Optical polarizing devices exploiting graphene embedded in waveguides have been demonstrated in the literature recently and both the TE- and TM-pass behaviors were reported. The determination of the passing polarization is usually attributed to graphene’s Fermi level , with, however, no direct confirmation of this assumption provided. Here we show, through numerical simulation, that rather than graphene’s Fermi level, the passing polarization is determined by waveguide parameters, such as the superstrate refractive index and the waveguide’s height. The results provide a consistent explanation for experimental results reported in the literature. In addition, we show that with an accurate graphene modeling, a waveguide cannot be switched between TE pass and TM pass via Fermi level tuning. Therefore, the usually overlooked contribution of the waveguide design is shown to be essential for the development of optimized TE- or TM-pass polarizers, which we show to be due to the control it provides on the fraction of the electric field that is tangential to graphene. For this reason, optical devices that incorporate graphene usually enhance the light-graphene interaction by embedding it longitudinally, close to the core of waveguides7891113et al. demonstrated a TE-pass polarizer in the optical fiber geometry, yielding up to 27 dB extinction ratio at 1550 nm wavelength, that used a ~3-mm length of grapheneet al. using a 5-mm graphene lengthet al., a TM-pass characteristic was observed.The polarizing effect in graphene-waveguide structures occurs because optical absorption in graphene takes place only for the fraction of the electric field that is parallel to graphene’s planeet al. measured the polarization dependent loss in integrated silicon waveguides with graphene and reported a TM-pass behavior with an extinction ratio of 40 dB/mm around 1550 nmPolarizing effects were also reported in integrated waveguides. Kou The operation of each specific reported device as TE pass or TM pass is usually explained in terms of graphene’s Fermi level. In some cases, the excitation of waves similar to surface plasmon polaritons (SPP) is said to occur at doping levels for which Pauli blocking takes place and intraband transitions dominate over the interband transitions111213111213This description of the guided light-graphene interaction has led to proposals of switchable polarizers213Here we show through numerical simulations that the waveguide design, rather than graphene’s doping level or coupling to SPP-like waves, determines the passing polarization in graphene-waveguide optical polarizers and that the refractive index of the superstrate (i.e. the layer beyond graphene) and the waveguide dimensions are crucial for the development of optimized optical polarizers. In addition, we show that the TE-pass to TM-pass transition cannot occur with the tuning of graphene’s Fermi level, but that it indeed takes place via tuning of the superstrate’s index or of the waveguide height.n1, of 2 (silicon nitride), a width , W, of 1.6 μm, and a height , H, of 0.8 μm. The waveguide is surrounded by a medium with refractive index, n2, equal to 1 and lies on the top of a substrate with refractive index, nsub, of 1.45 (silica). A single layer of graphene is placed on the top of the waveguide core and covers its whole width. Above graphene, a superstrate with a refractive index ntop and a 300-nm thickness is modeled. Sz, along the propagation direction (z axis), in color scale, and the electric field distribution, black arrows, for the TM-mode at a 1550-nm wavelength and for ntop = 1.45. The simulation computes the complex propagation constant of the guided modes; the absorption coefficient, α, was then calculated from the imaginary part of this constant.As several graphene-waveguide polarizers with differing structures and materials have been reported, it is instructive to, first, analyze a specific polarizer, in which the influence of different parameters can be analyzed. Therefore, simulations were initially performed by modeling a silicon nitride waveguide, a classic silicon photonics material that is of great interest for the development of graphene-based optical and electro-optical devicestg = 0.34 nmω is light’s angular frequency; Graphene was initially simulated, as commonly found in the literature, as a 3D isotropic material with a thickness 15 rad/s (corresponding to the 1550-nm wavelength), and EF, with the latter being varied. Graphene’s conductivity was calculated using the Kubo formalism, which takes into account graphene’s linear electronic dispersion, including both intra- and interband transitions22ħω = 800 meV and, as expected, in EF > ħω/2 due to Pauli blocking. Below this Fermi level value, interband transitions dominate. For EF > 505 meV the conductivity becomes predominantly imaginary and the intraband transition contribution increases. The ~505 meV Fermi energy corresponds to a minimum in the absolute value of graphene’s permittivity (εg = −0.26 + 1.12i), which is known as the epsilon near zero point, εNZ24xz plane) and j = a or b, being the electric displacement vector in medium j, near the boundary; E represents the electric field vector. This condition shows that the discontinuity in the permittivity, which is greater near The photon energy at 1550 nm is  εg = −0. + 1.12i,εNZ point that is shown in ntop = 1.75 and 1550-nm wavelength. εNZ point would make possible the fabrication of polarization switchable devices controlled by a gate voltage. Polarizers69εNZ point in graphene but the lack of experimental results raises questions about their feasibilityεNZ effect occurs solely if graphene is considered as a 3D isotropic materialεNZ peak in Furthermore, still within the isotropic graphene model, the local field enhancement would reportedly lead to an increase in the absorption for the TM mode, resulting in a TE-pass behavior around the y, out of graphene’s plane, direction is taken to be real and equal to 2.5, regardless of the Fermi levelH is the magnetic field vector. This modeling strategy is computationally less demanding and yields negligible differences relative to the anisotropic, 0.34-nm thick, graphene model. EF > ħω/2. Fermi-level induced polarization switching was not observed in any case when ntop was swept from 1 up to 1.8 and the waveguide height varied from 0.4 up to 1.2 μm; the same behavior was observed in simulations with the different waveguide geometries described in Methods. Results for a selection of waveguide parameters and for the optical fiber geometry are shown in the Hereforth, we, thus, proceed to model graphene as an anisotropic material whose permittivity in the ntop, for different waveguide heights, H. The Fermi level was fixed at 100 meV and the quantitative results are not significantly affected by its exact value up to ~300 meV, as can be noticed in ntop = 1). The TM-pass behavior is, on the other hand, achieved with higher superstrate refractive indices. However, when the waveguide height is lower than a given threshold, the TM-pass behavior is observed regardless of ntop due to the strong confinement of light in the y direction, which increases the TE-mode interaction with graphene. The inset in ntop = 1; an optimized TE-pass polarizer is obtained for H ~ 0.55 μm. The change in the waveguide width was also evaluated but a much smaller influence on the polarization dependent loss was noticed.Although the TM-pass or TE-pass behavior of the polarizer is not dependent on the Fermi level, it is determined by the exact waveguide design. E||, at the graphene layer for each mode and specific waveguide design. Only this field component has an effective interaction with graphene. Therefore, the mode that presents the highest fraction of its field parallel to graphene, at the graphene position, experiences the highest absorption. For comparing different modes, we define a normalized tangential field intensity, integrated along graphene’s width at the waveguide cross section:These results can be explained through the evaluation of the tangential electric field, E|| is dominantly composed of Ex, while for the TM mode E|| is mostly composed of Ez. U|| for the TE and TM modes as a function of ntop in a waveguide with H = 0.55 μm. The result shown is related to the blue curve of ntop = 1.17. This superstrate index is marked in U|| for the TE mode supersedes U|| for the TM mode. Therefore, it is shown that the polarization dependent loss is, indeed, determined by the fraction of the field that is parallel to graphene and that the waveguide design directly determines this fraction.with E|||2, across the waveguide is also shown. For the TE mode and for low superstrate indices, most of the parallel electric field is concentrated around the center of the waveguide; the increase in ntop leads to a weaker mode confinement and, therefore, to a shift of the mode distribution towards the superstrate. This trend increases the interaction of light with graphene and, consequently, increases absorption. On the other hand, for the TM mode, a low ntop means a high core-cladding index contrast, which tends to increase the fraction of the electric field that is parallel to the propagation direction and concentrated at the edges of the waveguide; as ntopincreases, the weaker mode confinement makes the field distributions tend to those of transverse electromagnetic (TEM) modes, thus, reducing the electric field along z. Consequently, a reduction in the absorption of the TM mode is observed. The attenuation for the TE (TM) mode is 29.79 dB/mm (36.47 dB/mm) for ntop = 1 and 58.93 dB/mm (10.50 dB/mm) for ntop = 1.8.In the extreme points of each curve of et al., showing a TM-pass behavior despite the absence of a superstrate can be explained by the small height (200 nm) of the silicon waveguide used in their experimentset al.  that are those of a single layer multiplied by the number of graphene layers. This is in agreement with the results reported by Lee Besides optical scattering caused by wrinkles, cracks and contamination on graphene, the polarizing effect can, in principle, be affected by scattering due to the waveguide roughness111314−2 have been reported to be required for saturation to be observed at 1550 nmGraphene-based optical polarizers can be affected by graphene’s nonlinear optical response at high intensities. At this regime saturable absorption3Finally, our analysis can be extended to different wavelengths; simulations performed at 532 nm and 1064 nm wavelengths resulted in the same dependence of the polarizing effect on the waveguide design, provided that the waveguides is properly scaled. This scale dependence can be exploited to create wavelength-dependent polarizers.In conclusion, we showed that the waveguide design, rather than graphene’s Fermi level, as widely believed, determines the polarization properties of graphene-based waveguide polarizers because it defines the fraction of the modal electric field that is parallel and superposed with graphene. More specifically, we showed that graphene-waveguide polarizers can work either as TE or TM pass devices depending on the waveguide dimensions and the superstrate refractive index. The development of optimized polarizers must, therefore, take into account a careful design of the waveguide, which is usually either overlooked or taken into account solely for increasing the light-graphene superposition. We stress that simply increasing this superposition does not suffice for maximizing the polarization extinction ratio, as the electric field perpendicular to graphene is not absorbed. The results presented here establish the foundations for the development of optimized graphene-waveguide polarizers and explain their operation.The modal analyses were performed in the COMSOL Multiphysics. The number of degrees of freedom solved was 1,047,000 using adaptive triangular meshes with maximum element size of 80 nm and minimum element size of 0.12 nm in the region around the graphene.et al.n1 = 3; n2 = ntop = 1; nsub = 1.45; W = 400 nm; H = 200 nm; and light wavelength 1550 nm. The convex polymeric waveguide reported by Kim and Choin1 = 1.39; n2 = ntop = 1 or 1.37, for the cases without or with the cladding respectively; nsub = 1.37; width at half the maximum height was assumed W = 5.75 μm with 68° wedge angle; height H = 4.8 μm and 1310 nm wavelength. The D-shaped optical fiber based polarizer reported by Bao et al.et al.The silicon waveguide reported by Kou How to cite this article: de Oliveira, R. E. P. and de Matos, C. J. S. Graphene Based Waveguide Polarizers: In-Depth Physical Analysis and Relevant Parameters. Sci. Rep.5, 16949; doi: 10.1038/srep16949 (2015).


In the current study, we describe investigations of a novel anti-PD-L1 mAb of the IgG1 isotype (MSB0010718). This mAb is currently in Phase I clinical trials for patients with metastatic or locally advanced solid tumors at the NCI, and is the first such mAb with the capacity to induce ADCC of PD-L1 positive tumor cells. We sought to investigate MSB0010718's ability to induce ADCC and to determine factors affecting tumor cell sensitivity to this mechanism.Expression of the immune checkpoint protein PD-L1 constitutes a major mechanism of tumor immune evasion. Multiple clinical trials in solid tumors have demonstrated that inhibition of tumor PD-L1 or immune effector PD-1 via monoclonal antibodies (mAbs) can produce dramatic clinical responses in many cancer patients. The main function of these mAbs is to inhibit signaling induced by ligation of PD-L1 on tumor cells with PD-1 on tumor infiltrating immune effectors. Antibody-dependent cellular cytotoxicity (ADCC) represents an additional mechanism of action for mAbs of the Iin vitro ADCC assays, we demonstrated that many cancer cell lines are sensitive to ADCC induced by MSB0010718. Sensitivity to ADCC positively correlated with PD-L1 MFI as determined by flow cytometry. Treatment of tumor cell lines with IFN-γ increased PD-L1 expression with concurrent increase in ADCC sensitivity in some cases. Isolation of NK cells for use as effectors significantly increased ADCC activity as compared to whole PBMCs, demonstrating NK cells as the major effectors of ADCC. ADCC activity could be significantly increased via activation of NK effectors with IL-12, suggesting potential synergy with IL-12 based therapeutics. Furthermore, a MUC1+ tumor cell line that was insensitive to CD8+ MUC1-specific CTL was demonstrated to be sensitive to the ADCC mechanism as a result of high surface PD-L1 expression.Using whole PBMCs as effectors in + CTL response. As cell surface PD-L1 density was an important predictor of ADCC sensitivity, this mechanism is expected to be most active against tumors with high density PD-L1 expression.As it is clear that not all patients respond to current PD-1 or PD-L1 based therapies, additional mechanisms of action may be needed to increase therapeutic efficacy. Our data demonstrate significant ADCC activity induced via MSB0010718. This mechanism of action represents a potential advantage for MSB0010718 over other anti-PD-L1 mAbs, as it can induce target cell lysis in the absence of an effective CD8


The emerging paradigm of ‘abundant-data' computing requires real-time analytics on enormous quantities of data collected by a mushrooming network of sensors. Todays computing technology, however, cannot scale to satisfy such big data applications with the required throughput and energy efficiency. The next technology frontier will be monolithically integrated chips with three-dimensionally interleaved memory and logic for unprecedented data bandwidth with reduced energy consumption. In this work, we exploit the atomically thin nature of the graphene edge to assemble a resistive memory (∼3 Å thick) stacked in a vertical three-dimensional structure. We report some of the lowest power and energy consumption among the emerging non-volatile memories due to an extremely thin electrode with unique properties, low programming voltages, and low current. Circuit analysis of the three-dimensional architecture using experimentally measured device properties show higher storage potential for graphene devices compared that of metal based devices. Increasing memory performance and density will require new breakthroughs in atomic-scale technology and three-dimensional device architectures. Here, the authors demonstrate a memory just 3 Å thick that can be stacked by exploiting the atomically thin edge of monolayer graphene. The rapid adoption of non-volatile memory technology such as Flash2Resistive random access memories (RRAM) based on metal oxide have shown considerable promise as a possible successor to Flash because of better endurance, retention, speed, lower programming voltages and a higher device density343Graphene, an atomically thin crystal lattice of carbon atoms, is known for its unique electronic properties78910In this work, the atomically thin (∼3 Å thick) edge of monolayer graphene was actively used as a SET electrode to form an atomically thin memory structure. We investigate the low energy consumption and the stacking potential of the device in a 3D architecture that is amenable to large scale manufacturing.x layer and the graphene electrode are depicted in yellow, green and black, respectively. The fabrication process is explained in x) is highlighted in red. We also fabricated RRAMs based on platinum electrodes (Pt-RRAM) as control devices. The Pt-RRAM  were stacked to build a 3D vertical cross-point architecture as illustrated in  Pt-RRAM , which w2 thickness of 6 nm, half-pitch of 22 nm and 1° of etch angle improvement—a maximum of 200 stacks will be possible for GS-RRAM as compared with the 60 stacks possible with conventional bulk-metal-based 3D RRAM (Such 3D architectures are part of an ongoing drive in the research community to adopt a bit-cost-effective architecture12141516 3D RRAM .x) similar to conventional metal oxide-resistive memories. The number and the size of the conducting filament (CF) paths determine the two resistance states of the RRAM: the high resistance state (HRS) and the low resistance state (LRS). In the Pt-RRAM structure  intensity is observed for HRS→LRS→HRS transition. During the SET process , oxygen ions are inserted into the graphene, doping the film. Consequently, a decrease in the 2D peak intensity is observed. During the RESET process , oxygen ions are pushed back into HfOx from the graphene film. This results in an increase in 2D peak intensity. The Raman peak intensity of silicon (520 cm−1) and the baseline are plotted in parallel to ensure that the references have not changed during measurement (see Methods section).In a GS-RRAM, however, a negative voltage is applied to the TiN electrode during the SET process, and the oxygen ions move towards the graphene . Unlike 20The spatially resolved Raman spectroscopy results for the change in 2D peak intensity during the HRS→LRS→HRS transition are shown in 202020The GS-RRAM offers significantly lower power consumption compared with Pt-RRAM due to three factors: low SET compliance current , low RESRseries from the graphene sheet compared with the Pt sheet, the difference of the TiN/oxide  and graphene/oxide interface, and the different sizes . From a transmission line measurement (x) dominated the total resistance change during the SET/RESET process.The low SET compliance current in GS-RRAM is possible due to a more resistive HRS and a larger memory window  comparednt sizes  of filamsurement , we founIt is known that in an RRAM structure, the resistance of HRS increases as the inverse of the cell area, roughly following Ohm's lawxNx1− film in the TiN layer, which works as a barrier against diffusion and carrier transportRint, TiN), and the total resistance at LRS becomes comparable to that of GS-RRAM.The LRS of these devices are related not only to the size of the filaments but also to the different effects of oxygen in the TiN and the graphene electrodes. The LRS of Pt-RRAM  is compaxNx1− diffusion barrier that is typically formed in TiN electrodes. The activation energy of diffusion for oxygen in graphene 25The low SET/RESET voltage is related to the thickness of the electrode and the oxygen migration mechanism. After the forming process , the tipThe result of the pulse-mode endurance test in V write scheme and the column parallel read schemeVaccess to the Vdd ratio) and the readout margin  as a function of total number of bits for the GS-RRAM and Pt-RRAM arrays are simulated under worst-case conditions assuming 200-layer stacks . For the imaging, we used an Tecnai TF-20 Field Emission Gun/TEM  operated at 200 kV in bright-field TEM mode or high-resolution TEM mode.The TEM-ready samples were prepared using the The images were taken with constant laser intensity right after the SET and the RESET programming. External perturbation was minimized with an oxide capping layer. For the purpose of Raman measurement, single-stack GS-RRAM (without the second stack) was fabricated and measured to eliminate any effect from the second graphene layer. A WiTec 500 AFM/micro-Raman scanning microscope was used for the 2D Raman raster scanning of graphene. A 532-nm wavelength was used for all measurements. A 30 × 60 μm area was scanned with an integration time of at least 4 s with a 1-μm resolution. Each measurement was conducted in <3 h.x, increasing the resistance  of the RRAM. The kinetics of this process can be described by the Arrhenius law.The temperature-accelerated LRS retention-time measurement can probe the thermal activation of oxygen ion migration, as shown in τreset is the characteristic time for RESET transition, τ0 is a constant, kB is the Boltzmann constant, Ea is the activation energy barrier and T is the absolute temperature. The linear fitting result of retention time in logarithmic scale versus reciprocal temperature provides a good estimation of the activation energy  with a temperature controller (Temptronic SA166550). All measurements were done inside the test chamber with the nitrogen gas flowing. The set-up was on an anti-vibration table with pneumatic vibration mount. The automated resistance measurement was conducted every 15 s to 3 min with 0.1 V bias using a semiconductor parameter analyser (Agilent 4156C).The pulse-mode endurance test was conducted with an Agilent Parameter Analyzer 4155C and an Agilent Pulse generator 81110A connected to a Keithley Switch Matrix 707B. Pulse width was 500 ns with 3 s time delay and ±0.2 V was the read voltage.Vaccess × Vdd−1) and the readout margin (ΔIread). The definition of Vaccess is the voltage across the accessed cell in the resistance network. ΔIread is defined as the difference in the current flowing through the read resistor (100 kΩ) when the RRAM cell is either in the HRS or the LRS. The HRS and the LRS values of GS-RRAM and Pt-RRAM were extracted from the experimental results of this work. Vdd, Vread and Vhalf-bias were set at 5, 3.5 and 2 V, respectively. The maximum total bits for an array were determined using these criteria. The sheet resistance of Ptx was 5 nm. Hence, the feature size was 2 × (5+3+12)+5=45 nm.We adopted the same resistance network and array simulation methodology for the worst-case selected cell of 3D RRAM as in refs How to cite this article: Lee, S. et al. Metal oxide-resistive memory using graphene-edge electrodes. Nat. Commun. 6:8407 doi: 10.1038/ncomms9407 (2015).Supplementary Figures 1-11, Supplementary Table 1, Supplementary Notes 1-2 and Supplementary References


In order to improve its supercapacitive properties, we have prepared a unique sandwich-structured graphene/polyaniline/graphene paper by in situ electropolymerization of porous polyaniline nanomaterials on graphene paper, followed by wrapping an ultrathin graphene layer on its surface. This unique design strategy not only circumvents the low energy storage capacity resulting from the double-layer capacitor of graphene paper, but also enhances the rate performance and cycling stability of porous polyaniline. The as-obtained all-solid-state symmetric supercapacitor exhibits high energy density, high power density, excellent cycling stability and exceptional mechanical flexibility, demonstrative of its extensive potential applications for flexible energy-related devices and wearable electronics.We reported a scalable and modular method to prepare a new type of sandwich-structured graphene-based nanohybrid paper and explore its practical application as high-performance electrode in flexible supercapacitor. The freestanding and flexible graphene paper was firstly fabricated by highly reproducible printing technique and bubbling delamination method, by which the area and thickness of the graphene paper can be freely adjusted in a wide range. The as-prepared graphene paper possesses a collection of unique properties of highly electrical conductivity (340 S cm Among the large family of carbon materials, graphene, consisting of a single-layer of sp2 bubbling delamination method. In order to increase the energy storage capacity of graphene paper, polyaniline (PANI) nanofibers were in situ electropolymerized on it2 fiber symmetric SC2@MnO2//H-TiO2@C asymmetric SC3-x/MoO3-x asymmetric SCIn this work, we present a facile method to prepare a new type of sandwich-structured graphene/polyaniline/graphene nanohybrid paper with desirable properties of high conductivity, good chemical stability and excellent mechanical robustness. Large-scale graphene paper used in this study was fabricated by a novel printing technique and subsequent CO2SO4 and CH3COOH, a lot of bubbles generated and provided a gentle force to delaminate GO film from the commercial A4 paper. These bubbles produced from the chemical reaction between the additions of the A4 paper and HCl are identified to be CO2 bubbles  and mass thickness (mg cm−2) of ten graphene papers display the relative standard deviation (RSD) less than 5.0%  of RGO, PANI/RGO and RGO/PANI/RGO paper electrodes calculated from their charg/discharge curves are illustrated in −1, the Cs of RGO, PANI/RGO and RGO/PANI/RGO paper are 55 F g−1, 522 F g−1 and 581 F g−1, respectively. RGO/PANI/RGO paper not only exhibits the highest specific capacitance values but also maintains them well at high current density compared to other electrodes. Its specific capacitance is preserved 64.4% (from 581 to 374 F g−1) as the current density increases from 1 to 10 A g−1, which is superior to many other graphene nanohybrid electrodes such as graphene/CNT24143−1 (T24143−1 . TherefoThe significant improved supercapacitive performances of RGO/PANI/RGO paper electrode in terms of high specific capacitance and good rate capability can be recognized as: i) RGO paper possesses highly electrical conductivity and mechanical strength, which acts as an ideal current collector for high-performance supercapacitor; ii) After in situ electropolymerization of PANI nanofibers on ROG paper, the PANI/RGO exhibits a better electrochemical capacitive characteristic and superior reversible redox reaction due to the incremental pseudocapacitive contribution the overall capacitance. iii) Although PANI exhibits poor electrical conductivity, this drawback can be overcome by further coating an ultrathin RGO layer on it. The strong π–π interactions between the highly conductive RGO layer and the PANI chains in the RGO/PANI/RGO could provide a good conducting network, which facilitates the redox reaction of PANI component and increases the pseudocapacitance of the nanohybrid paper electrode.The Nyquist plots of RGO, PANI/RGO and RGO/PANI/RGO paper electrodes have been demonstrated in 2SO4 for further applications of flexible SCs−1 (Rct) of the solid-state SC is about 4.5 Ω, which reveals the low resistance of the redox reaction of the solid-state SC device. The as-prepared device maintains its 88% areal capacitance (107 mF cm−2) as discharge current density increases from 0.2 to 2 mA cm−2 , which outperforms many previously reported solid-state SCs, such as polypyrrole-coated paper symmetric SC (1 mWh cm−3)2 fiber symmetric SC (0.22 mWh cm−3)2@MnO2//H-TiO2@C asymmetric SC (0.3 mWh cm−3)3-x/MoO3-x asymmetric SC (1.9 mWh cm−3)The Ragone plots for the electrode materials are shown in In summary, we reported a facile and scalable method to prepare freestanding sandwich-structured graphene/PANI/graphene nanocomposite paper and demonstrated its practical application in flexible all-solid-state SCs. The graphene/PANI/graphene nanohybrid paper exhibited three unique features: i) the constituent graphene paper fabricated by the simple and scalable printing method and subsequent HI treatment possessed high conductivity, light weight, mechanical robustness and chemical stability, which functions itself as an ideal current collector for SC architecture as well as the substrate for the further loading of active electrode materials on it. ii) PANI nanofibers synthesized by a green in situ electropolymerization method have effectively improved the electrochemical performance of the nanohybrid paper. iii) dip-coating of an ultrathin graphene layer on the surface of the porous PANI nanofibers has endowed the sandwich-structured graphene/PANI/graphene nanohybrid paper electrode with enhanced specific capacitance and increased cycling stability. The resultant flexible symmetric SC based on the graphene/PANI/graphene nanohybrid paper and polymer gel electrolyte exhibits high energy density, high power density and excellent cycling stability, which makes it promising for the future applications in flexible energy-related device and modern wearable/portable electronics. It is worth noting that this modular approach for the fabrication of freestanding sandwich-structured graphene nanohybrid paper is not just restricted to the graphene/PANI/graphene system, but also contributes to the design of hybrid nanoarchitectures with tailored compositions and functions, which provides new insights on designing high-performance paper electrode for flexible energy-related device and modern wearable/portable electronics.−1) was dropped onto a piece of commercial printing paper surface. Then, the rod was rolled back and forth to make the GO solution covered uniformly on the paper. After subsequent air-drying, a uniform GO paper are obtained. The GO paper was reduced by means of soaking in the HI solution (45 wt%) for 10 minutes at room temperature, during this process, bubbles generated from the cellulose papers prompt the thin film peeling off the paper in a self-releasing way. Subsequently, the RGO paper was rinsed with a saturated sodium bicarbonate solution, water and methanol and dried by vacuum drying at 80°C. RGO paper was then coated by a layer of uniform porous nanofiber network of PANI via in situ electropolymerization. In the typical procedure, the PANI nanofibers were synthesized by a two-step method on a conventional three-electrode system in a 1 M HCl electrolyte containing 0.3 M aniline monomer. The first step of the nucleation of PANI was performed at a constant potential of 0.8 V for 1 min at room temperature. In the second step, the nanowires were grown under a constant current condition with current density of 2 mA cm−2First, well dispersed GO solution (5mg mLScanning electron microscopy (SEM) images were taken on a FESEM instrument . X-ray photoelectron spectroscopy (XPS) measurements were performed on VG ESCALAB 250 spectrometer with monochromatic Al Kα (1486.71 eV) X-ray radiation (15 kV and 10 mA) and hemispherical electron energy analyzer. Xray powder diffraction (XRD) patterns were recorded using a diffractometer  equipped with a Cu Kα radiation source (λ = 1.5406 Å). Fourier transform infrared (FTIR) spectra were obtained on Bruker VERTEX 70 FTIR spectrophotometer (Germany).2SO4. In the case of all-solid-state SCs, two piece of RGO/PANI/RGO paper were immersed into a hot clear solution of PVA/H2SO4 electrolyte for 5 minutes, then taken out and assembled together with a piece of filter paper sandwiched in between the two electrodes. Finally, the device was solidified for 4 h at room temperature. The weight being used in the calculation of capacitance is referring to the weight of the entire device. The gel electrolyte was fabricated by mixing 6 g H2SO4 and 6 g PVA in 60 mL deionized water and heated up to 85°C for 1 h under vigorous stirring. CV measurements were conducted in a voltage range of 0–0.8 V with voltage sweep rates from 5 to 100 mV s−1. Galvanostatic charge/discharge measurements were carried out at different current density from 1 to 10 A g−1. EIS tests were performed from 10 mHz to 100 kHz with an amplitude of 5 mV referring to open circuit potentialThe electrochemical characteristics were evaluated by cyclic voltammetry, galvanostatic charge/discharge and electrochemical impedance spectroscopy using a CHI 660E electrochemical workstation . A conventional three-electrode system was adopted. The working electrode was RGO/PANI/RGO paper, and the counter and reference electrodes were platinum plate and saturated calomel electrode (SCE), respectively. The aqueous electrolyte is 1 M HF.X., S.X., S.W., H.F. and Y.Q. proposed, planned, and designed the project. F.X., S.X., Z.Z., L.W., J.W. and J.L. performed the material preparation, characterizations, and catalytic tests. All authors contributed to writing the manuscript.Supporting Information


However, targeted synthesis of either heterostructure remains a challenge. Here, via chemical vapour deposition and using benzoic acid precursor, we have achieved the selective growth of h-BN-G and G/h-BN through a temperature-triggered switching reaction. The perfect in-plane h-BN-G is characterized by scanning tunnelling microscopy (STM), showing atomically patched graphene and h-BN with typical zigzag edges. In contrast, the vertical alignment of G/h-BN is confirmed by unique lattice-mismatch-induced moiré patterns in high-resolution STM images, and two sets of aligned selected area electron diffraction spots, both suggesting a van der Waals epitaxial mechanism. The present work demonstrates the chemical designability of growth process for controlled synthesis of graphene and h-BN heterostructures. With practical scalability, high uniformity and quality, our approach will promote the development of graphene-based electronics and optoelectronics.In-plane and vertically stacked heterostructures of graphene and hexagonal boron nitride  and hexagonal boron nitride (h-BN) heterostructures remains a challenge. Here, the authors achieve the selective, CVD growth of h-BN-G and G/h-BN through a temperature-triggered switching reaction. Varying the concentration of h-BN in h-BN-G is a promising method to tune the graphene band gap89h-BN-G is very likely be used in the construction of atomically thin integrated circuits in next-generation graphene devices11h-BN and G in the hybridized atomic layer131415h-BN has been exploited to fabricate high-performance graphene-based transistors using h-BN as a gate insulator, owing to its atomically flat and dangling-bond-free surfaces, which ensure that no charge trapping occurs at the G/h-BN interface. The stacked structure of G/h-BN allows for the easy observation of several unique physical phenomena such as the Hofstadter butterfly effect1718Graphene (G), a single atomic layer of carbon, has attracted considerable attention since its isolation2h-BN-G heterostructures on copper foils by simultaneously introducing carbon and ammonia borane precursors in the growth processh-BN layers by applying the so-called etching-regrowth methods11h-BN, accomplished by directly growing graphene on exfoliated h-BN. In this case, plasma-enhanced CVD process was needed due to the weak catalytic activity of h-BNh-BN stacks by sequentially growing h-BN and graphene on Cu foils. However, the unavoidable etching of the pre-grown h-BN layer in the second growth step results in both in-plane and stacked heterostructures simultaneously, leading to severe degradation of G/h-BN device performance. The lack of stacking registry control in the currently available synthetic strategies usually bring about graphene sheets randomly oriented with regard to h-BN, hindering the exploration of the unique quantum phenomenon induced by minor interlayer rotation1718The technical challenge is to develop an effective synthetic route for these hybrid material systems, which determines how far we can go for practical applications. Recent efforts have been focused on the chemical vapour deposition (CVD) technique. Of particular importance is those by Ajayan and coworkers, who demonstrated the synthesis of randomly distributed h-BN-G and G/h-BN heterostructures on Cu foils, relying on a high carbon/hydrogen ratio molecule of benzoic acid as carbon source. Using atomically resolved scanning tunnelling microscopy (STM) and other methods, we systematically investigate the in-plane continuity and stacking configuration between graphene and h-BN for the synthesized h-BN-G and G/h-BN, respectively. Our work demonstrates the chemical designability of CVD process for deliberate control of graphene and h-BN heterostructures, offering a potential pathway for fabricating high-performance graphene-based electronic devices.In the present study, we develop a novel temperature-triggered reaction route in CVD process to achieve the selective growth of h-BN heterostructures. This process relies on the unique feature of the benzoic acid carbon source, which can decompose into carbon dioxide (CO2) and various hydrocarbon species on pyrolysis above 500 °Ch-BN monolayer is grown on a Cu foil by CVD, which serves as the first step of the whole process. On one hand, when the growth temperature for graphene exceeds 900 °C (Route 1) in the second step, the etching reaction of h-BN by CO2 is activated. As a result, the released CO2 partially etches away the h-BN monolayer, and the exposed Cu surface catalyses the subsequent graphene growth, leading to the formation of in-plane h-BN-G heterostructures. Note that, the by-products of the etching reaction can all be vaporized during the CVD process and can therefore be pumped away with the aid of the carrier gas. For confirming the etching reaction of h-BN with CO2 and the critical etching temperature, additional experiments were conducted for monolayer h-BN on both Cu foils and SiO2/Si substrates by directly using CO2 gas. The obtained results clearly indicate the occurrence of etching reaction at 900 °C, in which the underlying Cu plays an important role in inducing the etching reaction  height analysis after the sample was transferred onto SiO2/Si  and symmetric 2D peaks  with a 2D/G intensity ratio exceeding 1, suggesting its monolayer nature. Corresponding Raman mapping data also convinces the thickness uniformity and the monolayer feature of graphene regions in the two heterostructures (See −1) intensity of the graphene islands in h-BN-G is much larger than that of G/h-BN. This could be attributed to either the limited lattice substitution of graphene by B or N atoms introduced from the etching-patching growth process, or the small domain sizes of graphene and h-BNRaman spectroscopy provides further detailed information on the film structure and quality of samples after transferred onto SiOures See .. Howeveh-BN-G, the G peak is located at ∼1,600 cm−1, which is remarkably blue-shifted as compared with pristine CVD graphene on SiO2/Si substrates . Although the graphene in h-BN-G may have been slightly doped by boron or nitrogen during the etching growth process, no more than 10 cm−1 blue or red shift of G peak in Raman spectra can be induced as previously reported26p-doping effect of SiO2 or the adsorbed water layer between graphene and the hydrophilic substrate2829h-BN/G agrees well with that of pristine CVD graphene, with the G peak located between 1,585 and 1,590 cm−1. This is attributed to the clean interface formed between h-BN and graphene in G/h-BN, which is likely not contaminated during the wet-transfer process. These observations provide additional evidence of the monolayer nature of h-BN-G and stacked bilayer nature of G/h-BN.In addition, the G peak position in Raman spectra shows distinct difference between the two heterostructures. In h-BN monolayer (upper black curve), h-BN-G (bottom red curve) and G/h-BN (blue curve) on Cu foils. All spectra were normalized by the Cu 2p 3/2 peak intensities. For both h-BN-G and G/h-BN, the B 1s peak is centred at ∼190.9 eV while the N 1s peak is located at ∼398.2 eV, both of which are very close to those of pure h-BN. Note that, the intensities of both B 1s and N 1s peaks in G/h-BN are almost identical to those of pure h-BN, while they are significantly decreased in h-BN-G. This can be easily explained by their different film structures. Considering the XPS analysis depth, the entire h-BN layer underneath graphene in G/h-BN can be ‘seen', and nearly the same XPS signal as that in pure h-BN appears. Obviously, in the h-BN-G film, the surface coverage decrease of h-BN results in the intensity decrease of XPS signal.X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) were utilized for elemental analysis to obtain the chemical bonding information and spatial distribution of specific elements in the two-step CVD-grown heterostructures. h-BN-G and G/h-BN are present as similar isolated islands, consistent with the discrete island-shaped graphene SEM images shown in h-BN-G and G/h-BN shown in h-BN layer remains intact.h-BN structure by low-temperature two-step growth route, with the monolayer graphene islands being stacked on the pre-grown h-BN monolayer, forming a structural analogue of bilayer graphene. It is an interesting issue to understand the stacking order of two layers, which is directly related to the growth mechanism. In fact, recent studies utilizing 13C isotope labelling techniques prove that the second layer growth most likely occurs underneath the existing layer in the CVD bilayer graphene growth process on Cu foil31h-BN heterostructures. We designed an in-air oxidation experiment to assign the stacking order of G/h-BN as shown in h-BN film with a submonolayer graphene coverage was transferred onto a 300-nm-thick SiO2/Si substrate. Notably, the graphene flakes can be easily distinguishable from that of h-BN, as is shown in the AFM height image in h-BN sample was then annealed in air at 600 °C for 2 h, sufficient for completely etching away all the graphene flakes  than that of a h-BN-G film (h-BN/Cu and G/Cu).Electrostatic force microscopy (EFM) has been proven effective to evaluate the layer number of 2D materials37urements  reflectsh-BN regions can be calculated by plotting the EFM phase as a function of the tip bias voltage for h-BN-G and G/h-BN, as shown in h-BN-G and G/h-BN, graphene regions present a smaller work function than that of h-BN regions, with negative differences . This means that the graphene regions are electrostatically n-doped by the underlying Cu substrate, regardless of the presence of h-BN layer in between, consistent with the previous theoretical calculationsThe work function differences between graphene and h-BN would be much poorer than that of Cu for the dehydrogenation of carbon precursors. In fact, when using partly covered h-BN on Cu foil as the substrate, the subsequent graphene growth primarily occurred on Cu surface rather than on h-BN  surface exhibits a similar surface electronic property to pristine Cu(111), possessing the periodic standing wave patterns characteristic of metals in differential conductance (dI/dV) maps. A comparative experiment was performed on the thick h-BN for graphene growth. It is found that only amorphous carbons were obtained as seen in h-BN and Cu for the subsequent graphene growth in the h-BN/Cu bilayer structure. Recent experimental and theoretical works22h-BN would greatly facilitate the epitaxial growth of graphene on the h-BN/Cu surface. Moreover, the visible small Raman D peak at ∼1,353 cm−1 in G/h-BN  surfaceh-BN is also reported on Cu foils recentlyh-BN system, the high-resolution STM images are characteristic of a large-scale moiré pattern as clearly seen in STM has been employed recently to examine the stacking order of graphene and  of h-BN . In the λ) of a moiré pattern can be calculated using the following equation:According to LeRoy and coworkersδ and ϕ are the lattice mismatch and relative rotational angle between graphene and h-BN, respectively, and a is the graphene lattice constant. From the above equation, λ equals 12.9 nm when ϕ is set to 0°. The difference between the observed and theoretical periods is believed to arise from the perfect epitaxial growth mode of graphene on h-BN, which leads to three local stacking configurations, namely, AA stacking, nitrogen-centred and boron-centred AB stackings, respectively, as schematically illustrated in h-BN phase (without any misorientation), the three stacking configurations cannot be distinguished from each other in the STM image obtained directly on Cu, resulting in a change of the moiré period from 12.9 nm to et al.h-BN on Cu(111).where h-BN in the G/h-BN film on a large scale, high-resolution transmission electron microscopy (HR-TEM) combined with selected area electron diffraction (SAED) measurements were conducted. The TEM image of G/h-BN with submonolayer graphene coverage in h-BN. h-BN film with full graphene coverage, where the edges of the sheet breakage allow for the direct identification of a bilayer. The HR-TEM image in h-BN. Shown in h-BN heterostructure, which exhibits a similar sixfold diffraction pattern. Close-up views of the regions identified by blue circles show two separated spots along the radial direction. The outer spot should correspond to the graphene lattice due to its relatively shorter lattice constant of 0.246 nm. Meanwhile, the intensity profiles along the radial direction of the SAED spots are plotted in h-BN, is calculated to be 1.0166, consistent with the theoretical value (0.246/0.250=1.016).To confirm the stacking registry and twist angle between graphene and h-BN, a statistical analysis of the twist angles is given in h-BN, following a van der Waals epitaxial mode.To better understand the stacking registry of thus grown G/h-BN. As discussed above, at the initial epitaxial growth step of monolayer graphene on h-BN, discrete micrometer-scale flakes were usually evolved with relatively high nucleation density   and ethylene glycol (15%wt) as the electrolyte. In the electropolishing process, a 1.5 V DC voltage bias was applied between anodic Cu foil and cathodic Cu plate.h-BN growth. Using a rotary pump, a base pressure of 0.5 Pa can be achieved in our system. During growth, working pressure was held at 1.2 × 102 Pa, either with or without the introduction of benzoic acid.The pre-treated Cu foil was loaded into a 1-inch quartz tube and heated by a Thermal split tube furnace (Lindberg/Blue M HTF55347c) for graphene and h-BN-G growth, Cu foil was first heated from room temperature to 1,000 °C in 40 min, with 20 sccm (standard cubic centimetre per minute) H2 and 50 sccm Ar as carrier gas. Followed by 10 min annealing at 1,000 °C, ammonia borane, solid BN precursor, was introduced into furnace using a heating belt to grow monolayer h-BN . After that, furnace temperature was set to 900–920 °C for graphene growth, using solid benzoic acid as precursor, which was sublimated by a heating belt at ∼100 °C.With respect to the in-plane h-BN were identical with that of h-BN-G described above, except that the furnace temperature was set to 850 °C during graphene growth process.Growth parameters of stacked G/Characterizations were performed with SEM (Hitachi S4800), AFM , Raman , XPS , AES (PHI Quantera SXM), STM (Omicron UHV-VT-SPM-MBE System) and TEM .h-BN samples being transferred onto a 300 nm SiO2/Si substrate, the back-gated graphene FET was fabricated on a single graphene domain. With the help of electron beam lithography, graphene channels were patterned and (100 nm/5 nm) Au/Cr metal contacts were thermally evaporated. The transport measurements were performed at room temperature using a Keithley 4200-SCS semiconductor characterization system, equipped with a four-probe station. During measurements, a 0.1-V DC bias was applied between source and drain, with a linear sweep gate voltage from −100 to 80 V applied on back gate.After G/Z.L. and Y.Z. conceived and supervised the research project. T.G. developed and conducted the growth of graphene and boron nitride heterostructures, with X.S.'s assistance. H.D. and Y.Y. performed the EFM characterization. T.G., X.S., Y.N, Y.C., Q.J. and J.S. carried out SEM, OM, Raman, XPS, AFM, STM, TEM and transport measurements. T.G., Z.L., Y.Z., J.S. and X.S. co-wrote the manuscript and all authors contributed to the critical discussions and the revision of the final manuscript accordingly.How to cite this article: Gao, T. et al. Temperature-triggered chemical switching growth of in-plane and vertically stacked graphene-boron nitride heterostructures. Nat. Commun. 6:6835 doi: 10.1038/ncomms7835 (2015).Supplementary Figures 1-8, Supplementary Notes 1-8 and Supplementary References


Graphene oxide (GO) has become a well-established reinforcement for polymer-based nanocomposites. It provides stronger interfacial interaction with the matrix when compared with that of graphene, but its intrinsic stiffness and strength are somewhat compromised because of the presence of functional groups damaging the graphene lattice and increasing its thickness, and its tendency to adopt a crumpled structure. Although the micromechanics of graphene reinforcement in nanocomposites has been studied widely, the corresponding micromechanics investigations on GO have not been undertaken in such detail. In this work, it is shown that the deformation micromechanics of GO can be followed using Raman spectroscopy and the observed behaviour can be analysed with continuum mechanics. Furthermore, it is shown that the reinforcement efficiency of GO is independent of its number of layers and stacking configurations, indicating that it is not necessary to ensure a high degree of exfoliation of GO in the polymer matrix. It also demonstrates the possibility of increasing the concentration of GO in nanocomposites without sacrificing mechanical reinforcement efficiency.This article is part of the themed issue ‘Multiscale modelling of the structural integrity of composite materials’. AdditioThe Young modulus of monolayer GO has been shown to be of the order of 250 GPa . A compuωD) of GO as a function of strain ε has been used to follow the deformation mechanics of monolayer GO flakes to investigate the validity of continuum mechanics on the microscale. Based on the estimation of the effective Young modulus of GO [ωD/dε) is further used to compare the reinforcement efficiency of a series of different GO flakes.In this work, the deformation mechanics of mono-, bi- to multilayer GO structures with different stacking configurations have been studied. The shift of the Raman D band position The graphite (grade 2369) was supplied by Graphexel Ltd. All other reagents were of analytical grade and used without further purification.(b)−3 mg ml−1 and deposited onto poly(methyl methacrylate) (PMMA) beam followed by drying under ambient conditions. Prior to the GO deposition, the PMMA beams were treated using a UV–ozone plasma, facilitating the spreading of the GO dispersion and also the adhesion of the GO to the substrate.The GO was prepared using the modified Hummers method, and the details of preparation can be found elsewhere –23. Afte(c)α radiation source (λ=1.542 Å). Atomic force microscope (AFM) images were obtained using a Dimension 3100 AFM (Bruker) in the tapping mode in conjunction with a ‘TESPA’ probe (Bruker).Transmission mode Fourier-transform infrared (FTIR) spectrum was obtained from dried GO powder mixed with KBr, using a Nicolet 5700 spectrometer (ThermoFisher Scientific Inc.). X-ray diffraction (XRD) was carried out on dried GO powder using an X’Pert DY609 X-ray diffractometer (Philips) with a Cu-KRaman spectra were obtained using Renishaw 1000/2000 spectrometers and a Horiba LabRAM HR Evolution spectrometer equipped with HeNe lasers (λ=633 nm) with laser spot sizes of around 1–2 μm. The incident laser polarization was parallel to the strain, whereas the scattered radiation was randomly polarized. The specimens were deformed in a four-point bending rig, and the strain was measured using a strain gauge placed close to the region being analysed .3.(a)a, where a number of absorption bands, corresponding to the presence of functional groups, can be observed. For example, the band around 1710 cm−1 can be attributed to the C=O stretching of the ketonic species, and the band around 1620 cm−1 is assigned to alkene in the graphitic region. The epoxy C–O–C bonds generate a band at around 1230 cm−1, whereas the band resulting from the C–OH stretching is located around 1070 cm−1 [figure 1b), where the characteristic peak for the GO at 2θ=10.5° corresponds to an interlayer spacing of 0.84 nm, in good agreement with the reported value [The FTIR spectrum of the GO is shown in figure 1070 cm−1 . These f070 cm−1  and expa(b)As reported previously ,4,5,9, t−1, is due to the doubly degenerate E2g vibrational mode at zone centre [−1, results from the Ag1 symmetry K point phonons [−1 is from the PMMA substrate. The broadening of the D and G bands compared with those of graphene [2 carbon networks of pristine graphene by the strong oxidation [ID/IG, can actually be used as a measure of the degree of disorder of GO [′′ band (approx. 1520 cm−1) also needs to be considered, with a Gaussian function fitting needed to account for the broad shoulder between the D and G bands, which may be linked to the structural disorder of the GO [−1 is present [A Raman spectrum was then obtained from this typical monolayer GO flake as shown in e centre . The D b phonons . The smagraphene  is possixidation ,29, implxidation ,31. Thiser of GO ,32. It sf the GO ,34. Even present ,33, the ID/IG after four/five scans on both monolayer and bilayer GO flakes  [Another issue for concern is that GO, being chemically different from graphene because of the functional groups , may makgure S2) ,32.ωD can be seen during deformation of the GO to 1.0% strain ref) for supported graphene should be of the order of −30 cm−1/%.[3 hybridization of carbon atoms [ωD/dε for GO by a factor of 2 [ωD/dε determined here is in good agreement with the value estimated during the deformation of a variety of nanocomposites [ωD/dε in reference [From the knowledge of the Grüneisen parameter ,39,42, ithe form ,433.1wh is used , and ν iubstrate , the ref0 cm−1/%. The valu0 cm−1/%.,43, probon atoms  and wavion atoms . These ftor of 2  and the mposites . , the value of dtransfer ,9,40, ortransfer . This letransfer . There itransfer .(c)ωD with local value of strain ε in the GO flake [Similar to our earlier studies on monolayer pristine graphene , Raman sGO flake ,4,5,9. Iin situ, such as for epoxy resins, will lead to a stronger interface. It also explains the higher measured values of strength and toughness of GO nanocomposites than for nanocomposites reinforced with less functionalized graphene-based materials [There is, however, a major difference between the behaviour of GO and graphene near the flake edge. The deformation mechanics of graphene and GO can be described using the well-known ‘shear-lag’ theory . However, in this work, within the scatter in the data, the strain distribution appears to be constant with position along the flake, whereas in the case of pristine graphene, the strain falls over several micrometres to zero near the edge of the flake due to shear lag effects, enabling the ‘critical length’ of the graphene for good reinforcement to be determined ,9,50. Thaterials ,53. Furt(d)XL, where X is the number of layers, for multilayers denoted as its thickness in nm, assuming for simplicity that the thickness of the GO monolayers is approximately 1 nm), which were again identified using AFM. Their corresponding values of dωD/dε are shown in ωD/dε does not change significantly.It has been shown that in pristine graphene with no functional groups the reinforcement efficiency decreases as the number of layers increases , as a reωD/dε are still similar as highlighted with stars in As well as the number of layers, what is notable is the stacking configuration of the GO flakes. Some of the GO flakes display a stacked multilayer structure, whereas others have a re-stacked structure (prefixed as RS before the thickness as in (e)ωD/dε can be used to estimate the effective Young modulus of GO (EeffGO) in nanocomposites [ωD/dε measured in this present study was approximately −12.8 cm−1/% strain for monolayer GO. The effective Young modulus of GO compared with pristine graphene can be calculated from the reduced value of dωD/dε due to lattice damage and the increase in thickness as a result of the oxidation of the GO using the equationωD/dε)ref is the D-band shift rate for graphene (approx. −30 cm−1/%), tgra is the thickness of graphene (0.34 nm), tGO is the thickness of GO measured in this present study and Egra is the Young modulus of graphene. This equation yields a value of EeffGO∼180 GPa for the GO, in broad agreement with but slightly lower than the value reported for an indentation test on a GO membrane [It has been demonstrated previously that the value of dmposites . The avemembrane . This slEeffGO of GO of N layers (EeffGO(N)) derived from the Raman band shift data (EeffGO(N) values are calculated from equation  is used as the reference in equation /EeffGO(1) is plotted as a function of the number of GO layers with a series of values of k in k=1, EeffGO(N)/EeffGO(1)=1, the stress can be transferred completely through the bottom layer to the top layer, thus the effective modulus of GO would be identical, regardless of the number of layers. For all the other values of k, the ratio of EeffGO(N)/EeffGO(1) decreases as the number of layers increases, in accordance with a gradual loss of the interlayer stress transfer from layer to layer. Particularly, for k=0, EeffGO(N)/EeffGO(1)=1/N, only the bottom layer bears the load, and no stress transfer occurs to the upper layers, hence the effective modulus varies inversely with the number of layers (cross-section area). It can be seen in k equals unity, suggesting almost complete interlayer stress transfer in the GO flakes, regardless of the different number of layers and stacking configurations.As the number of GO layers increases, up to tens of layers, the ift data  still doequation , and k iThis behaviour of GO is remarkably different from that of graphene , for whiThis behaviour can be interpreted as being due to the hydrogen bonding controlled by the functional groups and also the water content , which tωD/dε, which would otherwise be expected to vary depending on the number of layers, as for graphene [In our samples, the GO forms after being oxidized and further separates in aqueous solution ,59. The graphene , where ographene ,11,12, wThese features of stacked and re-stacked GO flakes can be instructive for their use in GO-based nanocomposites. First, because of the strong interlayer adhesion, it is thought that there is less need to ensure a complete exfoliation of GO (less than 40 layers should suffice). As it is the GO flakes with large lateral dimensions that take longer times to be fully exfoliated , this re4.ωD/dε, has been used to map the deformation of a monolayer GO flake, so that the strain distribution of the GO flake could be revealed. Additionally, the measured value of dωD/dε has also been used to estimate the effective Young modulus EeffGO of GO flakes with different thickness. It is found to be almost constant, regardless of the number of layers or the stacking/re-stacking configuration. This was attributed to the good interlayer stress transfer provided by hydrogen bonding between the layers. It suggests that there is no need to exfoliate the GO completely when preparing nanocomposites. This also has the advantage of enabling flakes with relatively large lateral dimension to be prepared which will still have a significant positive impact on the reinforcement. Furthermore, it shows the possibility of preparing GO-based nanocomposites with a reasonably high GO content, without sacrificing mechanical performance due to re-aggregation effects.This work has presented a method to monitor the deformation mechanics of GO with different number of layers and stacking configurations using Raman spectroscopy. The stress/strain-sensitive Raman D band was used, and the measured value of the shift of the position of the Raman D band with strain, d


Diurnal flying animals such as birds depend primarily on vision to coordinate their flight path during goal-directed flight tasks. To extract the spatial structure of the surrounding environment, birds are thought to use retinal image motion  that is primarily induced by motion of their head. It is unclear what gaze behaviors birds perform to support visuomotor control during rapid maneuvering flight in which they continuously switch between flight modes. To analyze this, we measured the gaze behavior of rapidly turning lovebirds in a goal-directed task: take-off and fly away from a perch, turn on a dime, and fly back and land on the same perch. High-speed flight recordings revealed that rapidly turning lovebirds perform a remarkable stereotypical gaze behavior with peak saccadic head turns up to 2700 degrees per second, as fast as insects, enabled by fast neck muscles. In between saccades, gaze orientation is held constant. By comparing saccade and wingbeat phase, we find that these super-fast saccades are coordinated with the downstroke when the lateral visual field is occluded by the wings. Lovebirds thus maximize visual perception by overlying behaviors that impair vision, which helps coordinate maneuvers. Before the turn, lovebirds keep a high contrast edge in their visual midline. Similarly, before landing, the lovebirds stabilize the center of the perch in their visual midline. The perch on which the birds land swings, like a branch in the wind, and we find that retinal size of the perch is the most parsimonious visual cue to initiate landing. Our observations show that rapidly maneuvering birds use precisely timed stereotypic gaze behaviors consisting of rapid head turns and frontal feature stabilization, which facilitates optical flow based flight control. Similar gaze behaviors have been reported for visually navigating humans. This finding can inspire more effective vision-based autopilots for drones. When moving rapidly through a dense and cluttered environment like a forest, proximity information about tree trunks and branches is essential for negotiating a successful goal approach. To accomplish such navigational tasks, diurnal birds rely on vision. Remarkably, little is known about which visual cues birds actually use to orchestrate their rapid flight maneuvers during close range navigation in their habitat. Well-studied visual depth perception mechanisms for primate navigation and depth perception, in particular stereopsis, is very limited in birds and therefore not considered to play a role in rapid flight control . This moBetween rotational head saccades, head orientation is stabilized. Consequently, the time in which purely translational optic flow, and thereby proximity information, can be obtained is prolonged. Similar gaze behaviors have been reported for fast flying insects that rely on optic flow for depth perception . Birds tWe addressed the question of how birds change their gaze between maneuvering tasks by analyzing the flight and gaze kinematics of lovebirds, a small generalist parrot, performing a rapid turn maneuver during a simple goal directed task. Wild lovebirds roost and forage in small flocks of 5–20 birds in the scrublands of central Africa and Madagascar. We investigated the subsequent flight behaviors: take-off and fly away from a perch, turn on a dime, and fly back and land on the same perch. Such a sequence resembles natural flight maneuvers regularly conducted by this social bird when competing for perch locations on trees or feeding spots. Based on the reports that about 85% of the total visually induced gaze shifts in birds are accomplished by head rotations ,14 and eWe find that maneuvering lovebirds conduct among the fastest gaze shifts so far reported for vertebrates. These shifts are preferably timed at specific phases within a wingbeat and, thus, are specifically adapted in their speed and occurrence to the requirements of visuomotor control of rapid flight.agapornis roseicollis) to turn on a dime in our custom-built flight arena  on specific parts of the bird’s head  [One of our main interests in this study was to analyze the gaze behavior of maneuvering birds. The temporal resolution of the wingbeat related tracking procedure was not sufficient to analyze head and thereby gaze kinematics in detail. Therefore, we analyzed head data in multiple flights (at least three per bird), for each bird at full temporal resolution of 2000 Hz. The selection of flights was made based on following criteria: (i) the birds had to be in the camera view from beginning to the end of the flight. (ii) The flight contained not more than one complete U-turn. (iii) Changes in its flight altitude were less than 21 cm. (iv) Birds were facing into the flight direction (± 45°) before taking off. (v) Landing on the perch was successful on the first attempt, and to optimize marker tracking, (vi) head makers were not covered by the wires of the top mesh for long flight passages. 16 flight recordings of five birds fulfilled these criteria. Automated marker tracking for these recording was done with the open source software “ivTrace” (ivtools) . We callcoordinates = 100, cvecoordinates < 0.5; λhead = 5000, cvehead < 1; λbody = 1000000, cvebody < 2).Due to marker coverage by the wires of the top mesh and due to varying marker contrast levels for the differently colored birds, tracking could not be done completely automatically but was interspersed by sections in which we tracked manually. To account for noise introduced by our tracking procedures and digital jitter, we filtered the obtained marker point coordinates and obtained yaw orientations with a penalized least-squares smoothing algorithm based on a Whittaker filter ,25. Thishead) and of the normal of the vector connecting the birds’ shoulders (Φbody)  . As body (Φbody)  and inteSimilar to earlier studies addressing rotational head movements in free behaving animals ,21, we dThe quantitative saccade and landing analyses were based on high-resolution tracking data (2000 Hz) consisting of 3–4 flights for each of the five tested birds (see classification criteria above). To avoid strong effects of ascending or descending maneuvers and have better comparable flights, we only analyzed turn flights for the wingbeat analysis where height changes during the turn were minimal (< 21 cm) resulting in 9–10 flights per bird. Quantitative analysis of gaze orientation was done first with the smaller high-resolution data set, and after validating that found distributions are qualitatively similar, the analysis was extended to the larger low-resolution wingbeat related data set that included all above mentioned flights. This set contained 12–26 flights per birds. In all analyses, we averaged the results per bird before comparing or averaging across birds.ξ1+ ξ2= 1),1 and μ2 are the distribution means, σ1 and σ2 the standard deviations and ξ1 and ξ2 the mode weights. A fitted distribution was categorized as bimodal if the following conditions were true:Wingbeat and downstroke / upstroke periods were analyzed for bimodal distributions using the Gaussian mixture models algorithm (GMM) of the MATLAB statistics toolbox. The GMM algorithm assumes that the wing data are a mixture distribution, where the probability density function is a combination with coefficients that sum to 1 . Birds were housed in pairs in enriched cages, which included toys and artificial full-spectrum lights.n = 92 flights, N = 5 birds) lovebirds turned left towards the white sidewall. Only 10% of the analyzed flights contained right turns towards the dark sidewall  the straight flight phase after take-off and before the turning maneuver (before turn), (ii) the turning phase (during turn) and (iii) the straight flight phase after the turn until landing (after turn). The segmentation was based on the position of the wingtips during mid-stroke. When the turn was initiated, the tip position changed characteristically by deviating from its previously relative straight trace , showingManeuvering lovebirds had a relative constant head orientation before and after the turn , and fitDue to their visuomotor capabilities, birds are model organisms in the fields of animal locomotion and visual information processing. However, not much is known about how flight kinematics and visual behaviors are tuned to work in concert. When analyzing wingbeat kinematics and gaze behavior across all five birds we found that: (i) the wingbeat of lovebirds is intermittent during maneuvering flight, and (ii) saccadic gaze shifts are preferably initiated at the end of the downstroke.vs. normal frequency wingbeats in each phase, for each individual bird, and averaged the results. There is no general difference in intermittency between phases  but intermittent flaps (n = 153) tended to happen most often in the phase after the turn, while normal flaps (n = 528) tended to occur predominantly during the turn. Of all recorded intermittent flaps, 26.5% ± 12% occurred in the phase before the turn, 29.8% ± 9.9% during the turn and 43.7% ± 13.14% after the turn across birds. Of all normal flaps 30.8% ± 5% occurred before the turn, 40.9% ± 5.2% during and 28.7% ± 3.6% after the turn.We analyzed the wingbeat kinematic data with a Gaussian mixture model to determine if they are bi-modally distributed due to intermittency. The resulting Gaussian fits reveal that both the flapping frequency and the relative periods of the down- and upstrokes are bimodal distributed for each individual bird. Lovebirds almost halved their normal flapping frequency  during intermittent flaps . The advance ratio J, is a non-dimensional parameter defined as the ratio between forwards flight speed and the mean wingtip velocity (n = 697 wingbeats). This quantitative result confirms the general observation that rapid turning lovebirds were in slow-hovering flight throughout the maneuver [To see if flight speed varied between flight phases, we calculated the average flight speed per wingbeat and averaged it for each bird over each flight phase. Lovebirds flew 0.54 m/s ± 0.01 m/s slower during the turning phase than during the phase before the turn (1.05 m/s ± 0.16 m/s) and the phase after the turn . Head s2 = 0.78), suggesting that lovebirds have a preferred average head saccade velocity of 1500°/s. To evaluate this finding in a broader context, we extracted saccade amplitude and duration data from figures of earlier studies investigating gaze behavior in visual model organisms, specifically vertebrates (http://imagej.nih.gov/ij/index.html). Compared to the combined eye-head gaze shifts of rhesus monkeys, and the eye saccades of humans, rabbits and cats, the saccadic gaze shifts of maneuvering lovebirds are extraordinarily fast. There is a general trend that saccades in lovebirds are shorter than in others species, although their amplitudes are comparable to eye saccades in humans and rabbits. Horizontal head saccades in lovebirds are much larger than eye saccades of the cat. Combined gaze shifts in rhesus monkeys (head + eye) reach higher amplitudes than lovebird head saccades. The variability in saccade amplitude stimulated us to determine how much saccadic vs. intersaccadic gaze changes contribute to the total orientation of the head during a U-Turn. Hence, we summed all saccade amplitudes and compared it to the total head reorientation during the turn. We found that, on average, three quarters of the horizontal head reorientation was accomplished through saccadic gaze changes  [By combining head orientation and flight arena geometry , we foun, N = 5) . Due to , N = 5)  and sacc, N = 5) ,35, we f, N = 5) –15). Thin = 15 left turn flights, N = 5). We found that the results were quantitatively similar for a static and a moving perch , its relative retinal expansion velocity (RREV), and the estimated time-to-collision (tau), to initiate landing behavior on a static spot  analysis introduced by Wagner for landing houseflies  and subsThe dynamic swinging motion of the perch induced large variations in the relative approach speed , of which the function is interpreted to be similar to the fast resetting of the eyes; the optokinetic nystagmus (OKN). Brain systems involved in the control of both reflexes are the nucleus of the basal optic roots (nBOR), the pretectal nucleus lentiformis mesencephalic (LM) and the vestibule-cerebellum . The secBetween saccadic gaze changes, rotational head stabilization in space is likely coordinated by the vestibulo-collic reflex (VCR) ,66. VisuTo understand how the observed gaze behavior is used to fixate image features we need information about head stabilization, eye motion, and the actual focus of visual attention on the retina. So far only head orientation data has been obtained in freely flying birds ,51,77–80Whereas we can assume that eye orientation is coordinated with head saccades in free flight, we do not have retinal morphological data to determine how head orientation relates to fovea orientation. Temporal regions with higher visual acuity that are frontally oriented like in cockatoos  seem to n = 16 flights, N = 5), or rate of change of retinal expansion speed  [By scrutinizing the high-speed flight recordings, we found that the first behavior indicating landing is a downward pitch of the tail feathers that resulted in an upward pitched body posture accompanied by an extension of the legs. Therefore, and because the exact timing of leg extension was difficult to assess from our videos, we based our definition of landing initiation on the tail pitch behavior. The tail pitch analysis starts up to 50–100 ms earlier than the previous analysis based on leg extension  . On some, N = 5) ,39.The finding that lovebirds stabilize wall and perch edges before and during turning, and the perch center after turning to coordinate their landing, may indicate that birds and humans use similar features for navigation. Edges of obstacles that need to be avoided  are visually stabilized by lovebirds, similar to the center of the visual target they choose to approach (the perch) . Such a Flapping animals frequently conduct subtle adjustments to their wing movements, body posture, and head orientation to stabilize themselves . Our obsS1 MovieThe bird performed a saccadic gaze behavior timed within the wingbeat. Between saccadic turns, the head is rotationally stabilized. In contrast, the body turns continuously during the maneuver following the head. The bottom graph illustrates the estimated head and body yaw orientation relative to a horizontal in the arena. An orientation of 0° represents a straight flight away from the perch, 180° a flight to the right towards the perch. Vertical bars indicate the downstroke phases and thereby the wing beat timing. The video is 50x slowed down for illustration. The actual maneuver took 1.15 seconds. See (MP4)Click here for additional data file.


Different notations of graphene irreducible representations and optical modes could be found in the literature. The goals of this paper are to identify the correspondence between available notations, to calculate the optical modes of graphene in different points of the Brillouin zone, and to compare them with experimental data obtained by Raman and coherent anti-Stokes Raman scattering (CARS) spectroscopy. The mechanism of the resonance enhancement of vibration modes of the molecules adsorbed on graphene in CARS experiments is proposed. The possibility of appearance of the discrete breathing modes is discussed. Here,k ≠ 0. The shapes of the normal vibrational modes are computed in the K, M, and Г points of the Brillouin zone.The technique of quantum operator of projection on projective representations, which describes elementary vibration modes, is used to determine the shapes of the normal modes analytically in different points of the Brillouin zone, including 6h were modeled and optimized in the same basis and function. Parallelized implemented software at Gaussian 09 packages [Optical modes of single-layer graphene were investigated using density functional theory (DFT) with periodic boundary conditions (PBC) and STO-3G basis with the correlation functional VWN5. As an initial atomic configuration fragment, a flat hexagonal lattice of 40 carbon atoms Fig.  or 12 elГ (D6h), K (D3h), and M (D2h) points of the Brillouin zone were identified by using the apparatus of the quantum-mechanical projection operator. To optimize the search of the characteristic frequencies, the discrepancies between the experimental and calculated frequencies of the vibrational spectra were analyzed. Analysis of carbon atom movements in graphene lattice for the main observed bands of the vibrational spectra was done  or fundamental vibrational modes could be presented by irreducible representations of the D6h group in the notations [Phonon spectra of graphene in the Brillouin zone consist of six branches. Phonons with zero quasi-momentum at the otations  by the fГvib = B2g + A2u + E2g + E1u [+E1++E2− , Гvib = 2g + E1u  or \docuincluding the acoustic vibrationsand optical ones−1 correspond to a double degenerated state with −1, so-called the G-mode at the Г-point of the Brillouin zone and named by Dresselhaus as E2g mode. Calculated vibration at a frequency of 1455 cm−1 is associated with the K-point of the Brillouin zone and observed in the region of 1250–1380 cm−1 at different frequencies of laser excitation and called the D-mode . This process could be named resonant Raman in contrast to non-resonant Raman, where the virtual states of the electron and the hole are excited.The laser photon excites an electron with wave vector The electron comes to the other equivalent well and emits a phonon of i-TO branch of the dispersion curve (transition 2 → 3).ke), but its energy decreased by the energy of the emitted phonon. As a result, another phonon is emitted (transition 3 → 4).The electron returns to the state with approximately the same wave vector (≈The electron recombines with the hole (transition 4 → 1) and emits the Stokes Raman photon. Such process leads to a double electron-phonon resonance.Transitions occur simultaneously with the electron in the conductance zone (electron goes through the states 1–2–3–4–1). The hole goes through equivalent states in the valence zone.The electron goes through the states 1–2, 2–3 and transfers to the second well. The hole goes through the states in the valence zone simultaneously. Recombination of the electron and the hole occurs afterwards.The stages of electron-phonon processes leading to the appearance of 2D-band in the Raman spectroscopy (RS) of graphene are the following Fig. :Fig. 3RaThe transition 3 → 4 for D-band, which involves the structural defects, is a phononless. The sequence of transitions for D′- and 2D′-bands is the same, but the processes occur within one well.ke in the conductance zone and a hole with the wave vector kh in the valence zone . The electron emits a phonon with the wave vector q = 0 and recombines with a hole. All events occur in the frame of one valley.In the case of G-band, a laser photon excites an electron with the wave vector In order to calculate the wave vector of photons participating in the RS process, we computed the basic vector of reciprocal lattice Fig.  asFig. 4a1 = 24,562 Å and φ = 300. So, b1 = 29,539 Å−1. The wave vector in the M-point was determined as b1 divided by 2 . So, the value of wave vector at the M-point is k(M) = 14.7*107 cm−1. The value of wave vector at the K-point is −1. These values are the basis for the construction of the dispersion curve diagram of phonon states in graphene.where K-points and M-points were calculated from the energy of excited photons (1.96 eV for Raman scattering and 3.86 eV for CARS) from the electron dispersion curves  and in the vicinity of the M point (shown in green) (Fig. 5 was obtained in CARS experiment of thymine on graphene oxide [The values of electron wave vectors participating in Raman scattering and CARS in the vicinity of the ves Fig. . The theDiscrete breather is a spatially localized nonlinear vibrational mode in the defect-free lattice , 12. Dis−1 (Fig. Since the lattice dynamics in the first principle calculations is not limited to linear approximation, we can expect obtaining nonlinear excitations such as discrete breathers. In our calculations, they are observed near the frequency of 714 cmГ, K, and M points of the Brillouin zone were identified using the quantum mechanical projection operator. A correspondence between available notations for optical modes of graphene in the different points of the Brillouin zone are provided. Experimental data obtained by Raman and CARS spectroscopy are compared with calculations. The discrepancies between experimental and calculated frequencies of the vibrational spectra were analyzed. Based on these data, a resonant mechanism of RS and CARS in graphene was postulated. A possible existence of discrete breathers in graphene with the frequency of about 714 cm−1 was shown.Fundamental modes of graphene at the


LINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Ongoing L1 retrotransposition in the germ line and somatic tissues has contributed to structural genomic variations and disease-causing mutations in the human genome. L1 mobilization relies on the function of two, self-encoded proteins, ORF1 and ORF2. The ORF2 protein contains two characterized domains: endonuclease and reverse transcriptase.in vitro L1 cleavage assay, we demonstrate that the monoclonal anti-ORF2 protein antibody partially inhibits L1 endonuclease activity without having any effect on the in vitro activity of the human AP endonuclease.Using a bacterially purified endonuclease domain of the human L1 ORF2 protein, we have generated a monoclonal antibody specific to the human ORF2 protein. We determined that the epitope recognized by this monoclonal antibody includes amino acid 205, which is required for the function of the L1 ORF2 protein endonuclease. Using an Overall, our data demonstrate that this anti-ORF2 protein monoclonal antibody is a useful tool for human L1-related studies and that it provides a rationale for the development of antibody-based inhibitors of L1-induced damage.The online version of this article (doi:10.1186/s13100-014-0029-x) contains supplementary material, which is available to authorized users. L1 has shaped the evolution of the human genome through a copy-and-paste mobilization of itself , SINE-VN L1 mRNA -7 and arelements ,3,8. L1,elements ,10. Thiselements . The majelements -16.Figurin vitro and in mammalian cells using overexpressed tagged ORF2 proteins and polyclonal anti-ORF2p antibodies [L1 proteins are produced from the full-length L1 mRNA with significantly different efficiencies, mostly owing to the unconventional translation from the bicistronic L1 mRNA -20  of the purified hEN under these blotting conditions , which shares sequence homology with the L1-encoded endonuclease, was used to test the specificity of this effect. We observed no change in the APE 1 activity upon the addition of the highest amount of the L1 EN-specific antibody , NIH-3T3 (ATCC CRL-1658), and Ntera2 (ATCC CRL-1973) cells were maintained as previously described [FLP-In™-293 (Invitrogen) cells were cultured in HyClone Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Invitrogen) and maintained under 6% COescribed .6 cells per T25 flask and transfected 16 to 18 hours later with 2 μg of the human or mouse ORF2 or EN expression plasmids [6 and 2.5 × 106 cells per T75 flask, respectively, and transfected as previously described using 6 μg of plasmid [Western blot: 293 cells were seeded at 1.5 × 10plasmids , or 1, 2plasmids  (L1co) oplasmids . Plus re plasmid , 12 μL o2HPO4 (Sigma S3264), 2 mM KH2PO4 (Sigma P9791), pH = 7.4), 5 mM ethylenediaminetetraacetic acid (Sigma ED), and 0.02% sodium azide (Sigma S2002). Lysis buffer was supplemented with phosphatase inhibitors 2 and 3  and Halt Protease inhibitors at 10 μL/mL each. The samples were subjected to two freeze (−80°C)/thaw (25°C) cycles. The samples were sonicated three times for 10 seconds at 12 watts RMS using a 3 mm wide QSonica Microson homogenizer with Microson ultra sonic disruptor XL2000 (Misonix). The protein concentration of each sample was determined using 595 nm wavelength OD values against a bovine serum albumin standard.Total protein was extracted as previously described ,51 usingA 10 to 20 μg of total protein were combined with 2× Laemmli buffer and 1.6 μL (14.3 M) β-mercaptoethanol and boiled for 5 minutes prior to fractionation on Tris Acetate 3-8% Midi gels, Bis Tris 4 to 12% Midi gels (Invitrogen), and transferred onto nitrocellulose membranes . Membranes containing fractionated protein samples were blocked for 1 hour in PBS-Tween containing 5% milk and incubated with a 1:250 dilution of custom polyclonal antibodies against the mouse ORF2p endonuclease , a 1:500For SDS Tris Glycine gels , 1% dimethyl sulfoxide (DMSO), 0.1% triton, and 0.01% sodium azide.The LINE-1 EN was expressed and purified as described previously ,32. The Antibody − FIBufferControl)/(FIL1/APE1 EN − FIBuffer Control)).The effect of the monoclonal anti-ORF2p antibody on LINE1 EN activity was tested using three concentrations: 100 nM, 150 nM, and 200 nM. The antibody was diluted into the above described reaction buffer just prior to use. The same was done for the anti-hORF1p antibody . A buffeThe purified APE1 EN was purchased from New England Biolabs. The assay was performed using 0.01 and 0.1 units of enzyme and 200 nM of duplexed oligonucleotide containing an abasic site. The sequence of the oligonucleotide was based upon previously published work . The reaAll oligonucleotides were purchased from Integrated DNA Technologies. The oligonucleotides used in the assays were annealed by adding equivalent amounts of each complimentary nucleotide in annealing buffer (50 mM Hepes (pH 7.5) and 100 mM NaCl). The samples were incubated in boiling water for 5 minutes and slow cooled for 1 hour in the dark. The sequence for the LINE1 EN oligonucleotides used in the assay are as follows: 5′/AlexaFluor488/CCTTTTTTTTTAACCGC3′ and 5′GCGGTTAAAAAAAAAGG3′. The sequence for the APE1 EN oligonucleotides used in the assay are as follows: 5′/AlexaFluor488/GCCCCC_GGGGACGTACGATATCCCGCTCC3′ (where “_” represents an abasic site) and 5′GGAGCGGGATATCGTACGTCCCCCGGGGGC3′.Human L1PA family consensus sequences  and L1 SThe molecular weight of all proteins was calculated based on their amino acid composition using EditSeq software. The number of molecules detected by monoclonal anti-ORF2p antibody was calculated using the following formula:


TDP-43 (A315T)) under control of the murine prion promoter develop motor symptoms, but their use is currently hampered by sudden death. We aimed to understand and overcome the cause of sudden death in TDP-43 (A315T) mice. Since intestinal obstruction was suspected to be the cause, intestinal motility of TDP-43 (A315T) mice was studied in an ex-vivo pellet propulsion assay. The effect on the enteric and motor phenotype was assessed, both in animals on normal chow or on a jellified fiber deprived diet, aimed at preventing intestinal obstruction.Intraneuronal inclusions of TAR DNA-binding protein 43 (TDP-43) have been found in the majority of Amyotrophic Lateral Sclerosis (ALS) patients. Mutations in the gene encoding TDP-43 cause familial ALS. Transgenic mice expressing mutant TDP-43 with one such mutation (TDP-43 (A315T) compared to non transgenic (NTG) mice. Immunohistochemistry revealed significant enlargement in size and reduction in number of the nitric oxide synthase (NOS) neurons in the myenteric plexus of TDP-43 (A315T) mice. Prevention of intestinal obstruction by jellified food abolished sudden death, allowing the motor phenotype to develop and slowly progress with a more pronounced degeneration of upper and lower motor axons. A downregulation of endogenous TDP-43 mRNA and protein levels was observed prior to neurodegeneration.The frequency of the propulsive motor complexes was significantly reduced in the colon of TDP-43 (A315T) mice suffer from intestinal dysmotility due to degeneration of NOS neurons in the myenteric plexus. Feeding the mice jellified food prevents sudden death and allows the motor phenotype to progress. TDP-43 induced neurodegeneration, several transgenic mouse models have been generated . After 3 washes with PBS-T, slides were mounted with Vectashield with DAPI and analyzed using a Zeiss Imager M1 microscope .We performed immunostainings for total (mouse and human) TDP-43 and glial fibrillary acid protein (GFAP) on spinal cord of ES  To analyze the intestinal tract by immunostainings, segments of intestine (ileum and colon) were collected, opened along the mesenteric border and pinned flat in a sylgard lined dissection dish. Using fine forceps the mucosal and submucosal layers were removed prior to fixation in 4% paraformaldehyde (30 min). After rinsing in PBS, circular muscle layers were peeled in case of the small intestine; while for the colon the longitudinal muscle was removed. Tissues were treated in permeabilizing (0.5% triton-x) and 4% goat/donkey serum, prior to a 24 h (4°C) incubation in primary antibodies: specific human TDP-43 antibody , total TDP-43 , GFAP , NO-synthetase , ChAT  and HuCD . After rinsing, fluorescently labeled appropriate secondary antibodies were added for 2 h. Immunohistochemical staining was visualized under an epifluorescence microscope  with specific filter cubes (EX/DM/EM in nm) for blue (325-375/400/435-485), green (460-495/505/510-550) and red fluorescent probes (570-590/595/600-660). Images were recorded using Cell^F software on an XM10 (Olympus) camera.th slice placed on a slide . These were stained by Cresylviolet (Sigma). After taking photos of the ventral horns at 40x (10/mouse) with a Zeiss Imager M1 microscope (Zeiss), manual counting and analyses of the size of motor neurons was done with use of AxioVision (Zeiss). Six NTG mice, 5 ES TDP-43 (A315T) mice with normal food and 5 ES TDP-43 (A315T) mice with gel food were analyzed.For counting of motor neurons, the lumbar spinal cord was cut in 20 μm thick slices, with each 6TDP-43 (A315T) mice on normal food and 4 ES mice on gel food) was done as described by Wegorzewska et al. [Staining and quantification of the dorsolateral corticospinal tract axons in the distal thoracic spinal cord  or NTG mice (n = 3 for each genotype) were collected in Tripure isolation reagent  or tissue protein extraction reagents  with Complete  for qPCR and Western blot respectively.Spinal cord and brain from presymptomatic TDP-43 (A315T) and NTG mice was determined using Cyber green. The primers were: mouse Sort1 + Ex17b: 5′-AAATCCCAGGAGACAAATGC-3′ and 5′-GAGCTGGATTCTGGGACAAG-3′; mouse GAPDH: 5′- TGGCCTTCCGTGTTCCTAC -3′ and 5′- GAGTTGCTGTTGAAGTCGCA -3′.RNA extraction was performed as described previously . qPCR asTDP-43 (A315T) or NTG mice (n = 3 for each condition) were blotted using a precast gel  and following the protocol described in Van Hoecke et al. [TDP-43 (A315T) mice and NTG mice. Alfa-tubulin  was used as a loading control. Also, the total TDP-43 expression between 90 days old TDP-43 (A315T) males and females was compared by using the polyclonal rabbit-anti-TDP43-Ab  and normalized against mouse GAPDH .For Western blot, 40 μg of spinal cord of e et al. , allowinTDP-43 A3T or NTG Data are presented as mean ± standard error of the mean, except for Figure TDP-43: TAR DNA-binding protein 43; ALS: Amyotrophic lateral sclerosis; FTLD: Frontotemporal lobar degeneration; NTG: Non-transgenic; CK: Creatine kinase; ES: Endstage; GFAP: Glial fibrillary acid protein; Prnp: Prion promoter; NOS: Nitric-oxide synthase; ChAT: Choline acethyltransferase; NMJ: Neuromuscular junctions; ENS: Enteric nervous system; Sort1 + ex17b: Sortilin 1 including exon 17b.The authors declare that they have no competing interests. All experiments on rodents were approved by the local ethical committee of the University of Leuven, Belgium.SH carried out the breeding, genotyping, perfusions, qPCRs, Western blots, motor assessment, immunoassays and histopathology of the spinal cord and NMJs of the mice, as well as the quantifications and statistical analysis of these results. SH drafted the manuscript. CC has helped substantially to acquisition, analysis and interpretation of data on intestinal assays and in drafting the manuscript. PVD and PVB participated in the design and coordination of the studies and helped to analysis and interpretation of data and in drafting the manuscript. LVDB and WR have been involved in revising the manuscript. All authors read and approved the manuscript.Pieter Vanden Berghe and Philip Van Damme share last senior authorship.Movies 1–4: Motor phenotype of symptomatic and ES TDP-43 (A315T) mice. Difference in gait abnormalities between the TDP-43 (A315T) mouse on normal food , compared to one on gel food . The swimming gait of the gel fed mouse is much more pronounced and slowly proceeds till an ES situation, as is visible in movie 4 . The mouse is not able to turn from its side anymore due to the severe neurologic impairment, whereas the TDP-43 (A315T) mouse on normal food in movie 3  is about to die due to intestinal pseudo-obstruction.Click here for file