or even multiple modifications, hampering this experiment. Nonetheless, we were able to quantify 19 protein interactions including eight inter-protein interactions . If we cThese findings suggest a role for phosphorylation in stabilising the protein complex globally and for acetylation in stabilising protein subunit interactions and conformational changes locally, specifically in subunits ε and δ. Furthermore, we can conclude that the effects of removing both modifications simultaneously appear to be more significant than the sum of the effects observed when modifications are removed separately.pY196), all phosphorylation sites are located in the α/β-interface harbouring the catalytic nucleotide binding site  allows conclusions to be drawn on the impact of individual sites in the functionally active ensemble. We determined the occupancy of modified sites following an intensity-based approach (Methods)36The most abundant acetylation sites are located in key positions at β/α-interfaces. Interestingly the sites that show significant abundance  and are located in the C-terminal domains of α and β subunits and in close proximity to the regulatory nucleotide binding site in subunit α and the possible binding site of the extended ε subunit. Together with the observation that deacetylation effects nucleotide and ε subunit binding this PTM in particular may play an important role in allowing access to nucleotides in the regulatory binding sites.In our previous study271 head. It is possible therefore that the ε subunit could influence nucleotide binding by acting cooperatively with the α and β subunits to control depletion of nucleotides following deacetylation.In our study, disregarding N-terminal acetylation, most of the PTMs are located in structured regions of the proteins indicating that their functional role is orientated towards stability of the complex rather than controlling the cell cycleIn a previous study chemical modification revealed residues in close proximity to the nucleotide binding site which are important for the activity of the enzyme and its ADP/ATP binding propertiesOur results therefore corroborate and extend previous studies. N-terminal acetylation of the ε subunit was proposed as a regulatory factor of cATPase following proteomics observations that the abundance of non-acetylated ε was reduced in drought stressed leaves313233Arabidopsis thaliana42The relevance of the phosphorylation and acetylation sites identified here is also underpinned by their conservation in Interestingly, phosphorylation sites, and the most abundant of the acetylation sites, are located in catalytic (α/β) and regulatory (β/α) interfaces respectively. This concentration of different PTMs in distinct interfaces within the same complex is surprising and to our knowledge has not been described previously for cATPase or any other ATPases. Given the fact that cATPases have an additional requirement for activation/deactivation during light/dark/drought conditions, as well as the universal regulatory mechanisms needed for rotary ATPases, a more complex control system could be operative here. As in other cellular regulatory mechanisms, multiple PTMs play an important role in fine-tuning enzymes through the control of distinct phosphorylation and acetylation sites. We propose that their synergistic effects on local and global stability, as well as on catalytic and regulatory nucleotide binding sites, invoke a cross-talk between phosphorylation and acetylation sites that control rotary ATPase function under a variety of external conditions.The cATPase was purified from spinach leaves as previously described24The proteins were separated by gel electrophoresis using the NuPAGE system (Invitrogen) according to the manufacturer’s protocol.Proteins were digested in-gel after gel electrophoresis as described beforeGenerated peptides were separated by nano-flow reversed-phase liquid chromatography  formic acid (FA)/5% (v/v) DMSO; mobile phase B, 100% (v/v) ACN/0.1% (v/v) FA/5% (v/v) DMSO) coupled to a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The peptides were loaded onto a trap column  and separated with a flow rate of 200 nL/min on an analytical C18 capillary column , with a gradient of 7–30% (v/v) mobile phase B over 30 min and a column temperature of 45 °C. Peptides were directly eluted into the mass spectrometer.6. The ten most intense ions were selected for HCD fragmentation in the orbitrap at an AGC target of 50,000. Singly charged ions and ions with unknown charge states were excluded from the analysis. For cross-linking doubly charged ions were included or excluded in the analysis.Typical mass spectrometric conditions were: spray voltage of 2.1 kV; capillary temperature of 320 °C. The LTQ-Orbitrap XL was operated in data-dependent mode. Survey full scan MS spectra were acquired in the orbitrap (m/z 350−1500) with a resolution of 70,000 an automatic gain control (AGC) target at 3 × 10Spinacia oleracea) using MaxQuant36Raw data were searched against the SwissProt database  using the Mascot v2.4.1 search engine (Matrix Science) or against the Uniprot database  and an FDR of 1% was assumed.Prior to MS analysis cATPase aliquots in n-dodecyl-β-D-maltopyranoside-containing buffer were exchanged against 200 mM ammonium acetate using Micro Bio-spin 6 columns (Bio Rad). Mass spectra were acquired in replicates on a Q-ToF II mass spectrometer (Waters) modified for high masses20 μl cATPase (approx. 10 μM) was deacetylated by addition of 1.25 μg human recombinant SIRT3 enzyme (Cambridge Bioscience). The solution was incubated at 37 °C for 2 hrs. For analysis of intact complexes, the buffer was exchanged to 200 mM ammonium acetate using Micro Bio-spin 6 columns and the complexes were analysed as described above. For identification/quantification of acetylation sites the proteins were (i) separated by SDS-PAGE followed by in-gel digestion, or (ii) precipitated with ethanol followed by in-solution digestion using RapiGest surfactant (see above).http://www.pfindstudio.com/software/pXtract/index.html) and cross-links were identified and quantified as describedAfter deacetylation/dephosphorylation the cATPase was cross-linked by addition of 5 μl of 2.5 mM BS3-d0 or BS3-d4. The reaction solution was incubated at 25 °C and the aliquots processed under different conditions (i.e. after deacetylation/dephosphorylation or control) were pooled 1:1. The proteins were precipitated with ethanol and digested with RapiGest (see above). The mixture of tryptic peptides and cross-linked di-peptides was analysed by LC-MS/MS as described above. Raw data were converted into mgfs using pXtract (20 μl cATPase (approx. 10 μM) was deacetylated by addition of 1.25 μg human recombinant SIRT3 enzyme (Cambridge Bioscience). The solution was incubated at 37 °C for 1 hr. Then, 100–250 units of CIP (New England Biolabs) were added and the solution was incubated 37 °C for another hour. A control sample was processed in parallel. Instead of SIRT3 or CIP the respective buffer was added. cATPase was then cross-linked with BS3 or the buffer was exchanged for MS of the intact complexes.Acetylation sites were quantified using MaxQuant software v1.4.3.17 employing the described parameters (see above). For quantification of acetylation sites, a normalisation factor was calculated from unnormalised and unmodified peptide ratios of cATPase proteins. The normalisation factor was applied to unnormalised acetylated peptides.How to cite this article: Schmidt, C. et al. Acetylation and phosphorylation control both local and global stability of the chloroplast F1 ATP synthase. Sci. Rep.7, 44068; doi: 10.1038/srep44068 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Regulation of IgE concentration in the blood is a complex trait, with high concentrations associated with parasitic infections as well as allergic diseases. A/J strain mice have significantly higher plasma concentrations of IgE, both at baseline and after ovalbumin antigen exposure, when compared to C57BL/6J strain mice. Our objective was to determine the genomic regions associated with this difference in phenotype. To achieve this, we used a panel of recombinant congenic strains (RCS) derived from A/J and C57BL/6J strains. We measured IgE in the RCS panel at baseline and following allergen exposure. Using marker by marker analysis of the RCS genotype and phenotype data, we identified multiple regions associated with the IgE phenotype. A single region was identified to be associated with baseline IgE level, while multiple regions wereassociated with the phenotype after allergen exposure. The most significant region was found on Chromosome 4, from 81.46 to 86.17 Mbp. Chromosome 4 substitution strain mice had significantly higher concentration of IgE than their background parental strain mice, C57BL/6J. Our data presents multiple candidate regions associated with plasma IgE concentration at baseline and following allergen exposure, with the most significant one located on Chromosome 4. STAT6 (on Chromosome 12) and IL-13 (on Chromosome 5)  .Leishmania major infection in a BALB/c × STS/A recombinant congenic panel , and an ovalbumin induced model of allergic asthma. The AcB/BcA panel of RCS was derived from the parental strains A/J and C57BL/6J, two strains with significantly differing IgE phenotypes. To date, these strains have been used in studies on topics such as resistance to infectious diseases like influenza and ad libitum access to food and water. Only male mice aged 9–12 wk were used in all experiments. Animal protocols were approved by the Montreal General Hospital Facility Animal Care Committee, and were in compliance with the regulations set by the Canadian Council for Animal Care.Breeding pairs for A/J, C57BL/6J, C57BL/6J-Chr 4A/J/NaJ (CSS4), and C57BL/6J-Chr 12A/J/NaJ (CSS12) mice were purchased from Jackson Laboratories . The AcB/BcA panel of RCS were generated at the Montreal General Hospital Research Institute from A/J and C57BL/6J mice  adsorbed to 1.5 mg of aluminum hydroxide , in a total volume of 0.2 ml phosphate buffered saline (PBS). At 1 wk following the third sensitization, animals were aerosol challenged for 3 d consecutively with a 30-min exposure to either 1% ovalbumin solution or PBS. At 3 hr after the final challenge, mice were killed by COTotal IgE in the plasma was measured by enzyme-linked immunosorbent assay (ELISA) using the mouse BD OptEIA kit (BD Biosciences) following the manufacturer’s instructions.A marker-by-marker analysis was conducted to identify associations between genotype and phenotype, as previously described .P values ≤0.05 were considered to be significant.Unless otherwise specified, data were analyzed by GraphPad version 5.03 (GraphPad Software Inc.). One-way ANOVA followed by a Dunnet’s post-test procedure was used to compare each RCS to its respective major parental strains. All other comparisons between three groups was done by one-way ANOVA followed by Bonferroni post-test. File S1, contains the phenotyping data of the RCS and parental strains. Genotyping marker ID and location, along with genotype for each strain, has been previously published (File S1).Supplemental Material, Previous studies have demonstrated that A/J and C57BL/6J strains of mice have significantly different levels of plasma IgE concentrations  Moisan , after sTo identify the chromosomal regions associated with plasma IgE levels, we performed a marker by marker linear regression analysis using the phenotype data, and genotype data of each strain at 1215 markers distributed across the genome . The regBased on the marker association results, we hypothesized that mouse Chromosome 4 likely harbors one or more genes important in the control of plasma IgE levels. To test this hypothesis, we used CSS4 mice, which have a C57BL/6J background with Chromosome 4 from the A/J strain. Plasma IgE levels were measured at baseline, after ovalbumin sensitization, and after ovalbumin sensitization and aerosol challenge in the CSS4 mice. The results illustrated in We were able to confirm the polygenic nature of the plasma IgE phenotype in our ovalbumin allergen-exposed mice through the identification of multiple genomic regions associated with the trait. On the other hand, the analysis of baseline IgE levels in the RCS mice identified only a single associated chromosomal region on Chromosome 4 (81.46–86.17 Mbp). This region was associated with IgE levels under all three conditions. From our experiments involving CSS4 mice, we understand that the Chromosome 4 locus alone does not recapture the entire complex trait. At baseline, A/J and CSS4 mice have similar differences from C57BL/6J mice in their IgE levels; however, in the allergen-exposed models  A/J mice have a larger difference from C57BL/6J mice than CSS4 mice. These findings support the supposition that the genetic factors influencing baseline IgE levels, the increase in levels due to antigen sensitization, and the further increase in levels due to antigen exposure, affect the phenotype in a stepwise manner at each level of allergen exposure in the process of developing an IgE-mediated allergic response.In only two cases did we observe overlap between the IgE-associated regions identified in studies by other groups and our own. Bnc2 and Psip1, which are involved in immune system development, and are associated with other allergic immune disorders (Cer1, Ttc39b, Ccdc171, Cntln, Adamtsl1, Haus6, Gm12551, and Dennd4c, but these genes do not have any prior documented associations with allergy, IgE, or asthma (The human region homologous to mouse Chromosome 4 from 81.46 to 86.17 Mbp is found on Chromosome 9 from 13.31 to 18.60 Mbp. While this region has not been previously associated with atopy, human Chromosome 9 has been associated with IgE levels in a prior study (i.e., that AHR may be IgE independent since it can develop in B-cell and mast cell deficient mice, have also been observed by other groups (Plasma IgE concentration and airway responsiveness have often been shown to go hand-in-hand in allergic asthma (To our knowledge, no other studies have reported exploring the genetics of baseline IgE concentration, but instead have focused on IgE levels postallergen exposure (www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.042739/-/DC1.Supplemental material is available online at Click here for additional data file.


ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an  Thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) continues to serve as the primary treatment option for acute ischaemic stroke (AIS) within a confining therapeutic window of 3–4.5 h from stroke onset , 2. OtheRho-kinase (ROCK), a downstream effector of the small GTPase Rho, is a serine/threonine kinase , 10 whosin vitro that rt-PA affects the BBB via plasmin-dependent activation of ROCK in astrocytes, leading to their retraction  (Santa Cruz Biotechnology) antibodies, respectively, and quantitated by densitometry using ImageJ.2, 50mM Tris-HCl, pH 7.4, 150mM NaCl and 1% (v/v) Triton X-100) with gentle agitation. To inhibit MMP activity, the broad-spectrum MMP inhibitor GM6001 (10μg/ml) was added to the developing buffer. Bands of gelatinolytic activity were visualised by Commassie blue staining and quantitated by densitometry using ImageJ software.MMP-2 and -9 activity in conditioned medium was detected by electrophoresis of serum-free media samples (20–50μl) loaded under non-denaturing conditions onto SDS-polyacrylamide gels containing 0.03% (w/v) gelatine. Following 3 washes with Triton X-100 solution (2.5% v/v) to remove SDS, gels were incubated overnight at 37°C in developing buffer  and by the methyl-thiazole-tetrazolium (MTS) assay , respectively, according to the manufacturer’s instructions. Etoposide (100μg/ml) served as a positive control for cytotoxicity.BECs and SVG astrocytes were grown to confluence in 24-well plates and stimulated in serum-free medium for 6 h or overnight (17 h). Cell death and cell viability were then determined by the lactate dehydrogenase (LDH) release assay . Probability values under 0.05 were considered significant.P<0.01) and similar levels of ROCK-2 mRNA  compared to human BECs . Interestingly, KD025 applied alone also tightened the BBB compared to control conditions . As previously seen under normoxia  phosphorylation in mouse and human astrocytes. Notably, fasudil did not influence similar cellular events in brain endothelial cells . Becausevs. 1.92 ± 0.17 fold with KD025 at 20μM, P<0.01; P<0.01; selective ROCK-2 inhibition (which preserves ROCK-1 activity) is specifically required to antagonise rt-PA-induced detrimental responses in the brain endothelium.Importantly, however, a major difference between the activity of KD025 and fasudil was revealed in human BECs. KD025 potently and concentration-dependently blocked permeability rises induced over 6 h by rt-PA and plasminogen in monolayers of BECs alone  and MMP-2 and -9 activation by rt-PA and plasmin, which were not attenuated by KD025  production and activation in our BBB model under normoxia since MMPs, heavily linked to BBB disruption by rt-PA , were real cells  and astral cells . Our gelby KD025 . In contP<0.05) in human BBB permeability and did not block rt-PA-induced morphological changes in mouse astrocytes  and notably under OGD Figs . Importain vitro and in vivo [in vivo [Comparable observations by Beckers and colleagues in human lung and umbilical cord endothelial cells  support  in vivo . ROCK-2  in vivo . Since p in vivo , 51 (a c[in vivo ), it is [in vivo  could inet al. suggested that KD025 exerts its protective actions by overall improvement of ROCK-2-affected basal endothelial permeability (rather than direct antagonism of the thrombin effect) and that the two ROCK isoforms play opposite roles mainly in the context of basal barrier function [Additional mechanistic dissection by Beckers function . SimilarBased on these findings, an integrated model can be put forward where simultaneous utilisation of KD025 with rt-PA may rapidly improve basal barrier function via ‘tightening’ of BEC permeability. In parallel, early detrimental ROCK-2 activity triggered by rt-PA in BECs and astrocytes is eliminated by KD025. The improved selectivity of KD025 may also allow conservation of ROCK-1 activity in the brain endothelium, producing a potent BBB protective action, yet beneficial activities of ROCK-1 require further experimental support. A second wave of ROCK inhibition may be required since sICH can develop within 36 h post rt-PA [Importantly, the BBB is a complex structure and the cumulative effect of an inhibitor on all of its cellular components, for example in astrocytes and pericytes in addition to BECs, will determine the net benefit of the inhibition strategy during thrombolysis. While other approaches to counteract rt-PA involve a sole compartment (for example blockade of the astrocytic platelet-derived growth factor receptor α by Imatinib ), ROCK-2in vitro that ROCK-2 is the dominant isoform in cells of the BBB, playing an important role in barrier function loss during stroke-like conditions and particularly contributing to BBB impairment by rt-PA and plasmin. The use of a selective ROCK-2 inhibitor such as KD025 together with rt-PA strengthens all cellular layers of the BBB (unlike nonselective ROCK inhibitors which are less efficient in BECs) and may therefore effectively reduce sICH with minimal hypotensive side effects. Since intravenous thrombolysis with rt-PA is likely to remain a mainstay in AIS owing to its relative simplicity and proven cost versus benefit profile [In summary, we have shown  profile , such anS1 Fig(A) Standard curves of human ROCK-1 and ROCK-2 gene expression assays .To calculate qPCR reaction efficiency for human and mouse ROCK-1 and -2 gene expression assays, a dilution series (1 in 3) was made from human (SVG) and mouse (neurons) cDNA samples. The measured cycle threshold (Ct) values were plotted against the log of the relative cDNA dilution to establish a standard curve that permits the calculation of the efficiency (E) from the slope using the formula E = 10-1/Slope. Ideally, E values ~2 are considered efficient. Human ROCK-1 standard curve Y = (-3.296)X + 24.86. R2 = 0.9988. E = 2.010892.Human ROCK-2 standard curve Y = Y = (-3.301)X + 24.49. R2 = 0.9991. E = 2.008808.(B) Standard curves of mouse ROCK-1 and ROCK-2 gene expression assays .Mouse ROCK-1 standard curve Y = (-3.233)X + 25.84. R2 = 0.9946. E = 2.038363.Mouse ROCK-2 standard curve Y = (-3.472)X + 26.19. R2 = 0.9983. E = 1.941005.(TIF)Click here for additional data file.S2 FigA, B) and SVG astrocytes  were treated for 6 h or 17 h under serum-free conditions with rt-PA  and plasminogen , in the presence or absence of KD025 (20μM). Cell-death and cell viability were then determined using the lactate dehydrogenase (LDH) assay  and the methyl-thiazole-tetrazolium (MTS) assay , with total cell lysis and etoposide (100μg/ml) serving as positive controls, respectively  by one-way ANOVA with Tukey’s post hoc analysis. #P<0.05, ##P<0.01, ###P<0.001 against control by two-tailed student t-test.BECs (vely see . No cell(TIF)Click here for additional data file.S3 FigGelatine zymograms of conditioned medium from the endothelial chamber (top panel) and the astrocytic chamber (bottom panel) taken 7.5 h post stimulation under OGD with rt-PA (25nM) and plasminogen (100nM), in the presence or absence of KD025 (20μM). Medium from cells under normoxia is included as reference (left lane). KD025 displays similar activity towards MMPs under OGD as under normoxia Click here for additional data file.S4 Fig(A) rt-PA  and plasminogen  were added to the luminal chamber of the in vitro human BBB either alone or together with the broad-spectrum MMP inhibitor GM6001 (10μg/ml), and permeability was assessed 24h later. GM6001 had only a modest (but significant) inhibitory effect on permeability increases induced by rt-PA and plasminogen. This suggests that MMPs have only a partial role in this phenomenon. n = 3. Bars represent mean±SEM. Statistical analysis by one way ANOVA with Newman-Keuls post hoc. (B) Representative phase-contrast images of primary mouse astrocytes 24h after treatment with rt-PA (50nM) + plasminogen (40nM) without or with GM6001 (10μg/ml). GM6001 was inert on its own and did not attenuate rt-PA /plgn-induced shape changes of mouse astrocytes, suggesting that this effect is MMP-independent. n = 2. Scale bars represent 100μm and 40μm in the X10 and X40 images, respectively. (C) GM6001 (10μg/ml), supplemented into the developing buffer of a gelatine zymography, effectively blocks active MMP-2 in conditioned medium of mouse astrocytes as well as MMP-2 and MMP-9 produced by HT1080 human fibrosarcoma cells.(TIF)Click here for additional data file.