Molecular manipulation was used to reveal the surface below the adsorbed molecules allowing to achieve an atomic-scale measure of the interaction of molecules with doped graphene. The adsorbate’s frontier electronic states are downshifted in energy as the molecule approaches the doping site, with largest effect when the molecule sits over the nitrogen dopant. Theoretical calculations showed that, due to graphene’s high polarizability, the adsorption of porphyrin induces a charge rearrangement on the substrate similar to the image charges on a metal. This charge polarization is enhanced around nitrogen site, leading to an increased interaction of molecules with their image charges on graphene. Consequently, the molecular states are stabilized and shift to lower energies. These findings reveal the local variation of polarizability induced by nitrogen dopant opening new routes towards the electronic tuning of graphene.Graphene-based sensors are among the most promising of graphene’s applications. The ability to signal the presence of molecular species adsorbed on this atomically thin substrate has been explored from electric measurements to light scattering. Here we show that the adsorbed molecules can be used to sense graphene properties. The interaction of porphyrin molecules with nitrogen-doped graphene has been investigated using scanning tunneling microscopy and  Porphyrins belong to an important family of biomolecules and their properties at interfaces are of high current interest2TPP) adsorbed on gold, graphene and nitrogen-doped grapheneHere, we report on scanning tunneling microscopy and spectroscopy (STM/STS) measurements and ab initio calculations was still needed. In order to provide the lacking data and theoretical interpretation, we have used molecular manipulation to gain access to the surface below the molecules, thus revealing the link between the molecular states, energy shifts and the underlying nitrogen positions with unprecedented atomic scale precision. The results are interpreted with the aid of a density functional theory (DFT) molecular modeling of the adsorption of H2TPP on graphene and N-doped graphene flakes revealing that although a small charge transfer is found between the molecules and the graphene flake, a change of local polarizability around nitrogen can lead to the observed downshift of the molecular levels. DFT results are consistent with a binding mechanism involving charge polarization of graphene induced by the polar bonds of the porphyrin, such that the adsorbate interacts with its image charges on the substrate. The presence of nitrogen increases the local polarizability leading to an enhancement of the image charges and a stabilization (energy downshift) of the molecular states. This model reproduces the observed downshifts of the molecular resonances around a doping site and proves that nitrogen doping leads to a local increase of graphene’s polarizability over nanometer scale range. These findings show how heteroatomic doping can modify the polarizability of graphene opening new avenues to control the interaction of graphene with its environment.We found that the molecular resonances associated with the porphyrin frontier orbitals shifted to lower energy when the molecules were located nearby a nitrogen site leading to the appearance of bright molecules in the molecular islands. Using a statistical analysis of the density of nitrogen dopants and bright molecules we concluded that the effect of a nitrogen dopant extends over 0.8 nm leading to an average number of bright molecule 2.2 times larger than the number of nitrogen atoms. However a direct experimental observation of the location of nitrogen atoms below the molecular islands was still lacking. A theoretical interpretation based on 2TPP molecules on nitrogen doped graphene following the procedure previously described2TPPs on doped graphene. On the graphene area around the molecules, bright spots can be seen corresponding to the substitutional nitrogen atoms13We have deposited HIn order to link the distribution of bright molecules with the location of underlying nitrogen sites, we performed molecular manipulation with the STM tip to remove the molecular island. This was achieved by scanning the whole image area with a setpoint turned to 0.2 V and 1 nA. We then turned back the setpoint to a regular imaging condition of 2 V and 50 pA. The image obtained after this operation is shown in With a high-resolution image on a smaller area as shown in dI/dV spectra that have been recorded on different molecules as a function of the distance from N site. In dI/dV spectrum taken on the molecule centred above N dopant (molecule 3) reveals HOMO and LUMO resonances at −1.53 V and 2.02 V (measured at the maxima of the peaks), respectively, which are lower than those of molecule 1 that appears at −1.32 V for HOMO and 2.22 V for LUMO. These values are fully consistent with those reported in our previous workdI/dV spectrum recorded above molecule 2 shows intermediate energies at −1.38 V for HOMO and 2.09 V for LUMO. In order to gain more insight into this energy shift, we recorded dI/dV spectra as a function of the position along the lines crossing these molecules. The shift can be seen more clearly in the color spectra map shown in This is illustrated with ab initio calculations on model systems to understand the nature of the binding of porphyrin on graphene and the effects introduced by chemical doping. The graphene (G) model is a cluster including 286 carbon atoms built from a 12 × 12 graphene unit cells and saturated by hydrogen at the zigzag edges. In the N-doped graphene (NG) model two carbon atoms from G model, one at the cluster centre and the other near one corner, were replaced by nitrogen atoms.In order to gain further insight and rationalize the experimental findings, we performed π system, thus raising the Fermi energy  by 0.08 eV with respect to that of G. The combined effect of extra electrons and the presence of the polar C-N bonds is a charge oscillation that extends over a radius of ≈8 Å from the substitutional site, as illustrated in The ground state geometries of G and NG are planar and C-C bond lengths average to 1.425 Å, while C-N bonds are shorter (1.410 Å). The most important difference between these clusters is the charge distribution: the two substitutional nitrogen atoms in NG add two extra electrons into the 2TPP on G and on NG resulted in very similar geometries. In both cases the adsorbed porphyrin adopts a saddle-shaped geometry with its centre lying above a C-C bond of the substrate, at distances 3.47 and 3.41 Å, respectively, from G and NG. The dopant site of NG is between two neighboring nitrogen atoms of porphyrin. The phenyl ligands torsion angles decrease, from 58 in free H2TPP to 47 in the adsorbed state. Charge transfer toward the macrocycle is very small in both substrates, amounting to 0.06–0.07 of an electron  which indicates that the molecule is physisorbed. This small amount of charge transfer is consistent with the large energy distance between the LUMO state and the Fermi level preventing from a transfer of a full electron that would bring the LUMO state at the Fermi level. The binding energy is thus due to dispersion and to the electrostatic interaction coming from the charge redistribution on the substrates induced by the polar bonds of H2TPP. Dispersion contributions to the binding are nearly the same in G and NG, as expected. However, as the larger molecule-substrate distance of the G cluster indicates, the binding energy of the H2TPP-NG system is stronger by 0.14 eV and the origin of this difference is the increased electrostatic interaction between the formal charges on porphyrin atoms and their image charges on the substrate.Adsorption of Hα of a system can be written, according to the charge-dipole modelThe static dipole polarizability Pind is the induced dipole moment due to an external field E. In the above equation the induced polarization is expressed as a sum of the atomic contributions, namely, the induced charge ri and its induced dipole moment ab initio calculations and, as we show below, the induced charges on NG due to the electric field of the adsorbed porphyrin are larger than those induced on G. This means that the local polarizability of NG around the doping site is larger than that of pristine graphene and it extends further away from the doping site since adsorbed molecules nearby the doping site are also affected by a stronger electrostatic interaction.where 2TPP. A and B structures correspond to the lowest energy conformation of the macrocycle adsorbed on G and on NG, respectively. Notice the gold surface underneath the four nitrogen atoms of the macrocycle, which means an accumulation of positive charge on the substrate in response to the net negative charge of the nitrogen atoms above it. Similarly, an accumulation of negative charge occurs underneath the four phenyl ligands that point to the substrate and place the positively charged hydrogen atoms in close proximity to the substrate. The larger extension of the charge redistribution of B when compared to A translates to larger image charges. The panels below A and B figures show a side view including porphyrin density differences. The depletion of electronic density above graphene indicates that no covalent bonding occurs between the molecule and graphene and that the interaction is of electrostatic nature. We conclude that NG is locally more polarizable than G. Structures C and D were obtained by translating the macrocycle along the NG substrate without optimizing the geometry, but keeping the distance of the central part of the molecule at 3.41 Å from NG. In C the porphyrin is between two doping sites while in D its central ring is all above carbon sites but it is still close to the doping site at the center. These structures suffer the influence of the cluster edge bonds through the phenyl ligands, giving rise to larger regions of induced negative charges, however due to charge neutrality the positive charges are present in same amounts but spread differently from A and B.2TPP. In 2TPP system and the PDOS with respect to the cluster Fermi energy. The resonances corresponding to the molecular HOMO and LUMO levels are located at −0.93 eV and 1.57 eV, respectively, resulting in a gap of 2.50 eV. The experimentally measured values are −1.34 eV and 2.00 eV so that there is an almost symmetrical mismatch of ≈0.4 eV between theory and experiment that is due to a well known difficulty of Kohn-Sham eigenvalues of DFT theory in reproducing fundamental gaps2TPP are, within the B3LYP (see Methods) calculation, −4.86 eV and −2.22 eV, respectively, with a resulting gap of 2.64 eV. We calculated the total energy of positive and negative ions to obtain the so-called ΔSCF  estimation of ionization energy and electron affinity, which are 5.98 eV and 1.06 eV, respectively. Hence, the ΔSCF fundamental gap is 4.92 eV, in much better agreement with the value of 4.63 eV obtained from experimental ionization energy2829We now turn to the analysis of the electronic structures. In summary, combining STM/STS with DFT calculations we showed that nitrogen doping of graphene leads to a local enhancement of graphene polarizability. This enhancement was probed by measuring the energy shift of HOMO and LUMO states of molecular adsorbates. These shifts are due to the interaction between the molecules and their image charges. The larger is the local polarizability, the larger the image charges and the larger is the HOMO/LUMO downshift. The nitrogen atoms induce variations of the polarizability over a distance that spreads to several atomic sites and, as a consequence, depending on the respective molecule-nitrogen locations, one nitrogen atom can influence more than one molecule. Our findings show that nitrogen doping can be used to modify the polarizability of graphene and how this polarizability is modified with atomic scale precision.−10 mbar. All the STM and STS measurements reported here were obtained at 4.6 K. The dI/dV spectra were acquired using a lock-in technique at a frequency of ca. 670 Hz and a modulation amplitude of 35 mV. The measurements were performed with an electrochemically etched tungsten tip. Graphene samples containing multilayer (≈5–10) were obtained on SiC(0002TPP) molecules (Aldrich) were deposited using an effusion cell (Dr. Eberl MBE-Komponenten GmbH) under vacuum at 255 °C onto the samples at the STM stage maintained at 4.6 K. The graphene samples were then brought to room temperature in few hours, allowing molecules to self-assemble and form a 2D island on the surface.The experiments were carried out using an Omicron LT-STM operating at ≈4.6 K under UHV condition with a pressure lower than 1 × 102TPP molecule was optimized in three distinct situations: (i) isolated molecule, (ii) molecule adsorbed on graphene, and (iii) molecule adsorbed on nitrogen-doped graphene. Graphene (G) was modeled as 12 × 12 unit cells graphene cluster (286 carbon atoms) saturated with hydrogen at the edges. Nitrogen-doped graphene (NG) was built by substituting two carbon atoms (21.3 Å apart) by nitrogens, thus representing a doping level of 0.7%. The cluster geometries were also optimized prior to the adsorption of H2TPP for the analyses of charge distributions and adsorption energy calculations. Total densities of states (DOS) and projected densities of states (PDOS) were calculated by adopting a gaussian broadening (0.2 eV) of Kohn-Sham eigenvalues. DFT calculations were carried out within Gaussian 09 packageElectronic structure calculations of the present study were performed in the framework of Density Functional Theory (DFT). The hybrid B3LYP functionalHow to cite this article: Pham, V. D. et al. Molecular adsorbates as probes of the local properties of doped graphene. Sci. Rep.6, 24796; doi: 10.1038/srep24796 (2016).


The proposed approach leads to OLEDs exhibiting ultrahigh external quantum efficiency of 40.8 and 62.1%  for single- and multi-junction devices, respectively. The OLEDs made on plastics with those electrodes are repeatedly bendable at a radius of 2.3 mm, partly due to the TiO2 layers withstanding flexural strain up to 4% via crack-deflection toughening.Graphene-based organic light-emitting diodes (OLEDs) have recently emerged as a key element essential in next-generation displays and lighting, mainly due to their promise for highly flexible light sources. However, their efficiency has been, at best, similar to that of conventional, indium tin oxide-based counterparts. We here propose an ideal electrode structure based on a synergetic interplay of high-index TiO Replacing indium tin oxide with graphene in organic light-emitting diodes is a promising approach to enhance the flexibility of displays and light sources. Here, the authors combine graphene, TiO2 and low-index hole-injection layers to achieve high external quantum efficiency and good bendability. With the optical design that takes a full advantage of the synergetic collaboration between the high- and low-index layers controlling both cavity resonance enhancement and loss to surface plasmon polariton (SPP), the proposed graphene-based OLEDs exhibit ultrahigh EQE that is unprecedented in those using graphene as a transparent electrode. Furthermore, unusually high resistance of TiO2 to flexural strain is revealed that enables plastic OLEDs that are not only efficient but also highly flexible.To this end, we here explore a simple electrode architecture based on high-index TiOλ/4 cavity design, where λ refers to wavelength, is achieved with the open end of the cavity mode placed at the ITO/substrate interface18The efficiency of OLEDs is limited ultimately by a finite outcoupling efficiency that is typically around 20% at best for devices with isotropic emitters. Among various outcoupling enhancing schemes proposed to date16nH) underneath graphene layers as shown in 172021λ/4 cavity design can be achieved conveniently, for example, with the optical thickness of the organic plus injection layers and the high refractive index layer set at approximately 2λ/4 and λ/4, respectively  for light incident from organic layers, as can be confirmed in the graphs shown at the bottom of Rbot and concomitant resonance enhancement increase with nH (nHdH) can be achieved with low physical thickness dH, and because bending-induced crack of a given film generally forms at a lower onset strain with a larger physical thicknessnH as possible. Furthermore, it should be transparent, and, most of all, should not be damaged by a given graphene transfer process. Among various candidates, a sputtered TiO2 layer is transparent in a visible spectral range and has a relatively high refractive index (n=2.5). Unlike other high-index layers we tried, the TiO2 layer turns out to have chemical endurance robust against graphene transfer processes that use solvent, such as acetone or isopropyl alcohol (IPA), allowing for formation of a high-quality graphene electrode on its top  based on a thick organic stack may also be possible. In this work, however, a first-order cavity design was first chosen because it results in a stronger Purcell effect and fewer waveguide modes than the higher-order design, ultimately leading to larger outcoupling efficiency and EQE242528λ of 550 nm (n=1.8). With the synergetic interplay of high- and low-index layers sandwiching graphene layers, enhancement of the cavity-resonant effect and reduction of SPP modes may thus be done simultaneously so that the EQE can be significantly boosted even without outcoupling structures.It is noteworthy that a higher order micro-cavity structure  GraHIL layers, results in an ideal situation where resonance enhancement is large, yet loss to SPP/evanescent modes is mitigated. In addition, the combined use creates a situation similar to HL-stacks, typically used for multilayer thin-film coatingRbot (ηEQE(max)) as a function of organic (dorg) and TiO2 thickness (dTiO2) presented in ηEQE(max) can be as high as 44% in devices with both GraHIL and TiO2 layers while it would be limited to 38% without GraHIL or to 31% without TiO2. Optimal conditions do correspond to a situation where dH (with H being TiO2) gets close to λ/(4nH) (55 nm for λ=550 nm). It can also be noted that ηEQE(max) of 44% is higher than those expected for optimized, conventional thin-metal based cavity OLEDs with the same emitter. This can be attributed to the fact that the proposed architecture is less subject to the loss due to photon absorption and/or SPP mode excitation within semi-transparent electrodes phenyl-C)(acetylacetonato) iridium (III) (Ir(ppy)2acac) in configuration of glass/anode/(HIL)/OS1/LiF/Al with different anode/(HIL) structures of TiO2/graphene/GraHIL; graphene/GraHIL; and ITO (185 nm)/GraHIL, where OS1 refers to an organic multilayer stack defined in Methods. Experimental results presented in 2/graphene/GraHIL-based device are as high as 40.8%, 160.3 lm W−1 and 168.4 cd A−1, respectively, while those values are limited to 31.7%, 112.6 lm W−1 and 119.0 cd A−1 for the graphene/GraHIL-based device, and 27.4%, 104.3 lm W−1 and 106.2 cd A−1 for ITO/GraHIL-based device. The observed high EQE in the TiO2/graphene/GraHIL-based device confirms the synergetic role of both TiO2 and GraHIL layers, and is consistent with the simulation results. With a half-ball lens optically coupled to the back of a substrate, the TiO2/graphene/GraHIL-based device exhibits EQE, power efficiency and current efficiency as high as 64.7%, 250.4 lm W−1 and 257.0 cd A−1, respectively, further illustrating the promising potential of the proposed graphene-based OLEDs. We then further extended the proposed scheme to a tandem, multi-junction OLED, in which the net EQE is improved (at the expense of voltage) because it is eventually given as the sum of those of its individual OLEDs2/graphene/GraHIL-based multi-junction OLEDs containing a charge-generation layer (CGL) of 2, 9-dimethyl-4, 7-diphenyl-1, 10-phenanthroline (BCP):Li/MoO3 exhibit EQE and power efficiency as high as 62.1% and 120.8 lm W−1 , demonstrating the state-of-the-art performance and the versatile applicability of the proposed electrode structure. The simulation indicates that the observed value corresponds to the sum of EQE values of equivalent second-order cavity structures  with the electrical balance factor of 0.89 /graphene/PEDOT:PSS/OS2/LiF/Al on 50-μm-thick polyethylene terephthalate (PET) substrates (see Methods for the multilayer stack used for flexible OLEDs (OS2)). It is noteworthy that the commonly available PEDOT:PSS  can play essentially the same optical role as GraHIL, only with a little compromise in its optical effect due to its refractive index being slightly higher than that of GraHIL (see rC) as small as 2.3 mm. This can be considered remarkable because the proposed OLEDs contains oxide layers, which are often regarded as brittle and thus prone to bending-induced fracture even at relatively low strain.The success of the graphene-based OLEDs would not be complete unless the advantage of graphene electrodes in flexibility can be utilized to a significant degree. To test the suitability of the proposed structure for flexible OLEDs, OLED devices were made in configurations of  as shown in 2 can withstand flexural strain (ɛf) as large as 4% without developing bending-induced cracks, while ITO (200 nm) and indium zinc oxide  electrodes withstand ɛf of 1% but start to exhibit cracks at 2% , green (G) and blue (B) pixels simultaneously with a common TiO2 layers were placed underneath graphene to enable cavity resonance enhancement. With the optical thickness of TiO2 layers set at λ/4, the first-order 3λ/4 cavity design was realized with the thickness of other layers falling within a range practical for electrical properties. The TiO2 layer with the optical thickness of λ/4 played a role as a high-reflectance coating and thus improved the resonance enhancement. Conducting polymers of PEDOT:PSS or GraHIL adopted as hole-injection layers plays an additional yet substantial role as a low-index layer that further enhance the EQE by redistributing the optical power gained from reduction of SPP loss that limits the performance of first-order cavity OLEDs; and by increasing the reflectance from the bottom electrode assembly and thus leading to an additional improvement in Fabry–Perot resonance effect. With the proposed synergetic interplay between high- and low-index layers, the optimized OLEDs exhibited ultrahigh EQE and power efficiency of 40.8% and 160 lm W−1; and 62.1% and 120 lm W−1 for single- and multi-junction structures, respectively , which are unprecedented for graphene-based OLEDs. Furthermore, efficient flexible OLEDs were demonstrated that can be bent to a radius of 2.3 mm. Independent study revealed that the TiO2 layer has an excellent flexural strain resistance unusual for most ceramic materials due to its crack deflection toughening mechanism. When combined with its high refractive index, this unique property enabled highly flexible OLEDs with significantly enhanced efficiency. Given the level of efficiency and bendability realized in this work, we believe the proposed method can pave the way for graphene-based OLEDs to become a next-generation light source balanced with both efficiency and form factor advantages.In summary, an ideal optical structure was proposed for graphene-based OLEDs, which leads to maximized efficiency while retaining their advantages in flexibility. The inherent thinness of graphene electrodes being considered, high-index TiO−1; Shinan, Korea) were cleaned as reported earlier2 layers  or IZO layers were deposited using a DC pulsed (500 W) or RF (120 W) sputtering process. Formation of MLG on a target substrate was done in one of the following two ways: one involved quadruple repetition of growth of a SLG on a copper foil and subsequent wet-transfer process to a target substrate /Si wafer held at 750 °C in an inductively coupled plasma system, followed by a wet transfer process  of doped MLG layers from Methods 1 and 2 were 92.5±9.4 Ω sq−1 and 90%, and 330.3±16.1 Ω sq−1 and 88%, respectively.Polished glass substrates  and those with the pre-coated ITO films (<12 Ω sq2 (55 nm)/MLG, MLG or ITO electrodes to result in 70 and 50-nm-thick films, respectively. The spin-coating solution for GraHIL was prepared by mixing PEDOT:PSS (Clevios P VP AI4083) with PFI (Sigma-Aldrich) in 1:1 ratio2acac /CBP: Ir(ppy)2acac /TPBi (55 nm), where TAPC, TCTA, CBP and TPBi refer to 4,4′-cyclohexylidenebisbenzenamine); Tris (4-carbazoyl-9-ylphenyl) amine; 4, 4′-Bis(N-carbazolyl)-1, 1′-biphenyl; and 2, 2, 2-tris-(1-phenyl-1H-benzimidazole), respectively3 (5 nm)/CBP (20 nm)/1:1 co-host of CBP: B3PYMPM doped with 7% Ir(ppy)2acac (20 nm)/B3PYMPM (55 nm), where B3PYMPM refers to -2-methylpyrimidine). Note that, in the case of PEDOT:PSS-based devices, MoO3 (Alfa Aesar) was deposited on top of PEDOT:PSS to ensure efficient hole injection.GraHIL or PEDOT:PSS (mixed with IPA (3:1 by volume)) were spin-coated as a HIL on top of TiOn=1.52, Newport). Flexural strain measurement was done with a high-precision micromechanical test system  according to ASTM standard (D790). The bending-cycle test of OLED devices and TiO2/MLG on plastic substrates was done with a custom-made bending tester able to adjust the radius of curvature and the number of bending cycles.Fabricated OLED devices were evaluated either in ambient air after encapsulation done in an inert atmosphere  of 0.23±0.02, corresponding to ca. 77% horizontally oriented dipoles, the radiative quantum efficiency of 0.97 were used following the previous reports39Optical modelling was done with a custom-made MATLAB code following the formalism summarized by Furno The authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Lee, J. et al. Synergetic electrode architecture for efficient graphene-based flexible organic light-emitting diodes. Nat. Commun. 7:11791 doi: 10.1038/ncomms11791 (2016).Supplementary Figures 1-11, Supplementary Tables 1-6, Supplementary Notes 1-3 and Supplementary References.


Indeed, near-percolated liquid-crystalline graphene-based composites display unprecedented dielectric properties with a dielectric constant improved by 260-fold increase as compared with the polymer matrix, while maintaining the loss tangent as low as 0.4. This performance is shown to depend on the structure of monodomains of graphene liquid-crystalline phases. Insights into how the liquid crystal phase transition interferes with percolation transition and thus alters the dielectric constant are discussed.Graphene flakes with giant shape anisotropy are extensively used to establish connectedness electrical percolation in various heterogeneous systems. However, the percolation behaviour of graphene flakes has been recently predicted to be far more complicated than generally anticipated on the basis of excluded volume arguments. Here we confirm experimentally that graphene flakes self-assemble into nematic liquid crystals below the onset of percolation. The competition of percolation and liquid crystal transition provides a new route towards high- It is commonly believed that graphene flakes form electrical percolation networks at low concentration, and thus can be used as conductive materials. Here, Yuan et al. show in graphene polymer composites that the transition to liquid crystals hinders the formation of percolated networks, resulting in high-k materials. The concept of percolation, initially discussed in the context of scaling behaviour and universality in statistical physics, finds today a variety of applications in materials research as a powerful framework to account for the physical properties of heterogeneous materials245Percolation at very low concentrations has often been found in systems with rod-like particles of large aspect ratios in the macroscopically isotropic statek materials for various applications, including gate dielectrics, energy storage devices and electroactive materials1213The absence of percolation in equilibrated samples could still be exploited positively from a technological point of view, in particular for the development of new dielectric materials. There is a fast growing interest in developing graphene-containing composites that bear favourable dielectric properties1213However, experimental identification of the manifestation of competing percolation and LC phase transition requires the challenging development of graphene suspensions that can percolate or form LCs at rest without being affected by non-equilibrium mechanisms such as flow or volume changes in drying systems. Liquid crystallinity of monolayer graphene in aqueous media151719k materials.In this work, we have succeeded in producing LCs of graphene oxide (GO) in a non-polar organic solvent. Such suspensions can readily serve as precursors to prepare polymer composites with polydimethylsiloxane (PDMS) as matrix. This unique system allows the demonstration that large graphene flakes spontaneously form LCs before they percolate, confirming therefore the theoretical prediction that graphene flakes, in spite of their giant aspect ratio, do not exhibit a low percolation threshold. In addition to providing the first test of this prediction, the present materials have unique dielectric properties. They exhibit a very high dielectric constant of 753 at 100 Hz along with a low loss tangent of only 0.4. These findings are generic and expected in other composites manufactured from dispersions of highly conductive platelets with large aspect ratios, opening thereby a new route towards efficient high-b-PDMS copolymer (PAPMS-b-PDMS) under ambient conditions via electrostatic attractions . As shown in O flakes . As comp−1 by optical microscopy under crossed polarizers. At this concentration, the suspensions are found to be isotropic and appear homogeneous and black. However, more concentrated samples controlled by partial evaporation of the solvent display optical birefringence. The latter reveals a liquid-crystalline order . Such marginal improvement indicates that the 1.0 wt% sheet loading is well below the percolation threshold. However, at this rGO-PDMS mass ratio, the LC transition has already taken place  approaches the percolation threshold,ϕc, from below, the dielectric constant, ɛc, of the composite diverges as ɛmatrix is the relative dielectric constant of the polymer matrix and q is an exponent of about 1 . Here the volume of diethyl ether used is the same with GO solution. As the diethyl ether is immiscible with water and has a lower density than water, the copolymer solution in diethyl ether would form an upper phase with GO water solution as a bottom phase. This biphasic mixture was magnetically stirred for 12 h so that the modified GO flakes can be recovered in the organic phase as the phase transfer is achieved. Finally, the mixture will separate into two phase, that is, a clear aqueous phase and a thick, brown organic phase. It should be noted that the phase transfer can be well performed even in the presence of PDMS dissolved in the upper organic phase. In this way, the obtained GO/PDMS composite precursor can be used to prepare solid composites based on a typical solution casting method.A typical phase transfer process can be described as follows. A certain amount of selected copolymer was first dissolved in diethyl ether and subsequently mixed with the readily diluted GO suspensions . The dielectric properties of the rGO/PDMS composites were characterized as a function of frequency (0.1–106 Hz) at room temperature using an impedance analyser . Before measurement, silver grease has been applied on the sample surfaces for contacting.Surface morphologies of the GO flakes before and after copolymer modification were characterized by TEM (Hitachi H600). The Raman spectra were taken using an excitation of wavelength of 532 nm on Horiba Jobin Yvon Xplora. The laser spot size is 1 μm. Samples were prepared by depositing GO aqueous solution or modified GO organic solution on silicon wafers, followed by a completely drying process at room temperature for 12 h. GO and GO/PDMS suspensions  were taken to investigate their birefringence under a polarizing optical microscope (Leica DM 2500P) with × 10 and × 40 objectives. The morphology of as-prepared GO/PDMS composites was observed by a SEM using a field-emission SEM instrument (JEOL 6700FEG) after sputter coating with platinum. The GO and rGO-based PDMS composites are further examined under a TEM (Hitachi H7650). For TEM analysis, the composites were ultramicrotomed to slices of 40–60 nm thickness (Leica UC7). X-ray data were collected on a Rigaku Nanoviewer , with a 1,200-W rotating anode coupled to a confocal Max-Flux Osmic mirror  and a MAR345 image plate detector . Samples were put in quartz capillaries, which are exposed to the incident X-Ray beam. The detector is placed at a distance of 1,193 mm, providing access to 2How to cite this article: Yuan, J. et al. Graphene liquid crystal retarded percolation for new high-k materials. Nat. Commun. 6:8700 doi: 10.1038/ncomms9700 (2015).Supplementary Figures 1-6, Supplementary Note 1, Supplementary Discussion, Supplementary Method and Supplementary References


In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1–expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity  Recently, cancer has been a major cause of death in dogs and has surpassed infectious diseases. According to a recent report, 27% of overall dog deaths is due to cancer Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together can induce the “exhausted” status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells in vitro increased cytokine responses and enhanced immune cell function, leading to decrease in the viral load Recent studies in our laboratory have revealed that immunoinhibitory molecules including PD-1/PD-L1 play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus (BLV) infection Animal use throughout this study was approved by the Institutional Animal Care and Use Committee , Graduate School of Veterinary Medicine, Hokkaido University, which has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Peripheral blood samples were obtained from healthy 5- or 8-year-old Beagles kept at the Experimental Animal Facility, Graduate School of Veterinary Medicine, Hokkaido University. Clinical samples were surgically collected from dogs with tumors at the Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University in 2012–2013. For immunohistochemical analysis, tumor specimens kept at NORTH LAB  were used.2. Chinese hamster ovary-DG44 (CHO-DG44) cells were cultured in CD-DG44 medium (Life Technologies) containing GlutaMAX supplement  and 10% Pluronic F-68  at 37°C and 5% CO2. The canine melanoma cell lines CMeC −5 M 2-mercaptoethanol, 2 mM L-glutamine, 200 µg/mL streptomycin, and 200 U/mL penicillin at 37°C and 5% CO2. The canine mastocytoma cell lines CM-MC 3, 2 mM L-glutamine, 200 µg/mL streptomycin, and 200 U/mL penicillin at 37°C and 5% CO2. The canine osteosarcoma cell lines POS 2. To stimulate the cells, the canine tumor cell lines were treated with 100 ng/mL canine recombinant IFN-γ  and cultured for 24 h. Canine peripheral blood mononuclear cells (PBMCs) were purified from heparinized blood samples by density gradient centrifugation on Percoll  and cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 200 µg/mL streptomycin, and 200 U/mL penicillin at 37°C and 5% CO2. Concanavalin A (ConA)  or PMA  and ionomycin  were added to the medium to activate lymphocytes.Cos-7 cells (African Green Monkey SV40-transformed kidney fibroblast cell line) 5′-TTA TGT CTC TTC AAA TTG TAT ATC-3′ (PD-L1 inner reverse). The PCR cycling conditions were as follows: (1) initial denaturation at 94°C for 5 min, (2) 40 cycles of denaturation at 94°C for 1 min, annealing at 58°C (PD-1) or 50°C (PD-L1) for 1 min and extension at 72°C for 1 min 30 s, and (3) final extension at 72°C for 7 min. PCR amplicons were purified using the FastGene gel/PCR extraction kit , cloned into the pGEM-T Easy vector , and sequenced using the CEQ8000 DNA analysis system . 5′-RACE and 3′-RACE were then performed using the 5′-RACE system for rapid amplification of cDNA ends and 3′-RACE system for rapid amplification of cDNA ends (Life Technologies), respectively. The gene-specific primers for the canine PD-1/PD-L1 used for 5′-RACE were 5′-GTT GAT CTG TGT GTT G-3′ (PD-1) and 5′-TTT TAG ACA GAA AGT GA-3′ (PD-L1). The gene-specific primers for canine PD-1/PD-L1 used for 3′-RACE were 5′-CGG GAC TTC CAC ATG AGC AT-3′ (PD-1) and 5′-GAC CAG CTC TTC TTG GGG AA-3′ (PD-L1). Based on the sequences obtained, new primer sets were designed to amplify the full length of the canine PD-1 and PD-L1 cDNA. PCR was performed using primers 5′-GGG GGA GGC GAG CAG G-3′ (PD-1 ORF F), 5′-GAG TCG AGA GAG GAG AGC CAT GAG-3′ (PD-1 ORF R), 5′-GCC AGC AGG TCA CTT CAG AAC-3′ (PD-L1 ORF F), and 5′-GCT GAA CTC AAA CCA CAG GCC-3′ (PD-L1 ORF R) as described above, except that the annealing temperature used was 60°C. The resulting amplicons were sequenced as described. To confirm the polymorphisms of the PD-1 and PD-L1 genes among canine breeds, the blood samples of the Beagle, Samoyed and Labrador retriever, testis tissue of the Japanese Akita, and lung tissue of the Bernese mountain dog were collected and studied using ORF primer and each the cDNAs of each sample. The established sequences were aligned, and unrooted neighbor-joining trees were constructed using the Mega version 5 software Total RNA was isolated from the Beagle and the Samoyed PBMCs stimulated with ConA for 4 h, white blood cells of the Labrador retriever, testis tissue of the Japanese Akita, and lung tissue of the Bernese mountain dog, using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Residual genomic DNA was removed from the total RNA by DNase (Life Technologies) treatment. cDNA was synthesized from 1 µg of the total RNA using Moloney murine leukemia virus reverse transcriptase  and oligo-dT primer, as recommended by the manufacturer. To amplify the inner sequences of canine PD-1 or PD-L1, canine PD-1– and PD-L1–specific primers were designed based on the putative canine PD-1 and PD-L1 mRNA sequence reported in the GenBank database (XM_543338 and XM_541302). Canine PD-1 and PD-L1 cDNA were amplified from Beagle cDNA by PCR using primers 5′-AGG ATG GCT CCT AGA CTC CC-3′ (PD-1 inner forward), 5′-AGA CGA TGG TGG CAT ACT CG-3′ (PD-1 inner reverse), 5′-ATG AGA ATG TTT AGT GTC TT-3′ (PD-L1 inner forward), and XhoI restriction site), 5′-CGC GGA TCC TGA GGG GCC ACA GGC CGG GTC-3′ , 5′-GAA GAT CTA TGA GAA TGT TTA GTG TC-3′ , and 5′-GGA ATT CTG TCT CTT CAA ATT GTA TAT C-3′ ]. The amplicons were then cloned into the multicloning site of the pEGFP-N2 vector . These vectors were named pEGFP-N2–cPD-1 and pEGFP-N2–cPD-L1, respectively. For transient expression, Cos-7 cells (5×104/cm2) were transfected with 0.4 µg/cm2 of the expression vector pEGFP-N2–cPD-1 or pEGFP-N2–cPD-L1 or empty pEGFP-N2 vector as the mock, using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. The cells were cultured at 37°C and 5% CO2 for 48 h and harvested using a cell scraper. To confirm the expression of fusion proteins, the cells were observed under a confocal microscope LSM700 , and the subcellular distributions of EGFP were determined.To construct the enhanced green fluorescent protein (EGFP) fusion expression vectors, the ORF region of the canine PD-1 and canine PD-L1 cDNA that did not have the stop codons was amplified by PCR using the cDNA obtained from the Beagle PBMCs and gene-specific primers . The amplicons were then cloned into the multicloning site of the pCXN-2.1–rabbit IgG Fc vector  6 CHO-DG44 cells were transfected with 2.5 µg pCXN-2.1–rabbit IgG Fc-cPD-1 or pCXN-2.1–rabbit IgG Fc-cPD-L1 using Lipofectamine LTX (Life Technologies), as recommended by the manufacturer. Forty-eight hours later, the cells were collected and resuspended in the supplemented CD-DG44 medium containing 800 µg/mL G418 . Stably expressing cells were cloned using the limiting concentration technique, and high expression cell lines were obtained. The cell culture supernatant of these cell lines was collected after 7 days of the shaking culture. The supernatant containing Ig fusion proteins was concentrated by ultrafiltration using Centricon Plus-70  and the Ig fusion proteins were purified by Ab-Capcher Extra . Buffers were exchanged with phosphate buffered saline (PBS) using PD MiniTrap G-25 . The concentrations of the Ig fusion proteins were evaluated using the rabbit IgG ELISA quantitation set . To confirm the expression of these Ig fusion proteins, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis with the Immobilon-P transfer membrane (Merck Millipore) were performed as described elsewhere For the construction of the cPD-1–Ig and cPD-L1–Ig expression vectors, cDNA sequences of cPD-1 or cPD-L1 encoding the putative extracellular region of those proteins were amplified by PCR using the cDNA obtained from the Beagle PBMCs and the gene-specific primers , which were established in our laboratory 5) were incubated with various concentrations  of antibovine PD-L1 monoclonal antibody 6G7-E1 at 37°C for 15 min prior to incubation with 1 µg/mL  of cPD-1–Ig at 37°C for 30 min. The cells were washed twice, and cPD-1–Ig, which was bound to the cPD-L1–EGFP-expressing cells, was detected by flow cytometry, as described above. As a negative control antibody, rat IgM (κ) isotype control (BD Biosciences) was used.To confirm the blockade of canine PD-1/PD-L1 binding by anti-PD-L1 antibody, cPD-L1–EGFP-expressing Cos-7 cells  and side-scattered light (SSC), and the cell population with a higher FSC than lymphocytes was gated as the tumor cell population if there was no other population except for the population of tumor-infiltrating lymphocytes. To exclude any dead cells, the cells were stained with 7-amino-actinomycin D (BD Biosciences), and the live cell population was analyzed for PD-L1 expression. The canine PBMCs were used as a negative control under the same conditions.To obtain single cells from solid tumors, freshly excised solid tumor tissues were cut into small pieces and treated with 2 mg/mL collagenase D  in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 200 µg/mL streptomycin, and 200 U/mL penicillin at 37°C for up to 2 h. The single cell suspension was washed twice with PBS and cultured in supplemented RPMI 1640 medium at 37°C and 5% COFormalin-fixed and paraffin-embedded tumor tissues were cut into 4-µm-thick sections and dried on silane-coated slides. The dried sections were deparaffinized in xylene. Antigen retrieval was performed in citrate buffer (0.37 g/mL of citric acid and 2.4 g/mL of trisodium citrate dihydrate) by microwave heating for 10 min. Endogenous peroxidase activity was blocked by incubating the sections in methanol containing 0.3% hydrogen peroxide for 15 min. Primary antibody incubation was performed at room temperature for 30 min using antibovine PD-L1 monoclonal antibody 5A2-A1 (1.2 µg/mL) or rat IgG1 isotype-matched control antibody . The sections that were washed twice with PBS were incubated with Histofine simple stain MAX PO (rat)  at room temperature for 30 min, and positive staining was visualized with 3-diaminobenzidine tetrahydrochloride (DAB). The sections were observed under an optical microscope.g for 20 min, and the cell pellets were collected and resuspended in the supplemented RPMI 1640 medium. Then, the cell suspensions were again underlaid onto 1.077 g/mL Percoll separation solution and centrifuged. The tumor-infiltrating lymphocytes in the gradient interfaces were collected and washed with PBS three times. These lymphocytes were resuspended in the supplemented  RPMI 1640 medium.Single cell suspensions from fresh solid tumor tissues were obtained by collagenase treatment, as described, or by a mechanical method using the 100 µm cell strainer . The suspensions were underlaid onto 1.055 g/mL Percoll separation solution. The tumor cells were separated by density gradient centrifugation at 800×6/mL) were cultured with 20 µg/mL of antibovine PD-L1 monoclonal antibody 6G7-E1 at 37°C and 5% CO2 for 48 h, and the culture supernatant was collected. As a negative control, low-endotoxin, Azide-free rat IgM isotype control antibody  was used. The concentration of dog IFN-γ in the culture supernatant was evaluated by Duoset ELISA canine IFN-γ  according to the manufacturer’s protocol. Absorbance was measured using a microplate reader MTP-650FA .The tumor-infiltrating lymphocytes and PBMCs obtained from the healthy adult Beagles  and AB898678 (PD-L1).The complete nucleotide sequences and putative amino acid sequences of canine PD-1 and PD-L1 obtained from the Beagle dog are shown in in vitro model by transfecting Cos7 cells with canine PD-1 and PD-L1 and generated transient transfectants. Expression of canine PD-1 and PD-L1 was detected on cPD-1–EGFP and cPD-L1–EGFP transfectants  from canine mastocytoma, and the expression was enhanced by IFN-γ treatment , Table 3p<0.05). Finally, to confirm that PD-L1 antibody augments the production of cytokines from tumor-infiltrating cells, PD-L1 antibody was added to the infiltrating cell cultures from hepatocellular carcinoma, myelolipoma and seminoma at the beginning (To investigate the effect of PD-1/PD-L1 blockade by PD-L1 antibody in cytokine production, PBMCs from healthy dogs were cultivated in the presence of PD-L1 antibody or isotype control antibody. As shown in eginning . Intereseginning .Several immunotherapies against tumors have been recently developed in humans. Among these therapies, an immunotherapy targeting the PD-1/PD-Ls pathway would be considered as one of the most encouraging approaches because, in a Phase I clinical trial, treatment with PD-L1 antibody induced durable tumor regression and prolonged stabilization of disease in patients with advanced malignant cancers, including non-small-cell lung cancer, melanoma, and renal cell cancer PD-L1 was detected on canine cell lines from mastocytoma and on fresh cells from angiosarcoma, hepatocellular carcinoma, squamous carcinoma and breast adenocarcinoma. Furthermore, the increasing expression of canine PD-L1 on tumor cells was confirmed in clinical cases of melanoma, mastocytoma, and renal cell carcinoma by immunohistochemical analysis. These findings corresponded to those of human cancers. However, the expression of PD-L1 was not observed in some cases, such as osteosarcoma-derived cell lines and tumor tissues from clinical cases, melanoma, mastocytoma, and renal cell carcinoma. PD-L1 expression is induced by cytokines such as IFNs type I and type II In the present study, we detected significant PD-L1 expression on tumor tissues from dogs with mastocytoma and renal cell carcinoma, which originates from mast cells and renal tubular epithelial cells. Dogs have a unique risk of development of cutaneous mastocytoma, which accounts for up to 21% of all skin tumors; however, mastocytoma is rare in human and other species Melanoma is a common tumor in dogs; it is a locally invasive and frequently malignant type of cancer that can affect dogs in vivo. Furthermore, the level of PD-1 expression on the tumor-infiltrating T-cells is still unknown because of the lack of a specific antibody for canine PD-1. These preliminary results must therefore be investigated more in detail.As a preliminary test of the hypothesis that PD-L1 may be a therapeutic target for the treatment of canine tumors, we investigated the effects of blockade of PD-1/PD-L1 by PD-L1 antibody in tumor-infiltrating T-cells from dogs with seminoma, hepatocellular carcinoma and myelolipoma. Similar to previous findings in human or mice models, inhibition of the PD-1/PD-L1 pathway had upregulated the production of IFN-γ from lymphocytes from hepatocellular carcinoma and myelolipoma. However, although the effects of PD-L1 antibody on IFN-γ production seem to be profound, it still remains speculative whether the blockade of PD-1/PD-L1 by PD-L1 antibody will be sufficient for tumor regression In conclusion, we here presented aberrant expression of PD-L1 on tumors in dogs and discussed their potential as therapeutic targets for canine tumors. PD-L1 might contribute to the progression of canine tumors via antitumor T-cell dysfunction. In the clinical trials targeting the PD-1/PD-L1 pathway in humans, none of the patients with PD-L1–negative tumors had a positive response; however, reactivation of the antitumor immunity and subsequent regression of some tumors were induced in patients with PD-L1–positive tumors, including malignant cases


The transitions between turns at talk in conversation tend to occur quickly, with only a slight gap of ∼100–300 ms between them. This estimate of central tendency, however, hides a wealth of complex variation, as a number of factors, such as the type of turns involved, have been shown to influence the timing of turn transitions. This article considers one specific type of turn that does not conform to the statistical trend, namely turns that deal with troubles of speaking, hearing, and understanding, known as other-initiations of repair (OIR). The results of a quantitative analysis of 169 OIRs in face-to-face conversation reveal that the most frequent cases occur after gaps of ∼700 ms. Furthermore, OIRs that locate a source of trouble in a prior turn specifically tend to occur after shorter gaps than those that do not, and those that correct errors in a prior turn, while rare, tend to occur without delay. An analysis of the transitions before OIRs, using methods of conversation analysis, suggests that speakers use the extra time (i) to search for a late recognition of the problematic turn, (ii) to provide an opportunity for the speaker of the problematic turn to resolve the trouble independently, and (iii) to produce visual signals, such as facial gestures. In light of these results, it is argued that OIRs take priority over other turns at talk in conversation and therefore are not subject to the same rules and constraints that motivate fast turn transitions in general. In conversation opportunities to participate are organized by a system of turn-taking . The rulPrevious research on the timing of turn-taking has shown that the transitions between turns in conversation most frequently occur with only minimal gaps and overlaps. First documented systematically in a series of meticulous conversation-analytic studies , the timThis estimate of central tendency has clear implications for psycholinguistic models of turn-taking. As But an estimate of central tendency is by definition a simplification, a single value that ideally represents a more complex distribution. Research has also examined sources of complex and systematic variation in the timing of turn-taking, especially in question–answer sequences. The language and culture of the speakers, the deployment of gaze, and the type and modality of the response all have been shown to influence the timing of responses to polar questions . QuestioAt least one type of turn that does not conform to global generalizations about the timing of turn-taking has been identified in the conversation-analytic literature. Turns that deal with troubles of hearing or understanding prior turns , referred to as OIRs, have been reported to be systematically withheld . The traBased on a systematic qualitative analysis, longer than gaps before other turns. Thus while Robinson’s finding supports the claim by A second report of the phenomenon, made in passing by The observation of systematic variation in the timing of turn types  points to further questions. Is variation in the timing of OIRs also systematic? Do different types of OIRs, like different types of responses to questions, tend to occur after relatively shorter or longer transitions?The inventory of OIR practices in English is relatively well described see . A basicfield.” A: “Butterfield.”). Correction by someone other than the speaker of the error, known as other-correction, has been argued to be a dispreferred alternative to self-correction. A further distinction is whether an OIR constitutes a correction or not . An OIR A final set of questions concerns the motivations for and consequences of delay before OIR. Conversation analysts have argued that the timing of OIRs has a socio-interactional basis. A possible next speaker who encounters a problematic turn withholds an OIR in order to provide the current speaker with an opportunity to resolve the trouble on his or her own . If a neBut a priori one might propose complementary or alternative explanations for the phenomenon of delay before OIR. The private processes that speakers necessarily engage in to hear and understand a turn at talk occur in real time and, just like other actions, take time to complete. The high frequency of transitions with minimal gaps suggests that these processes very often occur quickly. But might they not take longer under certain conditions, say, when a next speaker has failed to hear, or understand a prior turn? Is the delay before an OIR for the benefit of the speaker of the trouble, to provide an opportunity for self-initiated repair, or is it  for the benefit of the recipient of the trouble, to allow a search for and a possible recovery of a hearing and understanding of the turn that may permit the sequence to move forward without repair?do in the transition spaces before OIR?One might also look to the embodied actions of the participants for an explanation. The private processes that participants engage in surely take time to complete, but so too do the physical actions they perform. A withdrawal of gaze from the speaker of a trouble-source turn, a rotation of the head and body to face a trouble-source speaker, a meaningful deployment of facial muscles into a gesture of puzzlement—embodied actions such as these all take time to produce. Through the filter of a text transcript, a silence between turns at talk, whether long, or short, can look like an absence of action. But the long tradition of multimodal conversation analysis Are the transition spaces before OIRs systematically longer than those before other turn types, such as answers to questions?(2)Are the transition spaces before some types of OIRs systematically longer than those before other types?(3)What are the motivations for the expansion of transition spaces before OIRs? That is, what do speakers use the extra time to accomplish?The investigation uses conversation analysis to identify and analyze OIRs and question–answer sequences, as well as to discover and describe a number of uses that speakers have for the expanded transition spaces before OIRs. The measurements of transition spaces and the comparisons of the distributions are done quantitatively. At the end of the article I return to the questions of the motivations for and consequences of delay before OIRs and consider the relationship between repair and the turn-taking system.The data for the investigation came from video-recordings of naturally occurring English conversation between friends and family members engaged in a variety of activities . The corpus consisted of 19 recordings, with a total duration of 9 h and 20 min, and included native speakers of English from the U.S., Canada, and the U.K. Informed consent was obtained from all participants.Table 1 along with examples  and Extract (2), respectively.(1) Virginia1 Wes:	    ◦Here you go◦2 Bet:	    (But) she4 Nix: →                       [you ] mean by his lawyer5 Nix:     [hhhhhhhhh]6 Hal:     [yeah or] (.) somebody else(15) CallHome 6079 1 A:      it was [so: nice] it was so nice =2 B:      [hhhhh @ ]3 A:      = that they came I can’t even tell you4         like.hhhh like (0.6) just seeing them5         like I was performing to them6         (.)7    →    like I was sm[iling at th]em like8 B: →                 [to who. ]9 A:      .hhhh Juliette, Sam: and (.) and TaraThe fact that OIRs win the turn without the need for competition and, moreover, that trouble-source speakers respond to OIRs with no delay is further evidence that repair is a priority activity in conversation .The data above also illustrate yet another intersection between turn-taking and repair. As noted previously, the imminent possibility that a current speaker may continue his or her turn if no one self-selects builds a motivation for fast transitions into the turn-taking system. Note, however, that the OIRs in Extracts (14) and (15) occur in overlap with a continuation by the current speaker (at the first arrowed lines). This demonstrates that OIRs supersede not only first-starters in self-selection, but also continuations by current speakers. The window of opportunity for OIRs is thus larger than for other next turns, which are subject to constraints on turn allocation and overlap that OIRs appear to out rank.this constraint, which operates for open OIRs, establish a motivation for fast turn transitions? It provides an outer bound for the timing of open OIRs, but given that an open OIR can in principle occur in overlap with a subsequent TCU (before its possible completion) and take priority, it would not systematically motivate a fast transition on the order of 100–300 ms between the trouble-source TCU and the OIR.This is not to say that the timing of OIR is without constraint. The organization of repair imposes certain constraints on the timing of OIRs vis-à-vis the selection of OIR practices see . An openavoid this covert signal , but rather exploit it. As a motivation for fast turn transfers, the potential for silences in conversation to be meaningful signals of interactional trouble is thus context-sensitive, operating only for next turns not themselves designed as signals of trouble.What, then, of the potential for silences to be meaningful, as signals of interactional trouble? Might this provide a motivation for fast transitions? The answer becomes clear once one registers that OIRs are themselves signals of interactional trouble. Although a covert signal of trouble like silence will be at cross-purposes with some incipient actions , it can also point in the same direction as an incipient action , in which case it is interactionally advantageous. An expanded transition space can indicate that a next speaker has encountered trouble, but it also provides an opportunity for the resolution of the trouble. There is reason to believe that speakers do not In sum, the rules and constraints of the turn-taking system that motivate fast transitions – concerning overlap management, turn allocation, and the semiotics of silence – neither rule nor constrain the timing of OIRs. The priority given, at the intersection of turn-taking and repair, to the resolution of troubles in hearing and understanding provides a systemic explanation for the observation that transitions before OIRs tend to be longer than those before other next turns, for which the rules and constraints of the turn-taking system remain operative.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using density functional theory calculations, we have investigated the effects of biaxial tensile strain on the electronic and magnetic properties of partially hydrogenated graphene (PHG) structures. Our study demonstrates that PHG configuration with hexagon vacancies is more energetically favorable than several other types of PHG configurations. In addition, an appropriate biaxial tensile strain can effectively tune the band gap and magnetism of the hydrogenated graphene. The band gap and magnetism of such configurations can be continuously increased when the magnitude of the biaxial tensile strain is increased. This fact that both the band gap and magnetism of partially hydrogenated graphene can be tuned by applying biaxial tensile strain provides a new pathway for the applications of graphene to electronics and photonics. HowevThe first mechanism can be achieved by substrate-graphene interaction ,6,35, apMotivated by the above results, we have carried out a systematic investigation to explore the stability and electronic and magnetic properties of partially hydrogenated graphene (PHG) by applying biaxial tensile strain using density functional theory. The calculated results indicate that the configuration with removing H-hexagon is the most energetically favorable in several types of HG configurations, while the appropriate biaxial tensile strain can effectively tune the band gap and magnetism of the partially hydrogenated graphene.3 code [All calculations in this study were performed using the spin-polarized first-principle method as implemented in the DMol3 code . The gen3 code , in comb3 code . All-ele−5 hartree, and that of maximum force in 0.002 hartree. The stability of hydrogenated graphene was determined from the formation energy Ef by the following:A hexagonal graphane supercell (7 × 7 graphane unit cell) was established with a lattice parameter of 2.54 Å. The modulus supercell vector in the z direction was set to 15 Å, which led to negligible interactions between the system and their mirror images. For geometric optimization, the Brillouin zone integration was performed with 10 × 10 × 1 k-point sampling, which brings out the convergence tolerance of energy in 1.0 × 10Etotal(n) is the cohesive energy of the system, n was the number of H atoms removed from a graphane sheet, and μH (μgraphane) is the chemical potential of the constituent H (graphane) at a given state. Here, we chose the binding energy per atom of H2 molecule as μH. And μgraphane was taken as the cohesive energy of a single graphane sheet.where According to the previous reports ,47, two Ef of the H vacancies on graphane can be calculated from the difference between the total energy of graphane and the sum of the total energy of dehydrogenated graphane and those of detached single H atoms. In Equation (1), positive Ef represents endothermic process of dehydrogenation. The calculations show that Ef of paired vacancies is always smaller than that of two unpaired vacancies  of the perfect graphane is about 4.41 eV. The Eg opening in graphene can be attributed to the changes of functionalized C atoms from sp2 to sp3 hybridization. However, after partially dehydrogenation of graphane, the Eg and the corresponding electronic structures can be changed. According to the unified geometric rule reported by previous works [Eg of partially dehydrogenated graphane decrease as the hexagon vacancies increase. Thus, the insulating graphane with a wide band gap becomes a semiconductor via appropriate dehydrogenation. Eg of PHG which are induced by the quantum confinement effect in PHG with increased vacancies. In other words, the trend of band gap narrowing can be attributed to the number of edge states in the PHG with the hexagon vacancies [Eg reduces to 0.52 eV and the magnetic moment (m) is about 0.98 μB  via selective dehydrogenation, whereas the band gap and magnetism moment of partially hydrogenated graphene can be enhanced by imposing a biaxial tensile strain.In summary, DFT calculations with biaxial tensile strain are carried out to investigate the effects of biaxial tensile strain on the electronic and magnetic properties of PHG structures. It is found that the configuration with removing H-hexagon is the most energetically favorable in the several types of HG configurations. In addition, the appropriate biaxial tensile strain can effectively increase the band gap and magnetism of PHG. Overall, tuning both band gap and magnetism of hydrogenated graphene by applying biaxial tensile strain provides a new perspective for wide applications of graphene in electronics and photonics.The authors declare that they have no competing interests.EHS carried out the design of the simulation and drafted the manuscript. GA, SHY, and QJ assisted in the simulation. SOC supervised the whole study. All authors read and approved the final manuscript.


Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disorder, and cytoplasmic inclusions containing transactive response (TAR) DNA binding protein (TDP-43) are present in ~90 % of cases. Here we report detailed pathology in human TDP-43 transgenic mice that recapitulate key features of TDP-43-linked ALS.WT) caused no clinical or pathological phenotype, while expression of Q331K mutant (TDP-43Q331K) resulted in a non-lethal age-dependent motor phenotype, accompanied by cytoplasmic TDP-43 aggregation, mild neuronal loss, with astroglial and microglial activation in the motor cortex and spinal cord at 24 months. However, co-expression of WT and Q331K mutant (TDP-43WTxQ331K) resulted in an extremely aggressive motor phenotype with tremor from 3 weeks and progressive hind-limb paralysis necessitating euthanasia by 8–10 weeks of age. Neuronal loss and reactive gliosis was observed in the spinal cord and layer V region of the cortex, with TDP-43, ubiquitin and p62 cytoplasmic inclusions and an increase in insoluble TDP-43. Nuclear clearance of TDP-43 was not observed in TDP-43Q331K mice but was seen in 65 % of aggregate containing spinal cord motor neurons in TDP-43WTxQ331K mice.Expression of human wild-type TDP-43  contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disorders characterised by the degeneration of motor neurons in the motor cortex and spinal cord, progressive paralysis and ultimately death due to respiratory failure.Transactivating response region (TAR) DNA binding protein TDP-43) has been identified as a major component of neuronal and glial inclusions found in ~95 % of sporadic ALS patients , 2. TDP- has beenPGRN [, valosin containing protein (VCP [UBQLN2 [FUS) [The identification of mutations in the glycine-rich C terminal region of TDP-43 that enhance protein cleavage, aggregation and neurotoxicity confirmed a pathologenic role for TDP-43 mis-accumulation in ALS and FTLD , 7, 8. TPGRN ), valosiein (VCP ) and Ubi [UBQLN2 ) and the [UBQLN2 , 13. TheN2 [FUS) , 15 suppN2 [FUS) , 17 are TDP-43 is a 414 amino acid protein, consisting of two RNA recognition motifs (RRM1 and RRM2) , 19, a nin vivo. Disruption or constitutive knockout of the Tardp gene in mice is embryonic lethal [A number of rodent models have been generated to assess the effect of mutation and TDP-43 expression levels c lethal , 27, 28,c lethal . In contc lethal , 31. Stuc lethal , 38, 39,c lethal  that mayc lethal , 41. In WT develop no overt physical or pathological phenotype, while those overexpressing mutant human TDP-43Q331K develop an age-dependent mild motor and pathological phenotype. Mice co-expressing TDP-43WT and TDP-43Q331K mutant TDP-43 develop marked tremor at 3 weeks and rapidly progressive paralysis requiring euthanasia by 8–10 weeks. Pathologically, TDP-43WTxQ331K mice demonstrate major neuronal loss, robust astroglial reactivity, increased cytoplasmic TDP-43 accumulation and detergent resistant TDP-43. We propose that mislocalisation and aggregation of the mutant protein seeds the rapid recruitment of wild-type TDP-43 that greatly accelerates the disease processes.Here we report the physical, pathological and biochemical characterisation of mice transgenic for human wild-type (WT) and ALS-linked Q331K mutant TDP-43. Mice overexpressing human TDP-43All experiments were performed under the terms of the UK Animals (Scientific Procedures) Act 1986, and were approved by the Kings College, London ethics review panel.WT (line 96) or TDP-43Q331K (line 31) has been previously described . Mice from the single hemizygous lines were identified using PCR with primers 5’- GGATGAGCTGCGGGAGTTCT and 3’- GTCAACCCCATACTACCCGT. Animals from the compound hemizygous lines were identified with direct sequencing using the primers 5’- ATGACTGAGGATGAGCTGCG and 3’- GGATGCTGATCCCCAACCAA.Construction of transgenic mice expressing TDP-43From 3 weeks of age, mice were regularly weighed and general health status was recorded. Animals showing signs of hind-limb paralysis were monitored daily, and disease end stage and death was defined as the time when animals could no longer obtain food or water, or had lost 25 % of their body weight, at which point they were euthanized.Motor strength and coordination were evaluated on the rotarod (Columbus Instruments) at multiple ages , using a 5 min accelerating protocol starting at 2 rpm, and rising to 30 rpm throughout the 5 min testing period. Mice were tested on multiple occasions, and all animals received an initial training session of 2 min at 2 rpm to acclimatise them to the equipment.WTxQ331K mice and their littermates were assessed statistically by one-way analysis of variance (ANOVA) followed by the Tukey test. All other data were assessed statistically by two-way ANOVA followed by the post-hoc Holm-Sidak test.Data from 5 week old TDP-43WTxQ331K mice, and 24 month old single hemizygous mice and their respective age-matched littermates were anaesthetised and transcardially perfused with PBS followed by 4 % paraformaldehyde (PFA) in phosphate buffer. Brain, spinal cord and gastrocnemius muscles were postfixed in 4 % PFA in 15 % sucrose for 5 h, cryoprotected in 30 % sucrose for 24 h and cut into 30 μm (brain and cord) or 40 μm (muscle) sections on a cryostat.Eight week old, end stage TDP-43For immunohistochemistry, the following antibodies were used: rabbit anti-TDP-43 , rabbit anti-mouse TDP-43 ), rabbit anti-ubiquitin , rat anti-myc , rabbit anti-GFAP , mouse anti-CD68 , rabbit anti-p62 (sequestosome 1) . For bright field imaging, sections were washed and incubated with the appropriate biotinylated secondary antibody , and then with an ABC kit (Vector). Sections were imaged using a Zeiss light microscope and Axiovision software. For fluorescence imaging, sections were incubated with rat anti-myc (1:200) and either rabbit anti-ubiquitin (1:200) or rabbit anti-p62 (1:5000), sections were washed and incubated with goat anti-rat Alexa Fluor 488 and goat anti-rabbit AlexaFluor 568 (Invitrogen), and imaged using a Leica confocal microscope and LAS-AF software.For motor neuron counts, perfused lumbar spinal cords from 3 to 4 animals per genotype were serially sectioned, and every 6th section (30 μm) was analysed. Sections were mounted, dried, incubated overnight in 1:1 ethanol/chloroform to de-fat the sections, stained for 10 min in warm 0.1 % cresyl violet, dehydrated and coverslipped. To compare the number of motor neurons, large neurons greater than 30 μm in diameter  in the anterior horn of the lumbar spinal cord were counted in 15 sections. Data were assessed statistically by one-way ANOVA, followed by the post-hoc Tukey test. For cortical neuron counts, perfused brains from 3 to 4 animals per genotype were serially sectioned, and every 12th section (30 μm) through the motor cortex was assessed. Sections were stained as for the lumbar spinal cord. To compare the number of neurons, cells greater than 5 μm in diameter  were counted in four images per section. Data were assessed statistically by one-way ANOVA, followed by the post-hoc Tukey test.-bungarotoxin  and rabbit anti-Synaptophysin 1 . Sections were washed and incubated in donkey anti-rabbit IgG DyLight 488 secondary antibody  and imaged on a Leica confocal microscope. To assess NMJ area, the total area stained by bungarotoxin was assessed in ImageJ, in 30–40 NMJs per animal. To calculate the number of intact NMJs, 70–100 were assessed per animal, and were considered intact if they demonstrated full colocalisation of bungarotoxin and synaptophysin staining. Data were assessed statistically by one-way ANOVA, followed by the post-hoc Tukey test.For muscle histology, gastrocnemius muscles  were dissected fresh, immediately frozen in isopentane cooled in dry ice, and cryostat sections were cut onto slides and stained with haematoxylin and eosin. For the detection of neuromuscular junctions (NMJs), perfused gastrocnemius muscle  was incubated in Alexa Fluor 555 αWTxQ331K mice (n = 3–4 per genotype), and 24 month old single hemizygous mice (n = 3–6 per genotype) and their respective age-matched littermates were anaesthetised and transcardially perfused with PBS followed by a mixture of 2 % PFA and 2.5 % glutaraldehyde in 0.1 M cacodylate buffer. The L5 ventral roots were removed and post fixed in the same fixative at 4 °C overnight. The roots were then further fixed in 1 % osmium tetroxide in0.1 M cacodylate buffer for 4 h. Fixed tissue was then dehydrated in ethanol and embedded in Epox 812/Araldite 502 (TAAB). semi-thin sections (0.65 μm) were cut using an ultramicrotome (Reichart-Jung Ultracut-E) and collected onto glass slides. The sections were stained with 1 % toluidine blue for 15 s before mounting for viewing and examined under a light microscope. Axon measurements were made using the integrated morphometry package on Metamorph 7.7  and α-motor axons, defined as those with a diameter greater than 3.5 μm, were counted. Data was analysed statistically by way of ANOVA followed by the post-hoc Tukey test.7 week old, end stage TDP-43WTxQ331K and 3–4 age-matched hTDPWT, hTDPQ331K and non-transgenic animals, were lysed in low salt buffer  with protease inhibitors . Total TDP-43 levels were also assessed in four 24 month old hTDPWT, hTDPQ331K and non-transgenic animals.To assess expression levels of full length TDP-43, the 25 and 35 kDa TDP-43 fragments, and phospho-TDP-43, whole brains of 3–4 end stage hTDP2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2 and protease inhibitors (10x vol/weight). After 10 min on ice, 2.5 M sucrose (0.5x vol/weight) was added. Tissue was homogenized and centrifuged at 6300 g for 10 min. The supernatant was collected as the cytoplasmic fraction. The pellet was washed four times in TSE buffer  and protease inhibitors 10x vol/weight), homogenized and centrifuged at 4000 x g for 5 min. Finally the pellet was resuspended in RIPA buffer with 2 % SDS (5x vol/weight) as the nuclear fraction.For cytoplasmic and nuclear fractionation, four brain samples for each age and genotype were prepared as described earlier . BrieflyFor insolubility assessment, four brain samples for each age and genotype underwent sequential extraction in buffers of increasing stringency, based on a modified protocol previously described . BrieflyProtein samples were then separated by SDS/PAGE using 10 % polyacrylamide gels, and transferred to nitrocellulose membranes. Total TDP-43 and the 25 and 35 kDa fragments were recognised by a rabbit polyclonal antibody to TDP-43 , and exogenous myc tagged human TDP-43 was recognised by a mouse monoclonal antibody to the myc tag . Phospho-TDP-43 was recognised using a rabbit anti-phospho-Ser409/410-TDP-43 , and in these analyses, total TDP was recognised using a rat monoclonal antibody to TDP-43  . Fluorescent secondary antibodies conjugated to Dylight 680 nm or 800 nm (Thermo Scientific) were used to detect protein levels, and results were visualised using the Odyssey Imager (Licor). Data were normalised to GAPDH , actin  or Lamin B1 . Quantitation of immunoblots was done using Image J software, and data were analysed statistically by way of ANOVA followed by the post-hoc Tukey test.WT) or Q331K mutant (TDP-43Q331K) TDP-43 were interbred to generate TDP-43WTxQ331K animals expressing both the WT and mutant protein. Initial breeding approximated a Mendelian distribution of genotypes on weaning. Because 3 week old TDP-43WTxQ331K animals struggled to compete for food against their single transgenic littermates we added wet mash food to their home cage prior to weaning which ensured survival.Mice expressing human WT  positive inclusions in the spinal cord of 8 week old hTDPQ331K animals , TDP-43Q331K (p < 0.001) and non-transgenic (p < 0.001) littermates  of α-motor neurons in the anterior horn  loss of α-motor neurons in the anterior horn of the lumbar spinal cord in TDP-43WTxQ331K mice showed group atrophy of muscle fibres  compared to their single and non-transgenic littermates . In contrast, young TDP-43WT single transgenic animals do not differ from their non-transgenic littermates  reveals a significant increase in levels in end-stage TDP-43es Figs. . This ises Figs.  providinWTxQ331K and TDP-43Q331K animals both displayed a significant increase in cytoplasmic TDP-43 levels compared to TDP-43WT mice indicating that the presence of the TDP-43Q331K mutation enhances this mislocalisation. In contrast, there was no significant increase in nuclear TDP levels between the various transgenic animals  and aged TDP-43Q331K mice (only cytoplasmic aggregation) one might conclude that while the loss of nuclear TDP-43 may accelerate disease, it is not essential, whereas cytoplasmic aggregation of TDP-43 into insoluble inclusions in cortical and spinal neurons, as seen in aged TDP-43Q331K and TDP-43WTxQ331K mice, is sufficient to cause neurodegeneration, as has previously been reported in another line of mice expressing Q331K (2).Evidence of “nuclear clearing” of TDP-43 from neurons containing cytoplasmic inclusions, similar to that seen in two-thirds of surviving aggregate containing cells in the TDP-43patients  and in opatients , 38. Corpatients  homozygopatients , 28. It patients . Similarpatients . By compWT were 1.4 above endogenous in non-transgenic littermates, while TDP-43Q331K mice were 2.2 fold and TDP-43WTxQ331K 3.3 fold. Caution must be taken when interpreting these data however, as the methodology used to assess protein levels was not consistent between each study, hence a general dose effect, regardless of mutation status, cannot be ruled out. In the current study, we show that the addition of a ‘dose’ of human WT TDP-43 appears to trigger massive TDP-43 aggregation in TDP-43Q331K mice and dramatically increases the toxicity such that 50 % of the mice are dead by 8 weeks. Assessment of the single transgenic animals used in this study have suggested that Q331K mutant TDP-43 has a greater tendency to aggregate in the cytoplasm than WT on its own, thus we hypothesise that Q331K aggregates provide a potent seed for the recruitment of human WT and endogenous mouse TDP-43 that results in nuclear clearing and rapid neurodegeneration. This hypothesis is supported by our finding that despite a general reduction in levels, endogenous mouse TDP-43 appears to be recruited to aggregates in the TDP-43WTxQ331K mice. Although there is no overt evidence of endogenous TDP-43 aggregation in the TDP-43Q331K single transgenic animals, the pathology seen in these mice is significantly reduced compared to the TDP-43WTxQ331K animals, and this, combined with the reduction seen in endogenous TDP-43 is likely to make detection of any potential recruitment problematic.The simplest explanation for differences in the severity of phenotype and pathology is a dose-threshold effect. A comparison of protein levels, phenotype and survival in mice transgenic for human TDP-43 under PrP promoter Fig.  shows thQ331K and TDP-43WTxQ331K mice while nuclear clearance was only seen in spinal cord motor neurons in TDP-43WTxQ331K mice. Thus while nuclear clearance of TDP-43 may accelerate disease it does not appear to be essential to cause neurodegeneration and loss of nuclear TDP-43 function may not be a primary or disease-critical event. A more detailed exploration of disease mechanisms in slow and rapid disease models of TDP-43 proteinopathy will help elucidate novel drug targets and provide a more informative platform for preclinical trials.Our mice recapitulate many aspects of the clinical phenotype and pathological features of TDP-43-linked ALS with slow and rapidly progressive motor phenotypes. Although the occurrence of ALS-causing TDP-43 mutations is rare in the patient population, the close correlation between the pathology observed in these mice and that seen in the majority of TDP-ALS patients suggests that these animals provide a viable model for studying the impact and progression of TDP-43 pathology in the central nervous system, and help to elucidate mechanisms that may underlie disease progression. The aggregation of TDP-43 into insoluble cytoplasmic inclusions in cortical and spinal neurons was a feature of both aged TDP-43


Kip1. Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension.It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27 Pulmomodeling . The dermodeling ,6. ThereAdenosine monophosphate-activated protein kinase (AMPK) is a key metabolic and redox sensor that has emerged as a critical regulator of cell proliferation and apoptosis . AMPK exRecent studies have employed AMPK activators such as 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin to demonstrate that activation of AMPK suppresses PASMCs proliferation ,18. Studp < 0.01 versus control). Non-targeting siRNA transfection did not reduce AMPKα1 and AMPKα2 protein expression. p < 0.05), while transfection of PASMCs with AMPKα1 siRNA or non-targeting siRNA did not affect cells proliferation. These results suggest that knockdown of AMPKα2 induces PASMCs proliferation.To specifically examine the effect of AMPK on PASMCs proliferation, siRNA-mediated knockdown of either AMPKα1 or AMPKα2 was performed. As depicted in p < 0.01), suggesting that knockdown of AMPKα2 causes unrestrained mTOR activation in PASMCs.Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that positively regulates cell growth, proliferation, and survival . SeveralKip1 is one of the cyclin-dependent kinases (CDKs) inhibitors that plays a critical role in regulating cell cycle progression in mammalian cells [Kip1 degradation underlies AMPK suppression of several types of cells proliferation [Kip1 are also associate with the effect of AMPKα2 knockdown on PASMCs proliferation. As shown in p < 0.01 versus control). Non-targeting siRNA transfection did not affect Skp2 protein level. Kip1 was reduced in AMPKα2 knockdown PASMCs, which was 0.50-fold decrease compared with control (p < 0.01), while non-targeting siRNA transfection did not change p27Kip1 expression. These findings suggest that knockdown of AMPKα2 upregulates Skp2 and downregulates p27Kip1 in PASMCs.p27feration ,28,29. HKip1 in malignant cells [Kip1 modulation in PASMCs, silencing of mTOR was applied. p < 0.01), while non-targeting siRNA did not affect mTOR protein expression. p < 0.01 versus control siRNA transfection) and reduced p27Kip1 protein level (p < 0.05 versus control siRNA transfection), while pre-silencing of mTOR decreased Skp2 expression from 1.87-fold to 0.89-fold over control siRNA and increased p27Kip1 level from 0.50-fold to 1.19-fold over control siRNA, respectively (p < 0.01 versus control siRNA + AMPKα2 siRNA transfection). We also observed that silencing of mTOR alone reduced Skp2 and increased p27Kip1 protein level (p < 0.05 versus control siRNA transfection), suggesting that there is a basal function of mTOR in PASMCs. These results suggest that activation of mTOR is one of major protein responsible for AMPKα2 knockdown-induced Skp2 upregulation and p27Kip1 downregulation.Studies have shown that mTOR plays an important role in regulating Skp2 and p27nt cells ,31,32. Tp < 0.05 versus control siRNA transfection), while pre-silencing of mTOR reduced PASMCs proliferation to 0.90-fold over control siRNA (p < 0.01 versus control siRNA + AMPKα2 siRNA transfection). Silencing of mTOR alone reduced basal PASMCs proliferation (p < 0.05 versus control siRNA transfection). Together, these observations indicate that mTOR mediates AMPKα2 knockdown-induced PASMC proliferation by regulating Skp2 and p27Kip1.To further confirm whether activation of mTOR mediates AMPKα2 knockdown-induced PASMCs proliferation, BrdU incorporation rate was determined in PASMCs transfected with non-targeting siRNA or mTOR siRNA followed with or without AMPKα2 knockdown. Kip1 ubiquitylation and degradation in various cell types [Kip1 reduction, depletion of Skp2 was applied. p < 0.01), while non-targeting siRNA did not affect Skp2 protein level. Kip1 protein level (p < 0.05 versus control siRNA transfection), while prior depletion of Skp2 abrogated the reduction of p27Kip1, which increased from 0.50-fold to 1.30-fold over control siRNA (p < 0.01 versus control siRNA + AMPKα2 siRNA transfection). Loss of basal Skp2 alone also increased p27Kip1 protein level (p < 0.05 versus control siRNA transfection). These results suggest that Skp2 specifically mediates AMPKα2 knockdown-induced p27Kip1 reduction.Skp2 is the main rate-limiting regulator of p27ll types ,29. To ep < 0.05 versus control siRNA transfection), while prior depletion of Skp2 inhibited PASMCs proliferation induced by AMPKα2 knockdown, which declined from 1.45-fold to 0.85-fold over control siRNA (p < 0.01 versus control siRNA + AMPKα2 siRNA transfection). Lacking Skp2 protein alone also reduced basal PASMCs proliferation (p < 0.05 versus control siRNA transfection). Collectively, these results suggest that Skp2-induced p27Kip1 reduction mediates AMPKα2 knockdown-stimulated PASMCs proliferation.To further verify the involvement of Skp2 in AMPKα2 knockdown-induced PASMCs proliferation, PASMCs were transfected with non-targeting siRNA or Skp2 siRNA followed with or without AMPKα2 knockdown. Kip1. The study suggests that activation of AMPKα2 might have potential value in the management of PAH by modulation of pulmonary vascular remodeling.In the present study, we have provided direct evidence that knockdown of AMPKα2 by siRNA stimulates primary cultured PASMCs proliferation; this effect is coupled to the particular activation of mTOR and subsequent upregulation of Skp2 and reduction of p27AMPK is a crucial energy sensor that regulates cellular and whole-body energy balance . AlthougKip1 [Kip1 levels and poor disease-free and overall survival [Kip1 degradation and suppresses proliferation of many cell types [Kip1 as a CDK inhibitor binds to and prevents the activation of cyclin E-CDK2 and cyclin D-CDK4 complexes, therefore blocks the G1-S transition of cell cycle and suppresses cell proliferation [Kip1 and proliferation of PASMCs. Silencing of mTOR attenuated the changes of Skp2 and p27Kip1 caused by AMPKα2 siRNA transfection. Our results further indicated that inhibition of Skp2 by siRNA abolished AMPKα2 knockdown-induced p27Kip1 reduction and therefore suppressed PASMCs proliferation. Taken together, these results suggest that Skp2/p27Kip1 pathway lays to the downstream of mTOR in mediating the effect of AMPKα2 on PASMCs proliferation. Encouragingly, our preliminary study in rat PAH model found that similar mechanisms were also existedin vivo (date not shown here).Mammalian cell proliferation is delicately regulated by cell-cycle regulatory proteins including CDKs and CDK inhibitors . Skp2, aKip1 . UpregulKip1 ,43. Moresurvival . Inhibitll types ,46. p27Kferation ,48. Our in vitro synthetic AMPK agonist which has been commonly used in clinic to treat type 2 diabetes with wide clinical experience and safety record [PAH is a common clinical syndrome with high mortality and requires a longer-term management . The prey record . We havey record , togetheet al. [2 overdose. Main pulmonary arteries were rapidly dissociated and cleaned under sterile conditions. Then, the adventitia and endothelium of pulmonary arteries were carefully scraped off and the remaining smooth muscle was minced (about 1 mm2) and placed into a culture flask. PASMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/High glucose  supplemented with 10% fetal bovine serum  and 1% antibiotics (complete DMEM) in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were fed every 2–3 days and subcultured by trypsinization using 0.25% trypsin  till reaching 80% confluence. All PASMCs used were from passages 4 and 6. The purity and identity of PASMC were determined by immunostaining with an antibody against α-smooth muscle actin  as described previously [Rat PASMCs were obtained from main pulmonary arteries from 4- to 5-week-old Sprague-Dawley rats using the method described by Wu et al. . All anieviously .2 humidified incubator. The silencing efficiency was determined by immunoblotting.Transfection of PASMCs with siRNA  was performed using Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacture’s protocols. PASMCs were seeded into 6-well plates and cultured till reaching 30%–40% confluence. Cells were then transfected with 100 μM siRNA plus 5 μL of Lipofectamine for 6 h in serum-free DMEM and sequentially cultured in complete DMEM for 48 h in a 37 °C, 5% CO3 cells were plated on 96-well plates and serum deprived (1% FBS in DMEM) overnight. After different treatments, cells were labeled by addition of BrdU for 2 h at 37 °C. After removing the labeling medium, cells were fixed and denatured by FixDenat solution and then incubated with anti-BrdU monoclonal antibody conjugated to peroxidase for 90 min at room temperature. Finally, substrate solution was added in each well and the absorbance at 370 nm was measured using a microplate reader . Absorbance was normalized to initial reading to verify equal cell numbers at the start of assay.Cells proliferation was determine by BrdU incorporation assay according to the instructions of BrdU ELISA Kit . Briefly, aliquots of 5 × 10Kip1 , AMPKα1 and AMPKα2  as well as glyceraldehyde-3-phosphate dehydrogenase . After incubating with primary antibodies, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies . Immunoreactive bands were visualized by SuperSignal West Pico Chemiluminescent Substrate  and quantified using Quality One software (Bio-Rad). Sample loadings were normalized by immunoblotting with GAPDH or related total protein.The cultured PASMCs were washed twice with ice-cold phosphate buffered saline (PBS) and lysed on ice in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride and proteinase inhibitors. The lysates were then centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant was collected as sample protein. Equal amount of protein from each sample was loaded and resolved on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad) using semidry transfer. The membranes were then blocked and probed with polyclonal or monoclonal primary antibodies against phosphor-mTOR, total-mTOR, Skp2, p27p < 0.05.Statistical analysis was carried out using the SPSS 13.0 software . All data were expressed as mean ± standard deviation. Group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. Statistical significant was determined at the level of