In this report, by combining particle-swarm optimization method with first-principles calculations, we present a detailed study of structural, electronic, mechanical, and dielectric properties of Al2O3 monolayer. We predict that planar Al2O3 monolayer is globally stable with a direct band gap of 5.99 eV and thermal stability up to 1100 K. The stability of this high-κ oxide monolayer can be enhanced by substrates such as graphene, for which the interfacial interaction is found to be weak. The band offsets between the Al2O3 monolayer and graphene are large enough for electronic applications. Our results not only predict a stable high-κ oxide monolayer, but also improve the understanding of interfacial properties between a high-κ dielectric monolayer and two-dimensional material.The miniaturization of future electronic devices requires the knowledge of interfacial properties between two-dimensional channel materials and high- BN11131416181920sp3 covalent bonds. Furthermore, 2D structures based on binary compounds of the group IV or group III–V elements, have been predicted to be stable by first-principles calculations22232423Two-dimensional 2D) materials present exceptional electronic, optical, mechanical, and thermal properties, as well as a high surface/volume ratio, all of which make them promising for applications ranging from electronics to energy storage. The discovery of graphene, a single layer of carbon atoms arranged in planar honeycomb lattices, has stimulated dramatic research interest on other 2D materials in order to realize novel functionalization and also further miniaturization of electronic devices1D materia345782 have been proposed by first-principles calculations2 with desirable reduced band gap can be formed on a rutile TiO2 (011) surface, for which the corresponding atomic structure has been refined by subsequent theoretical studies3132κ dielectric monolayers have also been found. A Y2O3 monolayer with a hexagonal lattice has been deposited on graphene2, Co3O4, and WO3, have been synthesized by rationally employing lamellar reverse micelles methodκ dielectrics based 2D structures. Al2O3 is an excellent electrical insulator, which has been widely used in current electronic devices. Thus, it is highly desired to explore the stability of its 2D form, and understand the related physical properties. In this study, via density-functional theory (DFT) based first-principles calculations, we predict that planar Al2O3 monolayer with a hexagonal lattice is stable. This oxide monolayer is found to be an insulator with a direct gap of 5.99 eV and a dielectric constant of 2.51. It is further found that the interaction between Al2O3 monolayer and graphene is weak, which leads to band offsets larger than 1 eV.More recently, many efforts have been made to realize oxide based crystalline 2D sheets. For example, a stoichiometric silica 2D sheet with bi-layer tetrahedral configuration has been grown using a Ru (0001) substrate as the support282O3 monolayer with the constrained stoichiometric formula of Al2O3. This method has been used to predict a number of 2D materials successfullyThe particle swarm optimization (PSO) method implemented in the CALYPSO372O3 monolayer, α-Al2O3 bulk, and hybrid structures of Al2O3 monolayer on the substrates of graphene and Al (111) was sampled by Γ centered 18 × 18 × 1, 12 × 12 × 4, 6 × 6 × 1, and 9 × 9 × 1 κ-point meshes, respectively. A vacuum layer with 15 Å thickness has been inserted normal to the Al2O3 surface for all slab structures to minimize the interaction between neighboring image surfaces. In order to get an improved electronic structure, the Heyd-Scuseria-Ernzerhof hybrid functionals (HSE06) were used−6 eV and 0.01 eV/Å, respectively. To explore thermal stability of Al2O3 monolayer, ab initio molecular dynamics (MD) simulations were performed with a time step of 1 fs, canonical ensemble (NVT), and Nosé heat bath on the 5 × 5 × 1 supercell. The dynamic stability was studied by calculating the phonon dispersion using density functional perturbation theory implemented in VASP with a higher electronic convergence criterion of 10−8 eV and analyzed by using the PHONOPY codeα-Al2O3 bulk are in good agreement with previous studies and experimental results48First-principle calculations, using DFT based VASP code40422O3 monolayer predicted by the PSO simulation is shown in h-BN monolayer and graphene. This honeycomb structure is a global minimum in the space of all possible 2D arrangements of Al2O3. Then, the predicted Al2O3 monolayer structure was optimized using first-principle calculations. The equilibrium configuration of Al2O3 monolayer can be obtained from the energy minimum in the equation of state curve in 2O3 monolayer with the optimized lattice constant of 5.84 Å and the Al-O bond length of 1.69 Å α-Al2O3 bulk. The contour plot of ELFs for the Al2O3 monolayer and α-Al2O3 bulk are visualized along the Al-O bond plane, as shown in 2O3 bulk, suggesting dominant ionic bonding character. In contrast, the electron density in the Al2O3 monolayer is slightly more delocalized. This indicates that the ionic bonding is still dominant in the Al2O3 monolayer, but it is weaker than that of α-Al2O3 bulk, consistent with the shorter bond length in the Al2O3 monolayer. The enhanced in-plane covalent bonding character can be seen also from the Bader charge analysis2O3 monolayer, less than To investigate the bonding character of the Al2O3 monolayer, its cohesive energy Ec can be calculated by the following equation:The stability of a material is indicated by the related cohesive energy, and a structure with negative cohesive energy is stable or metastable. For Al2O3 monolayer, and EAl and EO are the energy of isolated single Al and O atom, respectively. The calculated cohesive energy of Al2O3 monolayer is −5.97 eV/atom, which is slightly higher than that of the Al2O3 bulk (−6.56 eV/atom), but it is lower than that of recent experimentally confirmed 2D materials such as silicene (−5.16 eV/atom) and germanene (−4.15 eV/atom)192O3 is promising for an experimental realization.where 2O3 monolayer can also be examined from the calculated phonon spectra. As shown in 2O3 monolayer is dynamically stable and can exist as a free standing 2D structure.The structural stability of the Al2O3 monolayer, ab initio molecular dynamics simulation has been conducted at the temperature of 300 K with a time length of 10 ps. A (5 × 5 × 1) supercell of the Al2O3 monolayer is used to minimize constraints induced by periodic boundary condition. The averaged total energy of the monolayer is nearly constant, and the variation of the Al-O bond length is within 0.1 Å with respect to the average value at 300 K during the simulated time period, as shown in 2O3 monolayer is thermally stable at room temperature. Ab initio MD simulations were also carried out at higher temperatures of 800 and 1100 K, respectively, which show that this monolayer is still stable at the temperature of 1100 K . For the deformed state of Al2O3 monolayer, it is assumed that the contribution of bending to the strain energy density is insignificant, thus the vertical weight of elastic constants is zero. For all the deformed configurations, the positions of all atoms were optimized and the related energies were calculated. In this way, for each type of distortion, we could obtain the strain dependent energy. Then, the in-plane Young’s modulus Ys and Poison’s ratio v were calculated from the following relationship: h-BN monolayer and ZnO monolayer were calculated and summarized in 2O3 monolayer is 47.6 N/m, very close to that of ZnO monolayer (~47 N/m), but much smaller than that of graphene (~342 N/m), BN monolayer (~274 N/m), and Al2O3 bulk (~230 GPa). This clearly shows the relative softness of the Al2O3 monolayer. The softness of the Al2O3 monolayer is also reflected by the flat slope of the acoustic branch at the Γ point in the phonon dispersion2O3 monolayer is susceptible to external influence, with greater tendency to form ripples or out-of-plane deformations, as supported by the above MD simulation results , one of the most promising high-κ dielectrics. Thus, we believe that the band gap of the Al2O3 monolayer is large enough to be used as a dielectric material. From the PDOS and partial charge density . The formation of π bonds is believed favorable to stabilize the 2D planar structure, as shown in other 2D structuresAlsity see , we find2O3 monolayer is shown in εxx and εzz, the total dielectric constant ε can be determined by A dielectric material with high dielectric constant is preferred in order to achieve larger capacitance density. The dielectric constant decreases with the decrease of thickness, and thus it is important to study dielectric properties for a dielectric material with one atomic thickness. The calculated dielectric function of the Al2O3 monolayer is determined by the zero energy limit from the real dielectric function, which is about 1.27. With the inclusion of the ionic vibration contribution (~1.24), the overall dielectric constant is about 2.51. This value is much smaller than that of α-Al2O3 bulk (~10.5) due to the reduced thickness, changed bonding character, and also decreased band gap, but comparable to that of SiO2 bulk (~3.9). It is noted that the calculated dielectric constant of α-Al2O3 bulk is consistent with the experiment value (~9.0). From the imaginary part the dielectric function  graphene supercell matches 2O3 monolayer well. Since the Al2O3 monolayer is relatively soft, it was compressed by about 2.88% and placed on graphene supercell. This small strain on the Al2O3 monolayer yields a 0.18 eV increase in the band gap, but does not alter the electronic properties significantly, as shown in the 2O3 monolayer still favors planar structure, and becomes more stable as its energy is lowered by 0.19 eV per unit cell, compared with free standing Al2O3 monolayer.Experimentally, various substrates have been proposed for growing oxide monolayer such as SiO2O3 monolayer on graphene is shown in 2O3 monolayer, β-Si3N4, and g-C3N3 on graphene, and HfO2 on MoS2 monolayer255455562O3 monolayer planes remain planar. The shortest distance between Al2O3 monolayer and graphene is 3.26 Å, indicating weak interaction between them. The calculated adsorption energy is 13.62 meV/Å2, smaller than that of graphene on Al2O3 (0001) surface2O3 monolayer and graphene.The most stable configuration of the Al2O3 monolayer. From the charge density difference projected on graphene plane  and conduction band offset (CBO), respectively, as shown in 2O3 monolayer. Thus, the real VBO and CBO should be larger than the values in current studies.Due to the weak interaction, the charge inhomogeneity in graphene is insignificant with the presence of the Alne plane , we can 2O3 monolayer, thus it is a potential substrate to grow Al2O3 monolayer, similar to the growth of Y2O3 monolayer on graphene2O3 monolayer, the interface between the Al2O3 monolayer and Al (111) surface has been studied also , forming interfacial Al-O bonds with bond length of 1.9 Å. The strong interaction is also confirmed by the large adsorption energy (2.2 eV per Al2O3 unit) and significant charge transfer (see 2O3 monolayer on Al (111), and it can be expected that the 2D growth of Al2O3 monolayer is difficult on Al (111) substrate by a direct thermal oxidation process. Therefore, inert substrates with weak interaction, same surface symmetry, and small lattice mismatch such as graphene are favored to realize the 2D growth of Al2O3 monolayer via sputtering or other advanced deposition techniques.From the above discussions, we can see that graphene has same surface symmetry, small lattice mismatch, and weak interaction with Alsfer see . This st2O3 monolayer with a direct band gap of 5.99 eV and dielectric constant of 2.51. This Al2O3 monolayer can be further stabilized by the support of graphene. For the interface between Al2O3 monolayer and graphene, we find that the interaction between them is weak and band alignments are much larger than 1 eV. Moreover, the presence of the Al2O3 monolayer does not introduce interface states and electron-hole puddles in graphene. Together with high thermal stability, the Al2O3 monolayer might be a potential dielectric for future electronic applications. Due to similar surface chemistry between graphene and other 2D channel materials such as transposition metal dichalcogenide or phosphene, these results might be applicable to the interface between the Al2O3 monolayer and other 2D materials.In conclusion, based on PSO method and first-principles calculations, we find a stable AlHow to cite this article: Song, T. T. et al. The stability of aluminium oxide monolayer and its interface with two-dimensional materials. Sci. Rep.6, 29221; doi: 10.1038/srep29221 (2016).


We found no correlation between Id2 expression and other known prognostic factor in NBL patients. We assume that Id2 is not prognostic factor. However, due to its abundant expression in most of NBL cells and its role in cell cycle, it may be potential therapeutic target. Exact knowledge of expression time may be helpful in explaining mechanisms of oncogenesis.Id (DNA binding and/or differentiation) proteins occur physiologically during ontogenesis and negatively regulate the activity of other helix-loop-helix (HLH) proteins. Id2 protein causes block of cells differentiation in the S phase of the cell cycle and regulates the activity of Rb protein. The role of Id2 protein in physiological cell cycle progression and in neuroblastoma (NBL) pathogenesis was proposed by Lasorella. The aim of the study was evaluation of Id2 expression and its prognostic significance in NBL cells coming from primary tumors and evaluation of its prognostic significance, and correlation of Id2 expression with known prognostic factors. Sixty patients with primary NBL treated from 1991 to 2005 were included in the analysis. We found 50 patients with high and 10 patients with low intensity of Id2 expression. The median percentage of NBL cells with Id2 expression was 88 %. We found no correlation between the number of NBL cells or the intensity of Id2 expression and OS and DFS. In patients with stage 4 NBL, almost all patients had high expression of Id2 and it was significantly more common than in other disease stages ( Id (DNA binding and/or differentiation binding) proteins have a helix-loop-helix (HLH) structure Table . They ocThe aim of the study was evaluation of Id2 expression in neuroblastoma cells coming from primary tumors stored as paraffin blocks taken from patients at diagnosis, evaluation of its prognostic significance and correlation between Id2 expression and known prognostic factors. Sixty patients were included in the analysis, treated for NBL in the Department of Oncology and Hematology in Krakow from 1991–2005. Observation was finished in June 2012. The characteristics of patients are presented in Table Id2 expression was evaluated with immunohistochemistry. Staining was performed with rabbit anti-Id2 antibodies (Zymed Laboratories Inc.) on silanized slides. According to manufacturer guidelines, the optimal dilution of primary antibody was experimentally established as 1:100 and the time of incubation as 45 min. The detection system was based on peroxidase  for use with rabbit primary antibodies, for evaluation in light microscopy of paraffin embedded tissue. As the positive control NBL cell line SKNSH was used with known high Id2 expression. As the second control human testis was used, embedded in paraffin. The double control system was chosen because we wanted to compare staining procedures in the samples prepared under the same conditions in our laboratory . In negative control, no primary antibody was added. The percentage of cells with Id2 expression was evaluated in the light microscopy. The intensity of Id2 staining was evaluated by comparison with staining intensity of the positive control.t test and Pearson correlation test. For all analyses the p values less than 0.05 (p < 0.05) were considered as statistically significant. Statistica® software packages were used for statistical analysis.Estimations of the survival were performed using a Kaplan-Meier method and compared with a log-rank test. The comparisons between groups were performed by a chi-square test (with Fisher’s exact test if necessary), Student’s The influence of intensity of Id2 expression and the percentage of NBL cells with Id2 expression on mortality and therapy failures  was evaluated.Among 60 NBL patients evaluated, we found 50 patients with high and 10 children with low intensity of Id2 expression. The percentage of NBL cells with the Id 2 expression was 1–100 % . In 53 patients (88 %) the Id2 expression was found in more then 50 % of cells and in 28 (46.7 %) – in over 90 % . Three-year OS was similar for both groups . Five-year OS was higher in patients with low Id2 expression, but the results were not statistically significant (p = 0.3). Therapy failure defined as early progression or relapse occurred in 30 out of 50 children with high Id2 expression and 4 out of 10 children with low Id2 expression . No statistically significant differences were found (p = 0.15) for 3-year DFS (0.7 and 0.48) and 5-year DFS (0.7 and 0.4). In 41 evaluated patients over 1 year of age, the percentage of NBL cells with Id2 expression was 1-100 % . In 17 patients (41.5 %) the percentage of Id2 positive cells was over 90 %. In children younger than 1 year of age (n = 19) the percentage of cells with Id2 expression was 50 %-100 % . In 9 patients (47.4 %) Id2 expression was found in over 90 % of cells. In patients with stage 4 NBL, only 2 among 31 patients had low expression of Id2 – we did not perform comparative analysis. The percentage of Id2 positive cells was 1–100 % . In patients with stage 4, in comparison to other stages, we found significantly more common high expression of Id2 (p = 0.03) . The percentage of Id2 positive cells in patients in stage 2, 3 or 4 s was 1–100 % . In this group of patients neither intensity of Id2 expression nor percentage of Id2 positive cells had influence on treatment outcomes . Among 12 patients with MYCN amplification, only 1 had low Id2 expression. The comparative analysis was not performed. The percentage of Id2 positive cells in patients with MYCN amplification was 40–100 % . Percentage of Id2 positive cells had influence on neither NBL deaths (p = 0.75) nor therapy failure (p = 0.51) in this group of patients. In patients without MYCN amplification (n = 46) the percentage of Id2 positive cells was 1–100 % . The intensity of Id2 expression was not different in patients with and without MYCN amplification (p = 0.33). In the group with no amplification, 15/37 children with high Id2 expression and 2/9 children with low Id2 expression died of NBL (p = 0.17). Therapy failures occurred in 22/37 and 3/9 children, respectively (p = 0.15). In patients with no MYCN amplification, 3-year and 5-year OS and DFS were higher in the group with low Id2 expression. However, the difference was not statistically significant (p = 0.2 for OS and p = 0.13 for DFS). Percentage of Id2 positive cells had no influence on survival (p = 0.39) and on therapy failures (p = 0.2).As the main aim of the study was the evaluation of Id2 as a prognostic factor, we decided to exclude from the overall survival analysis the patients whose death was not caused by NBL. Deaths occurred in 29 out of 50 patients (23 died of NBL) and 3 out of 10 patients  with high and low Id2 expression, respectively , ferritin or neurospecific enolase (NSE).Extraordinary biological characteristics of NBL and diverse clinical outcome are the reasons why neuroblastoma is the focus of scientific interest –31. One In our study we found expression of Id2 protein in all evaluated tumors, with 83.3 % of patients with high expression in most of the cells. As neither the exact level nor the meaning of time of Id2 expression is confirmed to be informative, we assumed that each observed expression of Id2 will be defined as positive. There are some studies indicating the existence of neoplastic stem cells, in which the mechanisms connected with Id2 expression can play a potential role in creating and maintaining malignant phenotype of the cell. In our material the intensity of Id2 expression was comparable in all analyzed groups; however, and lack of statistical significance may be the result of a small number of patients. After analysis of the whole group and subgroups it cannot be confirmed that Id2 expression has prognostic significance in NBL. In most of univariate analyses, neither level of expression nor number of cells with Id2 expression had statistically significant influence on OS and DFS. However, almost all children with MYCN amplification had high intensity of Id2 expression and more children with strong Id2 expression were in stage 4 of disease.It has been proved that inhibition of Id2 function is possible in vitro, and it results in reduction of tumorigenic properties in neuroblastoma cells and promotion of cells differentiation –42. Addi


Mn2O3/Al nanothermite has been synthesized by magnetron sputtering a layer of Al film onto three-dimensionally ordered macroporous (3DOM) Mn2O3 skeleton. The energy release is significantly enhanced owing to the unusual 3DOM structure, which ensures Al and Mn2O3 to integrate compactly in nanoscale and greatly increase effective contact area. The morphology and DSC curve of the nanothermite membrane have been investigated at various aluminizing times. At the optimized aluminizing time of 30 min, energy release reaches a maximum of 2.09 kJ∙g−1, where the Al layer thickness plays a decisive role in the total energy release. This method possesses advantages of high compatibility with MEMS and can be applied to other nanothermite systems easily, which will make great contribution to little-known nanothermite research.Mn In recent years, the demand for miniaturization and integration of power generator has significantly increased in microelectromechanical system (MEMS). Although chemical battery technologies have been greatly developed, the energy density is still relatively low. It is impossible to attain large energy release from the micro-scale batteries22O3, MoO3, Bi2O3, NiO). Under the stimulus of flame, laser pulse, current or heating, an oxidation-reduction reaction breaks out and releases a large amount of energy. Owing to shorter mass transfer distance and larger contact area, MICs show better performance in energy release, ignition delay time and reaction rate compared to their bulk or micro counterparts3456891011121415171920222324Nanothermite, also termed metastable intermolecular composites (MIC), is a typical class of nEMs. An MIC generally consists of fuel  and a variety of metal oxides , respectively. 2O3/Al composite membrane. As expected, the diffraction peaks of Al are observed at 38.47°, 44.74° and 65.13°, corresponding to planes (111), (200), and (220) of Al. Simultaneously, the characteristic peaks of bixbyite Mn2O3 are kept in the same places without impure peaks, which reveals that phase transformation of Mn2O3 is avoided and the oxidation-reduction reaction between Al and Mn2O3 has not happened. These advantages are owing to the high-vacuum and the sustained low temperature during the aluminizing process. Over the Mn2O3/Al nanoparticle composites (NPC) sample, XRD pattern also indicates good crystallinities of Al and Mn2O3 after grinding with care.The phase of the crystalline components is identified by Powder X-ray diffraction (XRD) characterization. As shown in 2O3 structure, a high quality inverse replica of the template. Interconnected and ordered macroporous structure is observed after PS spheres are removed. A “honeycomb” structure is constituted by well-ordered air spheres and metal oxides walls in three dimensions with a thickness of 48 nm, and several layers within the skeleton are still visible in the surface image. The pore size is about 220 nm which corresponds to a pore shrinkage rate of approximately 18.5% compared with the original PS sphere. Some point defects and line defects are visible in the image. This is due to incomplete filling of the precursor and the transformation of the CCT during the calcination. Even so, we can consider that the integrity and consistency of the Mn2O3 skeleton are still maintained.Scanning electron microscope (SEM) images of polystyrene (PS) colloidal crystal template (CCT) prepared by vertical deposition are shown in 2O3/Al membrane with three different aluminizing times and a typical Mn2O3/Al nanoparticle composite. From surface view in 2O3 skeleton is strong enough to support the deposited Al, and there is no fracture or collapse phenomenon. With aluminizing time increasing, the coated layer on the Mn2O3 wall is getting thicker, and the pore size is reduced. 2O3/Al NPC. Mn2O3 particles are in the form of irregular morphology in nanoscales or macroscales, and the spherical Al particles are distributed among the Mn2O3 particles. One can see that Mn2O3 particles are severely agglomerated due to the high surface energy, and the dispersion homogeneity of Mn2O3/Al NPC is significantly lower than 3DOM Mn2O3/Al membrane. Furthermore, the cross-section view also has proved that Al is not only deposited on the surface, but presented in the interior of the Mn2O3 inverse template, see further discussion in the next section with X-ray photoelectron spectroscopy (XPS) results. In order to further exam the morphology of the Mn2O3/Al membrane, transmission electron microscope (TEM) images on cross section of Mn2O3/Al membrane with aluminizing time of 20 min are showed in 2O3 skeleton to form a core/shell structure, and the nanothermite membrane can be viewed as an assembly of multiple nanothermite units, which are in agreement with the results obtained by SEM. Since Al is coated on Mn2O3 uniformly and connected with the skeleton closely both on the surface and inside the structure, one can expect the significantly enhanced effective contact area and improved reactivity.For comparison, 2O3/Al membranes with corresponding SEM images. Al and Mn peaks are observed in the spectra, accompanying with some impurity peaks that should be ignored, such as Au and Si. Au is from the layer deposited onto samples prior to the SEM measurement in order to increase the electric conductivity, while Si signal is from the quartz substrate. It is visualized that the percentages of Al peaks are gradually increased exactly as shown in SEM images, see EDS elemental mapping in Energy dispersive spectrum (EDS) is used to obtain a further insight into the aluminizing time on the influence of the elemental ratio. 2O3 is then calculated, respectively. Due to the presence of impurities, only Al and Mn are taken into account, see 2O3 increases gradually with the increasing of aluminizing time, and they are linearly related because of the constant deposition rate of Al. Since the quantity analysis relies heavily upon the homogeneity of the samples, this is an indicative estimation in quantitation. One can see the molar ratio of Al to Mn2O3 is slightly larger than the stoichiometric value of the exothermic self-propagating reaction between Al and Mn2O3 when the aluminizing time is added to 30 min.The atomic percentages of the element (%) are obtained by integration of the peak areas, and molar ratio of Al to Mn+ sputtering on Mn2O3/Al membrane of aluminizing time of 30 min. The acceleration energy of ion beam is set to 3000 eV, corresponding to the etching rate of about 12 nm·min−1. A wide range survey, Mn2p and Al2p taken after each cycle of sputtering are shown in 2O3 skeleton in three dimensions, which is also confirmed by TEM examination, see In order to examine the elemental composition in the deeper layers within the nanothermite membrane, XPS depth profiling has been investigated with the aid of Ar2O3 would happen under the stimulus of flame, laser pulse, current, heating, etc. The reaction equation is described in the following equation (1),The self-propagating reaction between Al and Mn2O3/Al nanothermite membrane is characterized with differential scanning calorimetry (DSC). The DSC curves from Mn2O3/Al nanothermite membranes obtained with different aluminizing times are shown in 2O3 is only 0.73 calculated by EDS. When the aluminizing time is added to 20 min, see −1. All these peaks are with sharp profile, which suggests the energy release in a short period of time. Therefore, it implies that the thermite reaction between Al and Mn2O3 can be divided into two steps. First reaction is based on solid-solid diffusion mechanism, and second reaction triggers by liquid-solid diffusion mechanism after Al is melted, which is similar to NiO/Al system that has been reported before2O3/Al NPC under the same test conditions is shown in 2O3/Al membrane at aluminizing time of 10 min, which reveals that reactants are not able to react completely, although Al and Mn2O3 are mixed in the stoichiometric ratio.The thermite reaction of Mn−1, when the aluminizing time is 30 min. Its first energy release is not the largest, but the second one is five times larger than that from the sample with 20 min aluminizing. This is owing to the excessive Al on the interface. On the other hand, Al is insufficient in the sample with aluminizing time of 20 min. We draw the conclusions that the first exothermic reaction is mainly controlled by the coverage of the fuel. When it’s too thick, products cumulated on the interface may prevent the reaction to progress further, but the reaction is resumed after the residual Al is melted. Therefore, the total energy release is determined by the Al layer thickness deposited on the Mn2O3 skeleton. In addition, the maximum energy release is still lower than the theoretical value of 3.38 kJ·g−1, which may be attributed to the unavoidable oxidation of Al film during storage, and the fact that Mn2O3/Al membrane is not in stoichiometric ratio. Despite all this, the energy release is still considerably high, and the first onset temperature is as low as 480 °C, which is approximately 50 °C lower than Mn2O3/Al NPC. These characteristics make the Mn2O3/Al nanothermite membrane a remarkable nanoenergetic material.In order to compare the influence of aluminizing time on the DSC results, all the experimental data are collectively listed in 2O3/Al nanothermite membranes and Mn2O3/Al NPC after DSC test. From the sample with aluminizing time of 10 min, in 2O3. This means that Al has reacted with Mn2O3 unreservedly during heating process, and Al is insufficient to reduce Mn2O3 thoroughly. The oxidation–reduction reaction is expressed in the following equation (2):2O3/Al membrane with 20 min aluminizing, in 2O3/Al NPC, in 2O3 and Mn peaks are not presented. That is probably due to the product of Al2O3 and Mn are amorphous or nanocrystalline327Similar pattern is obtained from the samples of Mn2O3/Al nanothermite membrane has been tested with the sample of 30 min aluminizing. We assumed that one frame prior to the point of light appearing to be the initial time, and a sequence of test results with an interval of 20 μs between adjacent images are shown in 2O3/Al nanothermite membrane, a bright flash of white light surrounded by amaranthine glow burst out from the substrate at 20 μs, which indicated that the Mn2O3/Al membrane was ignited successfully. The whole ignition process lasted around 180 μs, and the maximum height of the flame was 8 mm at 20 μs. The high speed camera observations of ignition with other aluminizing time are shown in 2O3 releases more energy along with the increasing of aluminizing time, which is consistent with the DSC results. It can be definitely concluded that a thermite reaction between Al and Mn2O3 occurred under the stimulus of laser pulse.The ignition performance of 3DOM Mn2O3 is selected to combine with Al to achieve nanothermite of Mn2O3/Al. The 3DOM Mn2O3/Al nanothermite membrane is prepared via a simple and feasible procedure: vertical deposition of CCT, inverse template method for 3DOM Mn2O3 skeleton and magnetron sputtering for Mn2O3/Al nanothermite membrane. SEM results reveal that the Mn2O3 skeleton is highly ordered with interconnected and uniform macroporous structure, and Al is deposited uniformly onto the 3DOM Mn2O3 skeleton in order to form nanothermite membrane throughout the structure. With the increasing of aluminizing time, the Al layers get thickened. An optimized total energy release from nanothermite membrane of 2.09 kJ·g−1 with the onset temperature of around 480 °C is achieved when the aluminizing time is increased to 30 min. In addition, laser ignition test results show a bright flash of light lasting up to 180 μs. The 3DOM structural nanothermites possess the advantages of high energy density, large contact area, and short mass transfer distance that insure large energy release. Moreover, this method develops a new avenue to prepare various nanothermite membranes compatible with MEMS.In this work, Mn2 4H2O(2.0 mol·L−1) was selected as the Mn source, which was dissolved in a mixture solution of methanol and ethylene glycol (volumetric ratio = 2/3). Simultaneously, moderate Polyvinyl Pyrrolidone (PVP) was added to increase the viscosity of the mixed solution. The influences of PVP on morphology are shown in −1 and kept at 500 °C for 1 hour in an open atmosphere.The PS spheres were synthesized by emulsifier-free polymerization technology in our laboratory2O3 membrane was formed, Al was deposited onto the 3DOM Mn2O3 skeleton by magnetron sputtering to form core/shell structures with the 3DOM Mn2O3 as the core and Al as the shell. Prior to the Al deposition, the 3DOM Mn2O3 skeleton was rinsed by a small amount of anhydrous ethanol and dried under N2. All the sputtering process was under the vacuum condition of 5 × 10−3 Pa, the DC voltage 150 V, and ultrahigh purity Ar as functional gas with the flux of 30 sccm. On account of the high-vacuum and sustained low temperature, the oxidation of Al or the reaction of Mn2O3 and Al were mostly prevented during this process. In order to explore the influence of Al film thickness on the energy release from Mn2O3/Al nanothermite membrane, various amounts of Al was deposited by varying the thickness of Al membrane, through controlling the aluminizing time. Three different aluminizing times were designed: 10 min, 20 min and 30 min, respectively.After the 3DOM Mn2O3/Al NPC were also prepared via classic physical mixing. Aluminum particles with an average size of 50 nm were obtained from Aladdin Industrial Corporation , and Mn2O3 were purchased from Dk Nano Technology Corporation . Stoichiometric mixtures of the reactants were separately mixed in hexane for 5 min in an ultrasonic bath and then added to one beaker for continuing ultrasonic dispersion for 30 min. The suspension was then moved to a vacuum oven kept at 70 °C under 1.5 × 104 Pa in order to obtain Mn2O3/Al NPC.For comparison purposes, Mn2O3/Al nanothermite membrane was carried out using EDS. XPS were taken using an ESCALAB250Xi XPS instrument (Thermo Scientific). A monochromatic Al Ka x-ray source (1486.6 eV) was used with a spot size of 400 nm diameter. A pass energy of 100 eV and step size of 0.5 eV were used for survey spectra, and a pass energy of 20 eV and step size of 0.1 eV were used for high resolution spectra. Powder XRD  was used to analyze the component of the material at each stage. Prior to the measurement, all the samples were striped from the microslide and attached onto a piece of flat quartz. In addition, the exothermic reaction of the 3DOM Mn2O3/Al nanothermite membrane was performed by DSC  under N2 atmosphere in a temperature range of 200 to 900 °C with a flow rate of 30 mL·min−1, and the heating rate was set to 20 °C·min−1. A pulsed laser  was used to investigate the ignition performance of the 3DOM Mn2O3/Al nanothermite membrane. The laser was set to wavelength 1064 nm, pulse width 6 ns, beam diameter 700 μm, repetition rate 10 Hz, and the incident energy was 74 mJ per pulse. A piece of glass substrate with Mn2O3/Al nanothermite membrane on it was placed at the focal point of the laser and a high speed camera was placed at a position perpendicular to the laser direction to observe the ignition process. The whole ignition process was recorded by a high speed camera , and the data acquisition frequency was set as 50000 frames per second (fps).The morphology were characterized by a field-emission scanning electron microscope (FE-SEM)  and a TEM . Elemental analysis of the 3DOM MnHow to cite this article: Zheng, G. et al. Three-dimensionally Ordered Macroporous Structure Enabled Nanothermite Membrane of Mn2O3/Al. Sci. Rep.6, 22588; doi: 10.1038/srep22588 (2016).


The goal of this study was to compare significant SNP selection approaches in the context of complex traits based on SNP estimates obtained by models: a model fitting a single SNP (M1), a model fitting a single SNP and a random polygenic effect (M2), the nonparametric CAR score (M3), a SNP-BLUP model with random effects of all SNPs fitted simultaneously (M4). There were 46,267 SNPs tested in a population of 2601 Holstein Friesian bulls, four traits  were considered. The numbers of SNPs selected as significant differed among models. M1 selected a very large number of SNPs, except for a NRH in which no SNPs were significant. M2 and M3 both selected similar and low number of SNPs for each trait. M4 selected more SNPs than M2 and M3. Considering linkage disequilibrium between SNPs, for MY M2 and M3 selected SNPs more highly correlated with each other than in the case of M4, while for FY M3 selection contained more correlated SNPs than M2 and M4. In conclusion, if the research interest is to identify SNPs not only with strong, but also with moderate effects on a complex trait a multiple–SNP model is recommended. Such models are capable of accounting for at least a part of linkage disequilibrium between SNPs through the design matrix of SNP effects. Functional annotation of SNPs significant in M4 reveals good correspondence between selected polymorphisms and functional information as well as with QTL mapping results. For many years genome-wide association studies (GWAS) have been a useful tool for detecting genetic variants associated with traits in human genetics  introduced by Tibshirani  e.g., a, the elaOur study was focused on the comparison of significant SNP selection approaches in the context of complex traits. Significance of SNPs selected based on estimates obtained from a mixed model routinely used in genomic evaluation of dairy cattle in Poland was compared to three single SNP models. In particular, the genomic selection model, which considers all polymorphisms simultaneously by applying shrinkage on SNP estimates through fitting a normal distribution with a predefined variance, is compared with (i) a linear model with a fixed effect of a single SNP which accounts neither for SNP intercorrelation, nor for family relationships (ii) a linear mixed model with a fixed, single SNP effect and a random additive polygenic effect for capturing the family relationships, and (iii) the CAR score regression proposed by Zuber and Strimmer , which rThe material of our study consists of 2601 Polish Holstein-Friesian bulls genotyped with the Illumina BovineSNP50 BeadChip, which consists of 54,001 SNPs (version 1) and 54,609 SNPs (version 2). The applied SNP selection criteria comprised polymorphism, expressed by the minor allele frequency (MAF), with the minimum MAF of 0.01, and technical quality of a SNP expressed by the minimum call rate of 90 % within the analyzed sample of bulls. After quality control 46,267 SNPs were selected for further analysis.Four traits undergoing a complex mode of inheritance were considered in the study: somatic cell score (SCS) representing a trait with “pure” polygenic mode of inheritance, milk (MY) and fat (FY) yields representing traits with a polygenic mode of inheritance enhanced by single genes with large effects, and non-return rate for heifers (NRH) as a trait with a very strong environmental component expressed by heritability of 0.02. Deregressed proofs of bulls were used as pseudophenotypes. Deregression, which was performed in order to remove ancestral information from the conventional breeding values of bulls, was based on a method proposed by Jairath et al. . The cory = μ + Xβ + ε and (M2) y = μ + Xβ + Z1α + ε were solved using the ASReml3 software  where D is a diagonal matrix weighted by the effective daughter contribution for each bull and σε2 denotes a residual variance; μ denotes an overall mean. The CAR score regression (M3) proposed by Zuber and Strimmer Rempirical, where Rempirical is the empirical non-regularized correlation matrix among SNP genotypes. The CAR criterion was computed using an R package CARE. The genomic selection model (M4) y = μ + Z2g + ε, equivalent to the so called SNP-BLUP model, is routinely used for the prediction of direct genomic breeding values for the Polish Holstein-Friesian population  Strimmer  was seleStrimmer  this appβ in M2 the objective function log fy + log fα, where β and α leads to the mixed model equations  via Bonferroni’s approach, while for a multiple SNP model M4 a nominal P value was used as a selection criterion. In M3 the null distribution of the function of CAR scores κ − 1) degrees of freedom estimated by the R package fdrtool  no SNPs were identified by M1-M3. A very different pattern was observed for M4. Since all polymorphisms’ effects are estimated simultaneously with the underlying normal distribution shrinkage, the SNP selection procedure based on the 0.001 threshold chooses 0.1 % of the most significant SNPs regardless of a trait. As a consequence a very similar number of polymorphisms varying between 125 (NRH) and 182 (FY) for each trait was selected. Since, based on the very large number of SNPs selected, M1 does not seem to be a valid model for GWAS on complex traits, in further result description and discussion we confine ourselves to M2-M4.No marked differences between models were observed regarding the information content, expressed by MAF, between SNPs selected as significant by M2–M4. The average MAF of polymorphisms selected for MY by M2 amounted to 0.39, by M3 to 0.30 and M4 to 0.36 and was even more similar for FY with 0.37 (M2), 0.38 (M3) and 0.49 (M4). The average MAF in group of SNPs common between all considering models is 0.40 for MY and 0.38 for FY. On the other hand, considering MY, on average M2 and M3 selected SNPs which were more highly correlated with each other than was the case for M4 since the average LD expressed by pairwise correlation between significant SNPs was equal to 0.18 and 0.17 for M2 and M3, but only 0.08 for the genomic selection model. For FY, the average LD of 0.11 among SNPs selected by M4 was also lower from the corresponding values form M2 and M3 which were equal to 0.17 and 0.21 respectively. The highest average pairwise correlation was observed between significant SNPs which were common for models M2 and M3.Figure Genomic annotation was carried out for SNPs which were selected as significant by all three modles, i.e., M2, M3, and M3 Table , rangingAlthough some of the genes may represent spurious associations arising through high LD to DGAT1, those which are located in more distant regions of BTA14 are potential candidate genes. Special attention is to be focused on EEF1D which according to a recent study of Xie et al.  in dairyWe observed that for complex traits approaches which model the genetic component by a single SNP — here represented by M1, which are very common and successful in GWAS for disease traits, have methodological problems. Since such models do not account for the polygenic background differentiating between SNPs in high LD, especially in the neighborhood of genes with strong effects is problematic and the multiple testing correction is not sufficient to remove the SNP effect upward bias due to correlated SNPs. An intensive post-processing of SNP estimates and/or corresponding P values regarding LD is then required.p = 1/2Ne. Note, that here we used the estimated number of heterozygous QTL segregating for production traits as N = 10.73  or a multiple–SNP without any assumptions regarding the inheritance mode (like M3). In our study M2 and M3 for MY and for FY, above and beyond the M2-M3, also the genomic model (M4) selected a similar number of SNPs as the number 55 resulting from the estimator proposed by Hayes and Goddard  of the tHowever, if the research interest is to identify SNPs not only with strong, but also with moderate effects on a complex trait a multiple–SNP model – here represented by M4, is recommended. Such models are capable of accounting for at least a part of LD between SNPs through the design matrix of SNP effects. A fine tuning of SNP selection procedure is needed based on the expected number of SNPs influencing the variability of a given trait, e.g., based on heritability estimates.Recently, Wang et al.  applied


Based on first-principles calculations, the electrical conductivity of SLMoS2 is calculated by Boltzmann equations. The thermal conductivity of SLMoS2 is calculated to be as high as 116.8 Wm−1K−1 by equilibrium molecular dynamics simulations. The predicted value of ZT is as high as 0.11 at 500 K. As the thermal conductivity could be reduced largely by phonon engineering, there should be a high possibility to enhance ZT in the SLMoS2-based materials.Both electron and phonon transport properties of single layer MoS ZT) is utilized to evaluate the efficiency of the thermoelectric conversion, defined as: S is the Seebeck coefficient, σ is the electrical conductivity, T is the absolute temperature, and κ is the total thermal conductivity. The κ is composed of electrons’ contribution (κe) and phonons’ contribution (κp). The ZT value for most commercial materials are around one, which is far below the critical value of three that is comparable with the traditional energy conversion in efficiencyκp1314Thermoelectric materials are essential for converting waste heat to electricity and solid-state cooling, which have attracted much attention recently123456789S2 (SLMoS2) is a semiconductor and has a direct band-gapThe graphene, as the first two dimensional material, has extraordinary electronic property as well as super high thermal conductivity2. Eugene et al. have calculated the electronic structure of SLMoS2 which is compared with that of bulk MoS22 to the indirect band gap of bulk MoS2. Emilio et al. have shown that, after applying compressive or tensile bi-axial strain, the electronic structure of SLMoS2 transitions from semiconductor to metalet al. calculated the intrinsic electrical transport and electron-phonon interaction properties of SLMoS22 has been explored and a maximum ZT, at room temperature, is obtained as 0.5 by Huang et al.ZT. Fu et al. studied SLMoS2 ribbons and calculated the ZT value to be up to 3.4et al. has experimentally reported a value of S as 30 mV/K for SLMoS2Recently, some works have studied the electronic and phononic properties of SLMoS2. The SLMoS2 nanoribbon has a low thermal conductivity due to the size effect. Jiang et al. claimed that κp of SLMoS2 nanoribbon was around 5 Wm−1K−1 at room temperature by molecular dynamics (MD) simulationset al. reported three results for SLMoS2 nanoribbons which were 1.35 Wm−1K−1 by equilibrium molecular dynamics (EMD)−1K−1 by non-equilibrium Green’s function−1K−1 by Boltzmann transport equation2 with higher values. Li et al. predicts the κ as 83 Wm−1K−1 from ab initio calculationsκ of suspended few layers MoS2 has been measured as 52 Wm−1K−1−1K−1et al. claimed that the basal-plane thermal conductivity of single crystal MoS2 would be 85–110 Wm−1K−1κ of SLMoS2, and it needs more works on this issue.Besides electron properties, some works focused on the phonon properties of SLMoS2 are studied . When the Fermi level is in the band gap, ne and σ is much smaller. As the Fermi level moves up into the conduction band, ne and σ increases quickly . We calculated κp by EMD and show in κp of SLMoS2 exhibits a size dependence on the simulation cell and reaches a converged value when the simulation cell is larger than 8 × 8 × 1 units3 (8.66 × 7.50 × 0.616 nm3)  at 300K. In κp of SLMoS2 decreases with the increasing temperature , because there are more three phonon Umklapp scatterings for high temperature. A lower κp is good for enhancing thermoelectric properties.For semiconductors, the thermal conductivity is mainly contributed by phonons (616 nm3) . A weak κp of SLMoS2. Some of these works focused on the SLMoS2 nanoribbons2627402 is around 20 times larger than that of nanoribbon with 34.6 × 30 × 0.61 nm3 in size. Besides, due to the interlayer coupling by van Der Waals forces31272830κp of SLMoS2 is a little higher than the measurements of bulk multilayer SLMoS2−1K−1. Our value is comparable to the result predicted from ab initio calculationκp as 83 Wm−1K−1 at 300 K in the considering of phonon scatters and the simplification in calculation of BTE model. Another advantage for our results is that both the nonequilibrium Green’s function calculationComparing with previous results . The EMD is better than NEMD in predicting a bulk structure by applying the periodic boundary condition, because NEMD need impose artificial heat bath and use the extrapolating method. However, NEMD gets the advantage of EMD in predicting a structure with a finite size. There is a EMD report which showed a low value of κZT profiles can be obtained and are shown in ZT in the whole carrier concentration range. The optimized ZT values are 0.04, 0.07, and 0.11 for 300 K, 400 K and 500 K, respectively. These values get bigger as temperature increases because of the improved power factors and the reduced thermal conductivity. These optimized ZT values correspond to the situations where the Fermi level moves up to the first peak in the conduction band.With the above calculations of electron and phonon properties, 2 structures. The value of ZT is in the same order of Ref. 2 ribbon. Our results of κp is higher than others. As shown in κp overcome the size confinement effect and corresponds to an infinite SLMoS2 sheet. Moreover, different from NEMD, it does not need the assumption of linear relationship between 1/κp and 1/L.As shown in ZT trend and magnitude of these transport values. As shown in 2 is larger than that of nanoporous silicon. The large power factor of SLMoS2 comes from a larger intrinsic σ and a comparable S. It indicates that the SLMoS2 has comparable electron properties as the optimized nanoporous silicon. However, due to the high κe and κp, the SLMoS2 has a modest ZT value. It is also worth noting that the ZT value here is smaller than the prediction from ballistic models by Huang et al. where neglects the phonon scatteringsCompared to nanoporous silicon analyzed by LeeZT value of SLMoS2 is not over one, SLMoS2-based materials may be a good candidate for thermoelectric application. Our results show that SLMoS2 has a much higher thermal conductivity  than other thermoelectric materials (on the order of 1 Wm−1K−1)6144346ZT value as low as 0.003. Then, with phonon engineering, Si-based nanomaterials, such as Si nanowires45144346ZT. Another inspiration example is the graphene. The high pristine thermal conductivity of graphene can be reduced largely by phonon engineering4849ZT as high as 352Although the predicted ZT and power factor of SLMoS2 are much higher than those of silicon and graphene. With a reduced thermal conductivity and kept electron transport properties, the values of ZT of SLMoS2-based materials may be larger than one. Generally, a side-effect of phonon engineering is the reduction of power factor. However, the side-effect is not obvious because the mean free paths of electrons are around two orders smaller than that of phonons, such as what is shown in the recent thermoelectric results on Si phononic crystals47The values of 2 are explored using theoretical calculations. The electronic structure and phonon dispersion relation are calculated using DFT calculations. Combined with molecule dynamics simulations and Boltzmann equations, thermoelectric properties are predicted as a function of carrier concentration at room temperature. With the lattice thermal conductivity as 116.8 Wm−1K−1, 79.6 Wm−1K−1, and 52.9 Wm−1K−1, the optimized ZT of SLMoS2 is found to be of 0.04, 0.07 and 0.11 at 300 K, 400 K and 500 K, respectively. As SLMoS2 has a higher ZT than other pristine structure, like silicon and graphene, there will be a big room to enhance ZT in SLMoS2-based materials by the developing phonon-engineering.The thermoelectric properties of SLMoSTo calculate electronic properties, the first-principles calculation is implemented by QUANTUM ESPRESSO in the frame of density functional theory (DFT)2 yields lattice constant of about 3.13 Å, consistent with previous predictions of 3.12–3.16 Å20212 is assumed to be 6.16 Å – the same as that of the single-sheet in bulk MoS2The hexagon primitive cell is used to structure relaxation and property prediction in DFT calculation. Structure relaxation for SLMoSIn the calculations of transport coefficients, a k-point mesh as 28 × 28 × 1 (denser enough to obtain converged results) is used over the irreducible Brillouin zone. With the assumption of constant relaxation time, the transport coefficient for electrons can be calculated using BoltzTrap2, κp, is calculated by EMD with the Green-Kubo approachet al.2 film is constructed by periodic arrangement of supercell illustrated in 3 supercell corresponds to 1.083 × 0.938 × 0.616 nm3. To study the finite size effect on thermal conductivities, we calculated the simulation cells with the volumes from 2 × 2 × 1 to 32 × 32 × 1 units3 at room temperature .


An attempt has been made to synthesize metastable phase MgB6 crystal by electrolysis method. DTA/TGA studies were made to determine the eutectic point of the melt and it was found to be around 900°C. The electrolysis was performed at 900°C under argon atmosphere, at current density of 1.5 A/cm2. The electrodeposited crystals were examined using XRD, SEM, and XPS. From the above studies, the electrochemical synthesis method for hypothetical MgB6 from chloro-oxy-fluoride molten salt system is provided. Mechanism for the formation of magnesium hexaboride is discussed.The present work reports electrochemical synthesis of MgB Eg = 0.0150 Ry).Rare earth and alkaline earth metal borides belong to the group of nonoxide type metal-like compounds and have high melting point, high chemical stability, stable specific resistance, low expansion coefficient at certain temperature ranges, diverse magnetic orders, and high neutron absorbability , 2. They2: (1) MgB6, (2) unknown, and (3) MgB12 [4 and phase 2 to MgB6 and the formation of MgB12 (phase 3) was not confirmed [6 and MgB4 do not exist as individual phases and obviously are metastable with rather long equilibration times. According to Somsonov et al. [2, MgB4, MgB6, and MgB12. Mg–B system contains the phases of MgB2, MgB4, MgB6, MgB12, and Mg2B14. So the Mg–B system is known as multiphase system. The aim of the present work is to study whether the thermodynamically unstable MgB6 [Electrochemical synthesis of Mg–B system from molten salts is an economic feasible and environmental friendly way for the preparation of different binary phases . Particu3) MgB12 . Howeveronfirmed . MgB6 anv et al. , Mg–B syble MgB6  could be2O3 (22.27 mol%), and MgCl2 (17.14 mol%)  was taken as an electrolyte in high-density graphite crucible and acts as an electrolyte cell as well as anode for the electrolytic process. The Molybdenum rod of 1 cm diameter fitted to a stainless steel rod is used as cathode. The crucible was filled with the stoichiometric quantities of electrolyte salts, which were dried at 500°C under argon atmosphere. The whole assembly was placed in an inconel reactor, which was kept in an electrical heating furnace with thermocouple. The experimental setup for the electrosynthesis of magnesium hexaboride is described elsewhere [2 with the molar ratio of Mg : B as 1 : 6. After 5 hours of electrolysis the cathode was removed and the deposit was cooled in atmosphere. The deposit was then scraped off and the electrolyte adhering to it was leached with warm 5% HCl solution. Finally washing was done with distilled water for several times, the weight of the deposit was determined, and the nature of the powder was analyzed.The mixture of the salts LiF (12.95 mol%), BMgCl2 17. mol% (anlsewhere –17. The λ = 1.541 Å) radiation with 2θ value range of 20 to 90 using PAnalytical X'pert powder diffractometer. Differential thermal analysis and thermogravimetric analysis (TGA/DTA) of the reaction mixture was done using Rigaku Thermal—Plus TG 8120 with heating speed 20°C/min in a flow of air. The Fourier transform infrared (FTIR) spectra were recorded in the range of 400 to 4000 cm−1 using Perkin Elmer UK Paragon—500 spectrometer. Scanning electron microscopy (SEM) was employed for the morphological studies using JEOL JSM 3.5 CF Japan make model. UV Visible Spectrophotometer was employed for the absorbance study using JASCO Model 7800 UV Visible Spectrophotometer. Studying the binding energy of boron and magnesium was done using X-ray photoelectron spectroscopy Thermo Scientific UK Multilab 2000.The phase formation and the structural details of the synthesized compound were characterized by X-ray powder diffraction (XRD) using CuKα  [6 (JCPDS data card number 08-0421) existing in body centered cubic crystal structure (the space group Pm3m) [6 is difficult because the information on its lattice parameters and structure system is not available in 08-0421. The main building blocks of the MgB6 structure are B6 octahedra. Other than MgB6 some additional traces of MgO are also present at 2θ = 43 and 62.5° due to the partial oxidation of Mg [4.115 Å) –20 for Mk is the Scherrer constant usually taken as 0.9, λ is the wavelength characteristics of the Cu-Kα radiation (λ = 1.5406 Å), β is the full width at half-maximum (FWHM) in radiations, θ is the reflecting angle, and D is the crystal size. where 2O3, begins to turn into liquid (melting point 450°C) in the heating process. These reactants whether in the liquid or gaseous state play a crucial role in determining the shape of the final product. Further, this weight gain is mainly due to the oxidation of the reactants . The formation of 2MgCl2·3B2O3 is due to the solid-state reaction between residual B2O3 and MgCl2, turn into molten state (melting point of MgCl2: 708°C) is confirmed by an exothermic peak at 761°C. Finally, the reactants can be oxidized thoroughly at 761°C [The TGA/DTA curve for the reaction mixture is shown in at 761°C . Further6 in MgCl2–B2O3–LiF system; the main chemical reactions are The reaction was carried out for MgB The overall reaction is 6 as depicted from XRD pattern. Trace amount of the unreacted intermediate MgO was present in the synthesized compound as 0.6% and the remaining 99.4% was MgB3 and tetrahedral BO4 groups. The electron transfer would come from trigonal B 1s to B–O σ* orbital and from the unfilled tetrahedral B 2p orbital to B–O σ* [σ* resonance as expected for sp2-bonded boron incorporated in the crystal [6 at 1314 eV revealed that the auger spectral distribution over an extruded energy range far from the threshold; there is an extra energy for Mg rich compound [6 existing at 544 eV. This may be due to the core-hole Rydberg states containing O 1s → σ* resonance [π* molecular orbital [The XPS spectrum for B 1s is shown in  B–O σ* , 26. Thi crystal .  Figurecompound .  Figureesonance , 30.  Fi2+ cations in MgB6 complex species have Cv6 pyramidal structures interacting with a planar hexagonal B62− dianion. The bonding between these two may be due the electrostatic attraction [The Mgtraction .6 is shown in −1, respectively. The absorption at 2225 cm−1 is assigned to O–H stretching vibration of cluster of water molecules of crystallization, respectively. The characteristic peak of Mg–B is observed at 1642 cm−1 [−1; this MgO as impurity phases is also observed in the XRD pattern. The bending vibration for MgB6 is observed at 437 cm−1. The bending mode of Mg–B of BO4 anion is assigned to 499 cm−1. The asymmetric stretching vibration of Mg–B of BO4 anion is observed at 1021 cm−1. The asymmetric stretching vibration of B–O bond of trigonal BO3 units is observed at 1367 cm−1. The frequencies observed in the spectrum are in good agreement with the reported values [The FTIR spectrum of MgB642 cm−1 . Longitud values , 34.μm. From the microstructure, the molten regions are clearly visible, which give the clear indication of MgB6 formation. The white regions in SEM represent the impurity phase MgO present in the sample [6 phase as cubic crystal structure.The SEM image of the sample is shown in e sample , 36. The2+ metal cation and B62− dianion due to electrostatic attraction. They also suggested that the metal cations M2+ have definite role on stabilizing the B62− dianion [6 and SmB6, respectively [6 that the calcium and boron are reduced at the cathode to form submicron sized crystals [6 at 900°C as one of the secondary phases along with MgB4 due the volatile nature of Mg at this temperature resulted in Mg deficiency on in situ Cu doping of MgB2. The commonly accepted mechanism of boron deposition in molten salts is a single-step three-electron electrochemical reaction [6 [6 [6 [The mechanism of hexaboride formation was proposed by many authors , 37–47.  dianion . Kaptay  dianion . We repoectively , 16. We crystals . As repocrystals , the forreaction –43. Glooction [6 . Li et aon [6 [6 . Lee et on [6 [6  and post[6 [6 [6 , 47.6 cluster complex is postulated for the metastable magnesium hexaboride compound. Further experimental evidence is more needed to explore the thermodynamically unstable magnesium hexaboride.In summary, the electrochemical synthesis of hypothetical magnesium hexaboride by molten salt technology is presented. Various mechanisms for the formation of magnesium hexaboride are discussed. It is believed that the supersaturated MgB


SNAI1, a zinc‐finger transcription factor, plays an important role in the induction of epithelial–mesenchymal transition (EMT) in various cancers. However, the possible functions of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma have not been clearly identified. In this study, we investigated the effects and mechanisms of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma using clinical samples and cell lines. We found that SNAI1 is highly expressed in the tissues of liver cancer compared with adjacent nontumor tissues. SNAI1 is also highly expressed in the hepatoma cell lines HepG2, SMMC‐7721, and BEL‐7402 compared with the human normal liver cell line L02. We also observed that SNAI1 expression was correlated with distal metastasis, incomplete tumor capsule formation, and histological differentiation in hepatocellular carcinoma (HCC). Moreover, we demonstrated that knockdown of SNAI1 via lentiviral vectors of RNAi against SNAI inhibited cell proliferation by inducing G1 arrest, which was accompanied by the downregulation of cyclin D1 but not that of cyclin A. In addition, knockdown of SNAI1 promoted apoptosis by decreasing the expression of Bcl‐2. In conclusion, our findings revealed that SNAI1 is involved in the development of hepatocellular carcinoma via regulating the growth and apoptosis of tumor cells. DMEMDulbecco's modified Eagle's mediumEGFPenhanced green fluorescent proteinEMTepithelial–mesenchymal transitionHCChepatocellular carcinomaROCreceiver operating characteristicHepatocellular carcinoma (HCC) is a major worldwide health problem, particularly in China. It is the third leading cause of cancer‐related death in the world and the second leading cause of cancer‐related death in China The transition of epithelial cells to a mesenchymal phenotype, named epithelial‐to‐mesenchymal transition (EMT), has been recognized during the progression of various carcinomas, including HCC in vivo and in vitro. We found that the growth and migration of HepG2 cell lines were inhibited, and their apoptosis was enhanced after the silencing of SNAI1 expression in vitro. Mechanistically, we found that knockdown of SNAI1 decreased the expression of Cyclin D1 but increased the expression of Bcl‐2. Furthermore, our data demonstrated that this effect existed in vivo.In this study, we constructed lentiviral‐mediated RNAi vectors against SNAI1 and transfected them into HepG2 cell lines −1 penicillin, and 100 μg·mL−1 streptomycin. All of the cell lines were maintained at 37 °C in a humidified incubator with 5% CO2.The human hepatoma cell lines HepG2, SMMC‐7721, and BEL‐7402 and human live cell line L02 were obtained from the Shanghai Cell Collection . The HepG2 and SMMC‐7721 cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose  containing 10% (vol/vol) fetal bovine serum , and the BEL‐7402 and L02 cell lines were cultured in RPMI1640 medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U·mL−1). After 14–16 h, the supernatant, including Enhanced Infection Solution, was removed, and fresh, complete medium was added. The cells were cultured for another 24, 48, 96, and/or 120 h before subsequent experiments.All of the lentiviral vectors expressed enhanced green fluorescent protein (EGFP), which allowed the measurement of their infection efficiency in transfected cells. LV‐SNAI1‐RNAi #1, #2, #3 and scrambled control LV‐RNAi were obtained from GENECHEM . The LV‐SNAI1‐RNAi #1 sequence was 5′‐CCACTCAGATGTCAAGAAGTA‐3′. The LV‐SNAI1‐RNAi #2 sequence was 5′‐CCAAGGATCTCCAGGCTCGAA‐3′. The LV‐SNAI1‐RNAi #3 sequence was 5′‐GCAGGACTCTAATCCAGAGTT‐3′. The scrambled control LV‐ RNAi sequence was 5′‐TTCTCCGAACGTGTCACGT‐3′. These lentiviral vectors were transfected into HepG2 cells with a multiplicity of infection (MOI) of 20 in the presence of enhancer solution and polybrene (5 μg·mL™ (TaKaRa) with β‐actin as an internal normalized reference. The sequences of primers were 5′‐ ACAAGCACCAAGAGTCCG‐3′(forward) and 5′‐CCCTCCCTCCACAGAAAT‐3′ (reverse) for SNAI1 and 5′‐TGTTACCAACTGGGACGACA‐3′(forward) and 5′‐CTGGGTCATCTTTTCACGGT‐3′ (reverse) for β‐actin. Quantitative polymerase chain reaction (PCR) was performed according to the following steps: 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s, 58 °C for 20 s, 72 °C for 20 s, and 65 °C for 20 s, using the LightCycler Real‐time PCR System  as described previously Total RNA was extracted using TRIzol reagent  according to the manufacturer's instructions, and reverse transcription was performed using an RT‐PCR kit . The expression of SNAI1 was quantified using real‐time quantitative PCR using SYBR Premix Ex Tap™ Cell Lysis Reagent  supplemented with a protease inhibitor ‘cocktail’, the protein concentrations in the extracts were measured using the bicinchoninic acid assay , and then, the volumes were made equal using the extraction reagent. Equal amounts of extracts were separated by SDS/PAGE, and then, they were transferred onto polyvinylidene fluoride membranes for immunoblot analysis as described previously For western blot analysis, cells were lysed with the CelLytic3/well in 96‐well plates and then were transfected with scrambled control LV‐RNAi and LV‐SNAI1‐RNAi. After 0, 24, 48, 72, and 96 h, 10 μL of CCK‐8 solution was added to each well and incubated at 37 °C for 1 h. At the end of the incubation, the optical density was read at 450 nm using a plate reader . The average values were determined from different wells.Cell proliferation was evaluated using the Cell Counting Kit‐8 . Cells were seeded at a concentration of 2.5 × 10−1 RNaseA was added followed by incubation of the cells at 37 °C for 30 min. The cells were stained with propidium iodide (Sigma) to a final concentration of 5 μg·mL−1 containing 0.2% Triton X‐100 and incubated at 4 °C for 30 min in the dark. The cells were then subjected to flow cytometry  for cell cycle analysis. The apoptosis assay was performed on HepG2 cells after 96 h of transfection with either the control or three LV‐SNAI1‐RNAis using the Annexin V‐PE/7‐ADD Apoptosis Detection Kit  according to the manufacturer's protocol, and then the cells were analyzed by flow cytometry.For cell cycle analysis, cells were collected 72 h after transfection with scrambled control or three LV‐SNAI1‐RNA is and then were fixed with cold 75% ethanol for 2 h at 4 °C. The fixed cells were centrifuged and then washed 3 times with PBS. Next, the cells were resuspended in 1 mL of PBS, and 100 μL of 500 μg·mL™ Boyden Chamber was obtained from CellBiolabs . Cells transfected for 48 or 72 h were collected and washed with PBS. These cells were resuspended in 200 μL of DMEM without FBS and seeded to the upper chamber at a concentration of 1.5 × 105 cells per well. Next, 600 μL of DMEM with 10% FBS was added to the lower chamber. After incubation for 18 h, the cells that traversed and spread on the lower surface of the membrane were fixed with 4% paraformaldehyde, and nonmigrated cells on the upper surface of the filter were removed with a cotton swab and stained with hematoxylin. The average number of cells per field was counted by × 10 objective magnification from five microscopic fields.The CytoSelect7) were subcutaneously transplanted into the posterior flank of nude mice. After reaching a diameter of 0.5 cm, these mice were randomly divided into two groups, and 2 × 106 TU of control LV‐ RNAi or LV‐SNAI1‐RNAi #3 vectors, respectively, were injected into each mouse every 2 days for a total of 6–8 injections. The tumor size was monitored every 2 days and calculated as V (mm3) = width2 (mm2) × length (mm)/2 as described previously Male BALB/c nude mice at 3–4 weeks of age were purchased from the Animal Research Committee of the Institute of Biology and Cell Biology  and housed in a specific pathogen‐free environment. The animal room was kept at 20–22 °C under a 12‐h light/dark cycle. HepG2 cells  overnight in a humidified chamber and then incubated with SABC  for 40 min at 37 °C. Staining results were viewed under a light microscope , and pictures were taken with an imaging program. Written informed consent was acquired from each patient for this study. The study methodologies conformed to the standards set by the Declaration of Helsinki. The research protocol and consent program were approved by the Shandong Provincial Hospital Affiliated with Shandong University Medical Institutional Ethical Committee.Paraffin‐embedded tissue sections, obtained from the Department of Pathology of Shandong Provincial Hospital affiliated with Shandong University were deparaffinized in xylene and rehydrated through graded alcohol solutions. Antigen retrieval was performed for 15 min at 98 °C in citrate buffer (pH 6.0) in a water bath. Endogenous peroxidases were inactivated by immersing the sections in 0.3% Hspss 16.0 statistical software  was used for all data analyses. To evaluate significant differences between the groups, Student's t test, the Mann–Whitney U‐test or one‐way analysis of variance (anova) were applied. To determine significant differences between different treatments or time points, two‐way anova was used. The Chi‐squared test was applied to analyze the statistical correlation between the clinical parameters of HCC and SNAI1 staining levels in tissue sections. In all cases, a P value less than 0.05 was considered to be statistically significant.The To verify the potential role of SNAI1 in HCC, we first detected its expression by immunohistochemistry and real‐time PCR in 42 pairs of HCC and adjacent benign tissues. We found that the expression of SNAI1 was significantly increased compared with that of adjacent nontumor tissues  were constructed. First, we detected the expression of SNAI1 in the HCC cell lines BEL‐7402, SMMC‐7721 and HepG2. As shown in Fig. To explore the possible functions of SNAI1 in HCC cell apoptosis and death, three lentiviral siRNA vectors against SNAI1 or scrambled control vectors were transfected into HepG2 cells. Surprisingly, we observed under an optical microscope that HepG2 cells underwent obvious apoptosis and death after transfection for 96 h compared with controls Fig. A, particTo further investigate the effect of SNAI1 in HCC cell growth and metastasis, we also knocked down the expression of SNAI1 with the above lentiviral vectors. As an initial step, the capacity of proliferation was evaluated in HepG2 cells transfected with lentiviral vectors using the CCK8 assay. As shown in Fig. in vivo, nude mice were subcutaneously inoculated into HepG2 cells (1 × 107). After reaching a diameter of 0.5 cm, the tumors were injected with control LV‐ RNAi or LV‐SNAI1‐RNAi #3 vectors. As shown in Fig. in vitro. Taken together, these data show that the inhibitory effect of SNAI1 RNAi also existed in vivo.To further evaluate the influence of SNAI1 RNAi In this report, we demonstrated that the expression of SNAI1 is increased in the HCC sample obtained from surgical resection. Moreover, the abnormally higher expression correlated with invasion, metastasis, and poor prognosis. In addition, downregulation of SNAI1 expression led to increased HepG2 cell apoptosis and decreased proliferation and migration. Similarly, the role of lentiviral‐mediated RNAi vectors against SNAI1 existed in nude mice.et al. Epithelial–mesenchymal transition plays a pivotal role in cancer cell metastasis via increasing cell mobility In vitro, the lentiviral‐mediated RNAi assay demonstrated that SNAI1 knock down effectively reduces HepG2 cell proliferation, migration and invasion and promotes HepG2 cell apoptosis. Kurrey et al. et al. have shown that breast cancer cells can resist hypoxia through the SNAI1/beta‐catenin signal pathway Consistent with our previous findings, we found that the expression of SNAI1 is significantly increased in 34 of 42 cases by immunohistochemistry, and the positive rate of high expression was up to 80.95% in HCC. In addition, we analyzed the correlation between the expression of SNAI1 and certain clinical parameters. These results showed that SNAI1 expression was correlated with distal metastasis, incomplete tumor capsule formation and histological differentiation. in vivo. Fortunately, our data showed that the knock down of SNAI1 can significantly diminish the tumor volume and weight and occasionally induce necrosis. This result is consistent with that obtained in vitro.From another perspective, regarding whether SNAI1 is a target for the biological treatment of HCC, we observed the role of lentiviral‐mediated RNAi vectors against SNAI1 using a nude mouse tumor model In summary, our study has demonstrated an important role for SNAI1 in the regulation of growth and apoptosis of HepG2 cells. Moreover, the positive role of SNAI1 in the malignant behavior of HepG2 cells was also verified in nude mice. Given the correlation with the abnormally higher expression of SNAI1 and clinical features of HCC patients, SNAI1 may be a novel biomarker and may have therapeutic potential for HCC patients.WR and CQ conceived and designed the experiments. JQ, HB, and FL analyzed the data. YJ, SG, and JS contributed reagents/materials/analysis tools. JQ, TL, and HB performed the experiments and wrote the paper.


We have previously showed that the bundle–crossing region of the transmembrane domain constitutes the crucial segment responsible for the polyamine block. In this study, we demonstrated that the major blocking effect of intracellular Mg2+ on Kir2.1 channels is also closely correlated with K+ current flow, and the coupled movements of Mg2+ and K+ seem to happen in the same flux–coupling segment of the pore as polyamines. With a preponderant outward K+ flow, intracellular Mg2+ would also be pushed to and thus stay at the outermost site of a flux–coupling segment in the bundle–crossing region of Kir2.1 channels to block the pore, although with a much lower apparent affinity than spermine (SPM). However, in contrast to the evident possibilities of outward exit of SPM through the channel pore especially during strong membrane depolarization, intracellular Mg2+ does not seem to traverse the Kir2.1 channel pore in any case. Intracellular Mg2+ and SPM therefore may have a synergistic action on the pore–blocking effect, presumably via prohibition of the outward exit of the higher–affinity blocking SPM by the lower–affinity Mg2+.Inward rectifier K It has been reported that 0.5–1 mM Mg2+ 881415164891011151819202122242+ but not SPM block2+ in the molecular physiology of Kir2.1 channels, given the fact that the coexisting polyamines  are already very potent voltage– and flux–dependent pore blockers effectively making the inward rectification phenomenon. It is therefore desirable to study the biophysical as well as molecular attributes of Mg2+ block Kir2.1 channel pore in more detail, and to make a comparison with SPM to decipher the differential and combinational roles of these two physiological blockers of Kir2.1 channel pore.Inward rectifier K2+ on the Kir2.1 channel is correlated with K+ currents flow, albeit quantitatively much less marked than SPM. The coupled movement of Mg2+ and K+ ions also happens in the same flux–coupling pore segment at the bundle–crossing region as SPM. With preponderant outward K+ flow, Mg2+ is pushed to the outermost site of the flux–coupling segment in the bundle–crossing region of the Kir2.1 channel pore. Because Mg2+ is much less likely to exit to the outside upon very strong depolarization or preponderant outward flow than SPM, Mg2+ may effectively enhance the blocking effect of SPM, a blocker with an apparently much higher affinity than Mg2+ upon moderate depolarization, but an evident tendency of outward exit of the pore with large driving forces.In this study, we demonstrate that the blocking effect of intracellular Mg2+ block of the WT Kir2.1 channel. + solution by 10 and 100 μM intracellular Mg2+. The outward currents are inhibited by intracellular Mg2+ with the decay phase accelerated in a dose–dependent manner. The inward currents, on the other hand, are not evidently affected by intracellular Mg2+. 2+ has relatively prominent changes in the vicinity of 0 mV . The change, however, is far less abrupt if compared to the case of spermine (SPM) examined in a similar way2+ but is ~102,000 in the case of SPM2+ and SPM on non–Kir2.1 channel expressing membranes in symmetrical 100 mM K+ as a control. There is no discernible effect on the non–Kir2.1 channel expressing membranes whether Mg2+ and SPM are present alone or concomitantly   in the flux–coupling region. These flux coupling sites, but not the blocking site itself, thus seem to be saturated by 100–300 but not 20 mM ambient K+. We further investigated the effect of the intracellular Mg2+ with a EK+ of −40 mV. The binding constant (Kd) of the Mg2+ block in asymmetrical  K+ is shifted by only ~−20 mV (2+ than SPM in 2+ block of the Kir2.1 channel pore.We subsequently changed the ambient KmV (EK+) . Interes ~−20 mV . This is2+ at different membrane potentials is also larger in the E224G, E224Q, and E299S mutant channels than that in the WT channel .In our previous study, we demonstrated that intracellular SPM block of the Kir2.1 channel is composed of a strongly voltage–dependent unblocking (δ = ~0.58) and a voltage–independent (δ = ~0.06) blocking processes2+ unblocking is discernible not only in mutant, but also in the WT Kir2.1 channels at “appropriate” voltage ranges. 2+ in the WT and different mutant channels (single and double mutations involving E224 and M183). The monotonous deceleration at more positive membrane voltages indicates slower unblocking rates of the blocking Mg2+ with membrane depolarization. Despite quite evident variations in the absolute values of the off rates, the voltage dependence remained similar among the WT and different mutant channels, suggesting a significantly altered height but not location and shape of the major barrier to the blocking Mg2+ exiting the pore. These data are very similar to SPM2+ permeation between the blocking site and intracellular melieu.2+ in the E224Q mutant channel are substantially accelerated by concomitant mutations at the bundle–crossing regions of M183W, M183N, and A178C , but also at the same blocking site itself. In other words, although mutations involving E224 and the bundle–crossing region of TM2 are separated by >40 residues in the amino acid sequence, they seem to share the same loci of action for intracellular Mg2+ block. These features are once more very much reminiscent of that of internal SPM block of the Kir2.1 channel.To further examine the role of the residues located immediately between I176 and M183, we constructed a series of single mutations from I176 to M183, which presumably constitute the bundle–crossing region of TM2. n of TM2 . E224G a2+ and SPM on the Kir2.1 channel, it is intriguing why there are the two blockers in the cell. In other word, what would be the role of Mg2+ given the already very strong blocking effect of SPM? We examined the blocking effect of coexisting internal Mg2+ and SPM. 2+, the presumable “physiological concentrations” of the blockers in certain cells81415162+ or 10 μM SPM. Concomitant intracellular 1 mM Mg2+ and 10 μM SPM apparently could further reduce the residual currents in the presence of either blocker alone. It is interesting that the much weaker blocker (1 mM) Mg2+ could be so prominently increase the blocking effect of the much stronger blocker (10 μM) SPM. A closer look at 2+ block, but not in the case of SPM block  with stronger depolarization. This could be ascribed to the increasing tendency of outward exit (and thus decreased blocking potency) of the blocking SPM, but not Mg2+, when there is more and more preponderant outward flux of the permeating K+ ion. We therefore did the same experiments in a more physiological condition , which would result in even more preponderant outward K+ flux at the same membrane voltage  could also be kinetically additive in addition to the foregoing synergism based on a more steady–state consideration. To investigate the interactions during the onset of block, we reduced the concentrations of both blockers to submicromolar range for better resolution of the kinetics , should have its blocking effect roughly at the same pore segment as SPM, presumably a long molecule with 4 regularly space positive charges and 3 intervening hydrophobic segments. The fact that the two very different blockers act on the same region of the pore would strongly indicate that functionally there is only one flux–coupling region responsible for the flow–dependent blocking phenomenon of the Kir2.1 channel pore. Moreover, although this region very likely contains regularly or semi–regularly spaced ionic sites and intervening hydrophobic groups in view of the very high IR index of SPM, it probably is not a fixed narrow fit for the permeating ions . Instead, it caliber could be so wide that Mg2+ could also cross the hydrophobic segment between two ionic sites “rapidly” enough to make a discernible flux–coupling blocking effect (see below). The initial very fast phase followed by a much slower phase of the “slow tails” upon repolarization in the presence of either SPM or Mg2+  may also contribute to the much smaller flow–dependence of block. On the other hand, it is intriguing that the intrinsic voltage–dependence of approaching and leaving the flux–coupling segment is rather similar for intracellular SPM and Mg2+  than “functional” . In other words, the bundle–crossing point . A small outward K+ conductance may therefore persist to significantly contribute to keep the resting membrane potential close to the K+ reversal potential, a basic physiological function of the Kir channels (see below).Although Mgg region , 71213 wegion 7121213. Theand Mg2+ 12. In ott ~A18412. These f2+  to stay in the widened middle part of the central cavity due to the decreased preponderance of outward K+ flux . Intracellular Mg2+ and SPM (or polyamine) and the Kir channels in the cell membrane would therefore make an ideal combination to assure not only essentially undisturbed initiation of cellular activities  but also rapid “stabilization” of the membrane as soon as the intervening repolarization phase ensues.We have seen that SPM, even with its much lower physiological concentrations ~10 μM14151617 1415162+ 1213. How2+ 12. This isrization , either rization . Mg2+, win vitro T7 mMESSAGE mMACHINE transcription kits, Ambio, Austin, TX, USA).The mouse Kir2.1 DNA was subcloned into phagemid expression vector of Bluescript II SK11213271213Xenopus oocytes (stages V and VI) were isolated by partial ovariectomy from frogs anaesthetized with 0.1% (w/v) tricaine (3–aminobenzoic acid ethyl ester), defolliculated using 1.5% type IA collagenase  and then maintained at 18 °C in ND 96 solution which contains 96 mM NaCl, 5 mM HEPES, 2 mM KCl, 1.8 mM MgCl2, 1.0 mM CaCl2, titrated to pH 7.6 by 1 M NaOH. Xenopus oocytes were microinjected 40–60 nl wild–type (WT) or mutant cRNA  and were maintained in ND 96 solution at 18 °C for no longer than 7 days after defolliculation1213Xenopus oocyte was then used for recording right after wash in the external solution containing: 72 mM KCl, 2 mM KH2PO4, 8 mM K2HPO4, and 5 mM K2EDTA, titrated to pH 7.6 by 1 M KOH.All animal handling procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University College of Medicine (Permit number: 2006–2320), and were performed in accordance with animal welfare guidelines by the National Institutes of Health (NIH)1213Xenopus oocytes expressing WT or mutant Kir2.1 channels were conducted at room temperature (22–25 °C) using the pClamp 6.0 software and an Axopatch 200A amplifier . Data were filtered at ~2 kHz and digitized at ~10 kHz through Digidata 1200A interface . The pipettes were pulled from borosilicate glass  using a horizontal puller  and fired polished . The pipette resistance was 0.2–0.5 MΩ when filled with extracellular solutions. Both the extracellular and intracellular solutions contained (the 100 mM K+ solution): 68 mM KCl, 8 mM K2HPO4, 2 mM KH2PO4, 5 mM K2EDTA, and 4 mM KOH, titrated to pH 7.4 by 1 M KOH1213+, intracellular and extracellular solutions both contained: 8.6 mM KCl, 4 mM K2HPO4, 1 mM KH2PO4, 3 mM EDTA, and 2.4 mM KOH, titrated to pH 7.4 by 1 M KOH. For the experiments in symmetrical 300 mM K+, intracellular and extracellular solutions both contained: 270 mM KCl, 8 mM K2HPO4, 2 mM KH2PO4, 5 mM K2EDTA, and 2 mM KOH, titrated to pH 7.4 by 1 M KOH. The desired total concentration of Mg2+ was obtained by mixing an appropriate volume of 5 mM Mg2+ intracellular solution with Mg2+–free intracellular solution, with free concentration of Mg2+ calculated by the WinMAXC32 software 15+, the intracellular solution was not changed, but the extracellular solution contained 20 mM K+ (see above for the detailed component of the solution). The excised inside–out giant patch was moved in front of an array of square glass  emitting different intracellular solutions, and was always subject to continuous perfusion of the intracellular solution for at least 10 min to wash out endogenous intracellular blockers before actual experiments were carried out.Excised inside–out giant patch recordings from 2+ has been examined based on the apparent dissociated constant (Kd) values from the Hill’s equation (one–to–one binding reaction)152632332+ could be reasonably fitted with a simplest form of Hill’s equation at membrane potentials more positive than ~−60 mV in symmetrical 100 mM K+ or more positive than −100 mV in 20 mM external/ 100 mM internal K+ , and Sigmaplot 10.0 software 12132+, [Mg2+] is the concentration of Mg2+, and f (the “relative currents”) is the ratio between the steady–state current amplitude in Mg2+ and that in control. All data are presented as mean ± SEM. Statistical significance was assessed using Student’s independent t test.where Kd is the apparent dissociation constant of Mg+ ions and water molecules were free to move during the energy minimization, heating, equilibrium and production process1213+ ions and/or one Mg2+ or one SPM molecule were applied to the central cavity region of Kir2.1 channel, and the one with the lowest potential energy were selected from approximately twenty candidate models.The modeling and simulation were performed in a similar way to that described in previous studies121324252912131213How to cite this article: Chiung–Wei, H. and Chung–Chin, K. A synergistic blocking effect of Mg2+ and spermine on the inward rectifier K+ (Kir2.1) channel pore. Sci. Rep.6, 21493; doi: 10.1038/srep21493 (2016).