The formation of layered nanosheets relies on exfoliation driven by rapid evaporation of water and/or other gas molecules generated under annealing. Our route allows simple, efficient and inexpensive production of 2D oxides. As a demonstration, we evaluate Cr2O3 nanosheets prepared by our method as anodes in lithium-ion batteries and find superior performance in comparison with their microcrystalline counterparts.Two-dimensional (2D) nanoscale oxides have attracted research interest owing to their electronic, magnetic optical and catalytic properties. If they could be manufactured on a large scale, 2D oxides would be attractive for applications ranging from electronics to energy conversion and storage. Herein, we report facile fabrication of oxide nanosheets by rapid thermal annealing of corresponding hydrous-chloride compounds. By heating CrCl Oxide nanosheets are of interest for a range of applications. Here the authors demonstrate a simple route combining gas evolution and exfoliation of hydrated chloride precursors for making large quantities of oxide nanosheets of various compositions. These oe sample . Typicale sample  indicatee sample . The rea3·6H2O crystals on nickel foam and heated the crystals in the flame of an alcohol lamp for 30 s. We again observed marked volume expansion . The thickness of the amorphous Cr2O3 obtained by alcohol lamp heating reached 1.9 nm . We placed CrClxpansion . Most ofxpansion  and TEM xpansion . The nanxpansion  and the xpansion . EDS anaxpansion  indicatexpansion , we posid 1.9 nm . Howeverd 1.9 nm . In addi nm , the tap density of the expanded Cr2O3 was very low, ∼0.0332, g ml−1. Using this method, we could mass-produce Cr2O3 nanosheets as shown in 2O3 nanosheets in ∼2 h in the lab using only a small muffle furnace , low average charging voltage (∼1.2 V) and low electromotive force value (1.085 V versus Li/Li+)362O3 electrodes is poor because of a loss of electronic contact of active materials and poor electron and ion transport properties within the active material2O3 nanosheets as active materials could address these limitations. We fabricated graphene/2D Cr2O3 composite electrodes. The graphene and Cr2O3 were assembled layer by layer because of their similar 2D layered structure. The stacked 2D structure can provide good electrical contact between the graphene and Cr2O3 nanosheets, improving electron transport. The ultrathin, 2D layered structure can help access to the active material by the electrolyte and aid ion transport.Among TMOs, Cr2O3 (2D) electrodes and graphene/Cr2O3 (particle)/polyvinylidene difluoride (PVDF) electrodes, as mentioned in the Methods section. The cells were tested over a voltage range from 0 to 3 V versus Li+/Li. During the test, the initial lithiation and delithiation capacity of the graphene/Cr2O3 (particle)/PVDF electrodes was only ∼738 and 316 mAh g−1, respectively, and the delithiation capacity decreased to only 168 mAh g−1 after ∼10 cycles at the current density of 0.2 A g−1 (2O3 (2D) electrodes exhibited a high capacity and stable cycling performance. The graphene/Cr2O3 (2D) electrodes achieved an initial delithiation capacity of 974 mAh g−1 at a rate of 0.2 A g−1. After 297 cycles, the capacity was ∼986 mAh g−1, and the cell continued to function well (2O3 (2D) electrodes is excellent compared with the reported performance of other Cr2O3 electrodes383940412O3 electrodes are included in 432O3 (particle)/PVDF electrode were 12.3 and 56.9 Ω, respectively. However, the graphene/Cr2O3 (2D) composites showed lower SEI and charge transfer resistances, which were 6.6 and 21.4 Ω. The electrochemical impedance spectroscopy results indicated the interface of graphene/Cr2O3 (2D) electrodes is more stable and confirmed the 2D structure is attributed to the charge transfer and charge diffusion, which could support the good electrochemical and rate performances of graphene/Cr2O3 (2D) electrodes. Importantly, the graphene/Cr2O3 (2D) composites exhibited strong adhesion to the current collector (copper foil) and between the layers. The graphene/Cr2O3 (2D) electrodes and graphene/Cr2O3 (particle)/PVDF electrodes were immersed in the electrolyte solution; we then sonicated the electrodes in the electrolyte solution with ultrasonic cleaning  for 5 min. For the graphene/Cr2O3 (particle)/PVDF electrodes, the PVDF binder did not prevent the spalling of the electrode material from the current collector during the ultrasonic cleaning process (2O3 (2D) composite materials remained on the current collector (2O3 (2D) electrodes maintained structural stability in all three solutions, whereas the graphene/Cr2O3 (particle)/PVDF electrodes exhibited different degrees of spalling (2O3 (particle)/PVDF electrode was easily peeled off, the graphene/Cr2O3 (2D) electrodes remained intact and no stripping happened  electrodes. We suggest the strong adhesion has three potential causes. First, the 2D structure of Cr2O3 provides a high contact area with graphene and copper foil, and the calendaring process ensures good contact between Cr2O3 (2D), graphene and copper foil. Second, according to zeta potential measurements under neutral conditions, graphene was negatively charged (−9.26 mV) and the 2D Cr2O3 was positively charged (32.5 mV), suggesting that electrostatic attraction contributes to the adhesion between 2D Cr2O3 and graphene. Third, and finally, we speculate that some chemical bonding may occur at the interface between the electrode material and current collector, between amorphous Cr2O3 nanosheets and the copper foil due to the high-valence Cr and low-coordinated surface atoms, leading to robust adhesion to the copper foil45We fabricated graphene/Cr.2 A g−1 . By contion well . This el process . Howeverollector . We alsospalling . We usedhappened . The obs3·6H2O, we applied the rapid heating method to other hydrous chlorides to obtain metal oxides such as ZrO2, Al2O3 and Y2O3. The ZrOCl2·8H2O, AlCl3·6H2O and YCl3·6H2O crystals were not layered nanosheets before the rapid heating treatment  in water and painted the solution onto copper foil. The copper foil was then dried in an oven at 60 °C for ∼30 min to obtain a hydrous AlCl3 thin film. We heated the hydrous AlCl3 on the copper foil using the heating gun (∼500 °C) until the light-yellow solid appeared. We observed that the solid formed on the copper foil was exfoliated Al2O3 , but the crystallinity was poor. We measured the thickness of the metal oxide nanosheets using AFM. The thinnest layers of the ZrO2, Al2O3 and Y2O3 obtained by rapid heating methods exhibited layer thicknesses of 3.5, 2.7 and 4.0 nm, respectively  or without layered crystal structure (such as AlNO3·9H2O) were also treated by the rapid heating process, however the 2D oxides were not obtained in these cases  by rapidly heating hydrous chlorides; the 2D Cr2O3 showed excellent electrochemical performance in Li-ion battery and surprisingly strong adhesion to the copper foil substrate. We also demonstrated that this method of producing ultrathin nanosheets could be generalized to allow rapid production of other oxides such as ZrO2, Al2O3 and Y2O3. We believe that this concept provides a practically promising avenue for simple, efficient, fast and inexpensive production of large quantities of large-area, ultrathin 2D nanosheets.We produced the 2D Cr3·6H2O crystals were placed in a glass bottle and heated using a microwave oven for ∼5 min. Then, the obtained CrOCl materials were transferred and stored in an Ar-filled glove box. The CrOCl nanosheets were dispersed in benzene to prepare TEM samples.The CrCl3·6H2O crystals were placed on nickel foam and heated using an alcohol lamp for ∼2 min; or placed in a quartz crucible and heated in a muffle furnace at 400 °C for ∼15 min. The ZrOCl2·8H2O crystals were placed in a glass bottle and heated using a microwave oven for ∼10 min. The AlCl3·6H2O crystals were dissolved in deionized water at a concentration of 1 g ml−1, and the solution was painted onto copper foil. The copper foil was dried in an oven at 60 °C for 30 min to obtain the hydrous AlCl3 thin film. We heated the hydrous AlCl3 on the copper foil using a heating gun until a white or light-yellow solid appeared. The white or light-yellow solid was Al2O3 nanosheets. The YCl3·6H2O crystals were placed in an alumina crucible and heated in the rapid heat treatment furnace at 600 °C for ∼2 min. The obtained metal oxide products contain incompletely reacted intermediate products. We dispersed the obtained products in deionized water to remove the soluble impurities. The dispersion of products was centrifuged for 5 min at 12,000 r.p.m. to obtain the sediment. We repeated the centrifuging process three times to clean the products . The obtained sediment was dried in an oven at 80 °C.The CrCl2O3 nanosheets (90 wt%) in water at a concentration of 10 mg ml−1 . We also dispersed graphene (10 wt%), Cr2O3 particles  and PVDF (10 wt%) in N-methyl-2-pyrrolidone (NMP) at a concentration of 10 mg ml−1 . The composite slurry was dropped onto a flat copper foil and dried in an oven at ∼80 °C. The dried electrodes were pressed using a calendaring process (∼20 MPa). The mass loading of the electrodes was ∼0.5 mg cm−2. The electrodes were assembled into half-cells with Li metal foil (MTI) as a counter electrode in an Ar-filled glove box. We used a 25 μm-thick microporous polypropylene membrane as the separator (Asahi Kasei) and 1 M LiPF6 in ethylene carbonate/diethyl carbonate/fluoroethylene carbonate  as the electrolyte.We dispersed graphene (10 wt%) and Crα radiation source. The chemical states of the products were determined by XPS (Thermo Fisher ESCALAB 250Xi). The morphology of the samples was observed with a SEM . The chemical composition of the samples was analysed using EDS . HRTEM observation was carried out using JEOL-2100 TEM operated at 200 kV. The thickness of the nanosheets was determined by AFM . The electrochemical performances of the batteries were measured by a BS-9300R/10V2A MTI 8 channels battery analyser.The X-ray diffraction patterns of the rare materials and products were evaluated using a D/max-2500 diffractometer  equipped with a CuKThe data that support the findings of this study are available from the corresponding author on request.How to cite this article: Zhao, C. et al. Mass production of two-dimensional oxides by rapid heating of hydrous chlorides. Nat. Commun. 7:12543 doi: 10.1038/ncomms12543 (2016).Supplementary Figures 1-183˙6H2O starting material undergoes large volume expansion upon microwave heating.CrCl3˙6H2O crystals directly placed into a furnace at 400 C and heated for 5 minutes to obtain porous 2D Cr2O3 nanomaterials.CrCl2O3 nanosheets and copper foil substrate.Tape test showing strong adhesion between Cr


We propose a nonvolatile terahertz (THz) switch which is able to perform the switching with transient stimulus. The device utilizes graphene as its floating-gate layer, which changes the transmissivity of THz signal by trapping the tunneling charges. The conventional top-down electrode configuration is replaced by a left-right electrode configuration, so THz signals could transmit through this device with the transmissivity being controlled by voltage pulses. The two electrodes are made of metals with different work functions. The resultant asymmetrical energy band structure ensures that both electrical programming and erasing are viable. With the aid of localized surface plasmon resonances in graphene ribbon arrays, the modulation depth is 89% provided that the Femi level of graphene is tuned between 0 and 0.2 eV by proper voltage pulses. Various approaches, including those based on active metamaterials5 and two-dimensional materials8, have been investigated over the past decade to manipulate the propagation of electromagnetic waves from RF to THz frequencies. However, most of them are volatile devices, as they return to original states once external stimuluses are removed. Nonvolatile photonic devices, which can sustain the transformed properties with short triggering stimuluses rather than persistent signals, hold an apparent advantage of saving the energy in the advent of photonic technologies. More importantly, the persistent photonic memory effect of these devices lend themselves to a variety of applications including protective optical/terahertz circuitry, adaptable transformation-optics devices, dynamically reconfigurable optical/THz networks or metamaterials, electrically controllable photonic memory15, and so on. Furthermore, most planar lightwave circuits, especially those built on the silicon-on-insulator substrate that have high refractive index contrasts and strong optical confinements, are sensitive to fabrication tolerance. Therefore, many properties of practical photonic circuits such as the resonance wavelength would inevitably drift from their design values. Nonvolatile devices then can be incorporated into these photonics circuits as a post-trimming technique to compensate permanent errors.The terahertz technology has gained increased research attention due to its industrial and academic applications16, phase-change materials13, organic materials17, ferromagnetic materials18, have been well developed for data storage, few of them turn out to be a feasible solution to implement the nonvolatile THz switching. The reason is that most of those devices contain functional materials or top-gate electrodes that are nontransparent at THz frequencies. For example, the floating-gate (FG) shown in Fig. 20 graphene is employed as the FG layer to store charges, which exhibits improved performance in terms of endurance, writing speed and operation voltage. However, THz signals cannot propagate through this configuration, since it demands a metallic control gate on the top, which is highly lossy at THz frequencies. Although metamaterial structures12, for example, a periodic hole array patterned on an Au film as shown in Fig. Although a lot of nonvolatile devices, e.g., those based on floating gate structures2), electrons from the drain electrode can tunnel through the HfO2 layer and be stored in the FG (graphene) layer under a negative drain voltage. Meanwhile, since the tunneling barrier under the source electrode between graphene and HfO2 is higher than that between the drain electrode and HfO2, electrons that tunnel out to the source electrode from the FG can be neglected. Therefore, electrons can accumulate in the FG. Similarly, under a positive drain voltage, the electrons stored in the FG can tunnel back to the drain, while electrons that tunnel into the FG from the source is negligible. Electrons therefore can be erased from the FG also due to the unequal tunneling barrier heights at the two electrode terminals. The nonvolatile switching is finally realized by taking advantage of different tunneling barriers at the two terminals.In this paper, we theoretically propose a nonvolatile THz switch by employing the graphene as the floating gate layer. The extraordinary optoelectronic property of graphene allows stored carriers to significantly tune the transmissivity of the THz signal. The conventional control-gate layer that covers the full device is replaced by source and drain electrodes made of different metals at the two sides as shown in Fig. 22 or plasmonic waveguides25. We note that active graphene layers in many of these modulators can be readily replaced by the graphene-based floating-gate in Fig. Although the proof-of-principle design of the THz nonvolatile switch here is based on free-space optics with the THz wave normally incident on the graphene layer, this scheme is also well adapted to various waveguide-based switches operating at THz and optical wavelengths. A lot of graphene-based photonic modulators have been reported on either dielectricψg = 4.6 eV19) vertically stacking on a 300-nm-thick SiO2 layer works as the floating-gate layer. Another 4.5-nm-thick HfO2 layer (ψHfO2 = 2.5 eV26) acts as the tunneling/blocking layer. The drain and source electrodes on top are made of aluminum (ψAl = 4.08 eV) and gold (ψAu = 5.1 eV27), respectively. To reveal the operation principle, the finite elements methods (Comsol Multiphysics) is utilized to model the electrostatic potential distribution in this device. The distance between the two electrodes is set to be 50 nm to better display the vertical configuration. It should be noted that the width of this transmission window for THz beams is on the scale of centimeters in practical devices. However, the electrostatic potential distribution in practical devices is similar to that presented in Fig. 20. Therefore, the potential difference from drain to graphene almost equals to that from graphene to source as shown in Fig. A schematic diagram of the nonvolatile switch is shown in Fig. 28, which is far less than the distance between the two terminals of our device. Therefore, the band bending is neglected to simplify the band diagrams. Resultant directions of tunneling currents and band diagrams at different operation states are shown in Fig. VD < 0) is applied on the drain. As already analyzed in Fig. 2 (ϕB1 = ψAl − ψHfO2 = 1.58 eV) is lower than that between graphene and HfO2 (ϕB2 = ψg − ψHfO2 = 2.1 eV). Since the tunneling probability strongly depends on the barrier height, the tunneling current cross the right barrier is greater than that cross the left barrier (J1 ≫ J2), and then electrons accumulate in the FG layer. In the retention mode  in the FG layer, potential and Femi level of graphene rise by ~0.08 V and ~0.2 eV, respectively. Tunneling currents from the FG layer to the source and drain is negligible as a result of the weak electrical field in the blocking layer. Therefore, electrons could be stored in the FG layer for a long period before completely leaking out. In the erasing mode of Fig. J3 ≫ J4 as shown in Fig. To explain the necessity of using different metals for source and drain, we at first examine the case of both electrodes being made of the same metal, for example, Aluminum (Al). It is known that in a graphene MOS structure, charges will accumulate in graphene provided that work functions of graphene and metal are different. This localized metal-induced doping effect leads to internal built-in electrical fields inside the graphene, and hence bend its energy band. However, these fields only exist in narrow regions ~0.2 μm) adjacent to drain/graphene and source/graphene interfaces μm adjacode Fig. , the volϕB3 > ϕB4) as shown in Fig. J3 is accordingly insignificant as compared with J4. Therefore, the stored electrons could be erased by a positive voltage due to the asymmetric-electrode configuration.To solve this issue, the Al source is replaced by a Gold (Au) source. The different work functions of Al and Au lead to an asymmetrical energy band structure as shown in Fig. 30. However, a strict calculation based on this approach is much more cumbersome than that based on the Fowler-Nordheim (FN) theory31. The FN theory is initially built on the free-electron theory of metals and leads to a very simple relationship between the density of emission current and the applied electric field. Strictly, the FN model applies only to bulk systems, e.g., metal/insulator/crystalline solid. However, FN equations are also frequently used as an approximation to describe low dimensional systems, including nanocrystals33 and two-dimension materials35. For example, the FN model captures measured tunneling currents in a graphene/insulator/metal system quite well with proper modifying factors in ref.35. Based on these considerations, we employ the FN theory without considering the linear energy-momentum relation of graphene. This greatly simplifies the calculation with acceptable accuracy. Regarding the applicability of the FN model to the graphene/insulator/metal interfaces in our device, we discuss how the density of states of graphene affects the tunneling current density in the Supplementary Information. However, a rigorous treatment of the tunneling current is still suggested for our future work.A quantitative calculation is provided below. Considering the two-dimensional character and band structure peculiarities of graphene, a rigorous treatment should resort to the Bardeen transfer Hamiltonian approach, as it has been utilized to explain tunneling currents in relevant two-dimensional systemsED > qϕB1/d). It can be used to calculate tunneling currents in programming/erasing modes, but is not suitable for the retention mode (VD ≈ 0) of very weak electrical field. Generalized formulas are derived by Simmons which enable the calculation of tunneling currents for several different voltage ranges36. According to Simmons’ formulas, tunneling current densities J at programming/erasing and retention modes can be calculated asA and C are: A1 = q2(2 m*)1/2/(4d π2ℏ2) and C1 = 2d(2 m*)1/2/ A2 = q3/(16π2ħϕB) and C2 = 4(2 m*)1/2(ϕB)3/2/3ħq, here m* denotes the effective mass of the electron, ϕB is the barrier height and q is the electron charge. The electrical field between source and FG (ED) and that between drain and FG (ES) are determined by the potential of the floating gate layer VFG, which depends on the amount of stored electrons QFGVS, VD, and VG are the potentials of source, drain and ground, respectively. As shown in Fig. CS, CD, and CG represent the capacitances (per unit area) between graphene/source, graphene/drain, and graphene/ground, respectively; and CT is their sum (CT = CS + CD + CG)37.The conventional FN formula of tunneling current describe the field-assisted emission of electrons under a very high electrical field  of graphene. A part of the applied voltage drops in the graphene layer itself, and this effect can be captured in the quantum capacitance Cq of graphene, which can be calculated as38EF = 0, the quantum capacitance reaches its minimum value of CGox = 11.5 nF/cm2, which is far less than the quantum capacitance. Hence, the influence of the quantum capacitance on CG can be neglected. On the other hand, as the thickness of the HfO2 layer is very thin, its capacitance is comparable with the quantum capacitance. Therefore, both CS and CD should be considered as series connections of their respective oxide capacitances and quantum capacitances: 1/CS = 1/CSox + 1/CSq and 1/CD = 1/CDox + 1/CDq.According to the MOS theory, W1/W2 is the width ratio of the electrode and the FG layer as shown in Fig. VG can be neglected due to the fact that CG is far less than CS and CD.The stored-charge density in the FG layer is related to the tunneling currents across the two barriers. It can be calculated asentclass1pt{minima41. If these effects need to be reflected, VD should be replaced by corresponding effective voltages: VD_eff = VD − VD0. Here VD0 is drain voltage at the Dirac point without external voltages, whose role is very similar to that of the flat-band voltages in conventional MOS transistors. Hence electric fields ED and ES can be approximately written as, ε0, εr are the vacuum permittivity and relative permittivity of HfO2, respectively, d is the thickness of the tunneling layer.The discussion above has not considered the influences of different work functions between metal and graphene, and the resultant inevitable doping of grapheneJ1, J2) and erasing  modes. Parameters in the calculation are: εr = 1742, W1 = 1 mm, W2 = 1 cm, d = 4.5 nm, VD = −5.5 V and +6 V in programming and erasing modes. In Fig. J1 ≫ J2) in the programming mode. However, in the erasing mode the electron density in the FG layer keeps on growing as J4 ≪ J3, and therefore they cannot be erased. In Fig. With the aid of equations –5, we cad of the HfO2 layer. We calculate time responses of tunneling current densities in programming mode, electrical field strength between drain and graphene, density of stored charge, i.e., J1, ED, and QFG, in Fig. d as a variable. The drain voltage in the calculation is −5.5 V. It can be seen that reducing the thickness d could enhance the electrical ED and consequently the amplitude of tunneling current density J1, which implies a reduced programming time for storing the same amount of electrons in graphene. However, reducing the thickness d would deteriorate the retention time, and even the programming process forbidden. As shown in Fig. J2 becomes equal to J1 at t = 0.5 μs for d = 3.5 nm. Therefore, a very thin tunneling layer is not advisable. Based on the trade-off between the programming speed and the retention time, a 4.5-nm-thick HfO2 is chosen as the tunneling layer.A critical parameter which decides the device performance is the thickness 2 (17 MV/cm in42) and the stability of device. The drain voltages in the programming and erasing modes are finally set as VD = −5.5 V and +6 V, respectively. The absolute amplitude of VD in the erasing mode is higher and operating time is longer than those in programming mode. This is due to the fact that tunneling electrons experience a higher barrier height in the erasing mode as shown in Fig. −10 A/cm2 provided that the charge density in graphene layer is ~3 × 1012/cm2. This order of magnitude is in accordance with the calculations in refs44, and implies a very long retention time in theory. However, since insulating abilities of practical thin dielectric layers cannot be ideal, leakage currents in realistic nonvolatile devices are usually much larger than this theoretical estimation. Here RC time constant is not considered in calculation, since it is usually on the order of picoseconds (ps)7.In Fig. QFG) change the transmission of THz signal by varying its optical conductivity (σg(ω)). Since charges can be stored for a long period before an opposite erasing voltage comes, the device hence acts as a nonvolatile THz switch. Optical absorption in graphene includes interband and intraband transitions. However, in THz range the intraband transition dominates, so its optical conductivity can be described by a Drude-like dispersion formula45νF = 106 m/s is the Fermi velocity of Dirac fermions. The transmissivity of THz signal can be calculated as46ε1 and ε2 are the permittivities of covering layer and substrate, respectively.In this part, we study the switching characteristic and the dynamic response of this nonvolatile device. The charges stored in graphene (11/cm2. EF of graphene then rises by 0.1 eV. The modulation depth (MD), defined as |T(ns) − T(n0)|/T(n0), is calculated to be 9.5% at 5 THz according to equation (12/cm2) are stored, and thus the MD is enhanced to 20%. By further prolonging the programming and erasing time , QFG can reach to (~7.4 × 1012/cm2), meanwhile, its MD is increased to 27%.According to the analysis above, the amplitudes of the programming/erasing (negative/positive) pulses are chosen to be −5.5 V and +6 V. Waveforms of the programming/erasing pulses applied on the drain and the corresponding responses of the device are shown in Fig. QFG) relies on the duration time of the programming/erasing pulses as long as amplitudes of driving pulses are fixed. Enhancing the MD by using long pulses is in contradiction with improving the operation speed. In order to overcome this tradeoff, we can take advantage of graphene-based plasmons. As collective oscillations of charge carriers, plasmons can occur in patterned graphene47. For example, the graphene-ribbon array shown in Fig. 52. The simulation result shows that this structure helps to enhance the MD remarkably. The modeled resonance curves are presented in Fig. EF from 0 eV to 0.1 eV and to 0.2 eV, respectively. The power consumption is proportional to the total stored charges and hence the area of the floating-gate layer, the corresponding power consumptions per unit area are 0.0068 J/cm2 and 0.0085 J/cm2 to tune the Femi level from 0 to 0.1 eV (programming mode) and from 0.1 eV back to 0 eV (erasing mode), respectively. These values become 0.026 J/cm2 and 0.033 J/cm2 for tuning Femi level from 0 to 0.2 eV and from 0.2 to 0 eV, respectively. The simulation in Fig. 2 substrate according to data in52. It is observed that both impurities and phonons affect scattering rates of practical graphene layers55, so the value of scattering time is not fixed but depends on the particular processing condition. To investigate the impact of the scattering time, we also carry out the simulation in Fig. 51. It can be seen that a high-quality graphene layer of long scattering time is favorable to realize a narrow resonance curve with high resonance depth, since the conductivity of graphene is proportional to iτ−2.In accordance with Figs EF of graphene between 0 and 0.1/0.2 eV with voltage pulses of appropriate amplitude and duration. Moreover, the MD can be further improved to 89% by patterning the graphene layer into a ribbon array. The performance of this configuration can be further improved by different manners. For example, since charged impurities in oxides introduce disorders of graphene, h-BN can be used to replace HfO2 so as to increase the quality of graphene56; using multi-layer graphene instead of graphene monolayer is able to enhance the effective refraction index variation19, and so on.In conclusion, we propose a graphene-based nonvolatile THz intensity switch with asymmetric electrodes. If a uniform graphene layer is used as the floating gate layer, the modulation depths are 9.5%/20% by nonvolatile tuning 9. The width of electrodes (drain and source) is W1 = 1 mm; and that of the floating-gate layer is W2 = 1 cm. Since W1 and W2 are several orders of magnitude larger than the thickness of graphene, they are scaled down to 5 nm and 50 nm in the simulation for a better eye-capture.The numerical simulation of electrostatic potential distribution and the corresponding electrical distribution as shown in Fig. 41; the results are shown in Figs VD_eff is calculated via the equations in refs41. The voltage at the Dirac point is normally related to work function difference between metal and graphene as well as interface trap states. In this study we assume the graphene is ideal, so only the effect of different work functions is considered. The transmissivities through the monolayer graphene in Fig. The tunneling processes of electrons in programing/erasing modes are calculated by Matlab based on the model in refsy direction. A plane-wave source is normally incident (z-direction) on graphene. The periodic boundary condition is used along the y direction, while the anti-symmetric boundary condition is used along the x direction. The perfectly matched layer (PML) is set at the z-direction. The graphene material type (temperature of 300 K) together with the 2D rectangle geometry described in modeling methodology are employed to model the graphene layer following the surface conductivity approach. The grid size for graphene layer is Δx = Δy = 2.5 nm and Δz = 0.25 nm.The numerical simulation of the THz transmissivity through graphene ribbons is performed with the 3-D finite-different time domain method . The result is shown in Fig. Supplementary Information


TDP-43 pathology was investigated in the anterior temporal pole cortex (ATPC) and orbital frontal cortex (OFC), regions often degenerated in frontotemporal lobar degenerations (FTLD), in aging and Alzheimer’s disease (AD). Diagnosis of dementia in the 1160 autopsied participants from 3 studies of community-dwelling elders was based on clinical evaluation and cognitive performance tests which were used to create summary measures of the five cognitive domains. Neuronal and glial TDP-43 cytoplasmic inclusions were quantitated in 8 brain regions by immunohistochemistry, and used in ANOVA and regression analyses. TDP-43 pathology was present in 547 (49.4%) participants in whom ATPC (41.9%) was the most frequently involved neocortical region and in 15.5% of these cases, ATPC was the only neocortical area with TDP-43 pathology suggesting not only that ATPC is involved early by TDP-43 but that ATPC may represent an intermediate stage between mesial temporal lobe involvement by TDP-43 and the last stage with involvement of other neocortical areas. To better study this intermediary neocortical stage, and to integrate with other staging schemes, our previous 3 stage distribution of TDP-43 pathology was revised to a 5 stage distribution scheme with stage 1 showing involvement of the amygdala only; stage 2 showed extension to hippocampus and/or entorhinal cortex; stage 3 showed extension to the ATPC; stage 4 – showed extension to the midtemporal cortex and/or OFC and finally in stage 5, there was extension to the midfrontal cortex. Clinically, cases in stages 2 to 5 had impaired episodic memory, however, stage 3 was distinct from stage 2 since stage 3 cases had significantly increased odds of dementia. The proportion of cases with hippocampal sclerosis increased progressively across the stages with stage 5 showing the largest proportion of hippocampal sclerosis cases. Stage 5 cases differed from other stages by having impairment of semantic memory and perceptual speed, in addition to episodic memory impairment. These data suggest that of the regions studied, TDP-43 pathology in the ATPC is an important early neocortical stage of TDP-43 progression in aging and AD while extension of TDP-43 pathology to the midfrontal cortex is a late stage associated with more severe and global cognitive impairment. The transactive response DNA-binding protein 43 (TDP-43) was first localized in brains of cases with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) , 20. SubFew studies have investigated the regional distribution of TDP-43 pathology in the brain. In the behavioral variant FTLD, 4 patterns of TDP-43 distribution were reported  with earTDP-43 pathology is considered central to the pathogenesis of FTLD/ALS whereas its role in other neurodegenerative diseases is less clear. Given the propensity for TDP-43 pathology to affect the anterior and/or orbital frontal cortex (OFC) and temporal lobe structures with prominent neurodegeneration in FTLD-TDP, the hypothesis that TDP-43 pathology may also preferentially involve the anterior temporal pole cortex (ATPC) and OFC in aging and AD was tested. ATPC is the most rostral neocortical area of the superior and middle temporal gyri and is highly interconnected with the amygdala and the OFC.Our results show that the ATPC, but not the OFC, appears to be a prominent and early neocortical site of involvement in TDP-43 pathology associated with aging and AD and that this stage is related to dementia. To better study this early neocortical stage, and to integrate with other staging schemes, we propose a new 5 stage system of TDP-43 distribution that includes TDP-43 in ATPC. The association of all 5 stages of TDP-43 pathology with dementia, memory, and other cognitive domains was studied in participants of 3 longitudinal studies of aging and dementia: the Rush Memory and Aging Project (MAP), the Religious Orders Study (ROS) and the Minority Aging Research Project (MARS).n = 1160) were from 3 longitudinal clinical-pathologic cohort studies of aging and dementia, Rush MAP (n = 636), ROS (n = 501) and MARS (n = 23), each approved by the Institutional Review Board of Rush University Medical Center. All data collections (antemortem and postmortem) were similar in these studies allowing combined analyses of the cohorts. A signed, informed consent was obtained from each participant for an annual clinical evaluation and for brain donation. Fifteen cases having a pathologic diagnosis of FTLD in accordance with previous FTLD classifications ) Fig. . In analThe National Institute on Aging-Reagan criteria  were useThe age, volume and anatomic location of all macroscopic infarcts and the age and location of microscopic infarcts were documented. Only chronic macro and microinfarcts were included in the analyses as dichotomous variables.Atherosclerosis was assessed in basal cerebral arteries while arteriolosclerosis was assessed in the basal ganglia and both vessel pathologies were graded using a semiquantitative scale from 0 (none) to 6 (severe) as described previously . CerebraThese were assessed in 6 regions  as described previously  and recoWhere pertinent, immunohistochemistry was done for anti-phospho-PHF tau pSer202/Thr205  and ‘fused in sarcoma’ (FUS) protein  to exclude FTLD cases. All immunohistochemistry was done using a Leica-Bond Max autostainer . Antigen retrieval was done using heat-induced epitope retrieval (HIER) solution 1 (citrate-base) (Leica Microsystems) for 10 min for α-synuclein, and 30 min for FUS and HIER solution 2 (EDTA-base) (Leica Microsystems) for 20 min for phosphorylated TDP-43. Sections were pretreated with formic acid prior to immunostaining for β-amyloid. In case of α-synuclein the Bond polymer alkaline phosphatase red detection kit was used while for the other antibodies the Bond Polymer Refine Detection Kit was used which produced a brown reaction product.2 or the analysis of variance (ANOVA). Age, education, MMSE and cognitive domains were further compared by post hoc pairwise comparisons between TDP-43 stages 2 and 3, stages 3 and 4 and stages 4 and 5 cases with application of a Bonferroni Correction (α = 0.05/3) to adjust for multiple testing.Demographics, clinical characteristics and age-related pathologies including macro and microinfarcts, HS, LBs and AD pathologies and vascular pathologies , were compared between subjects without and those in the five TDP-43 stages using χp < 0.05 was used for statistical significance throughout except for the post hoc pairwise comparisons mentioned above.Multivariable linear regression analyses were used to determine the association of the 5 TDP-43 pathology stages with the outcome measures of episodic, semantic, and working memory, perceptual speed and visuospatial skills. Multivariable logistic regression analyses evaluated the association of the five stages of TDP-43 pathology with dementia as a binary outcome. In both multivariable linear and logistic regression analyses, Stage 0 (no TDP-43 pathology) was used as the reference group and all models controlled for age, sex, education and the age-related pathologies listed above. All analyses were carried out using SAS software, . Model assumptions were examined graphically and analytically and were adequately met. A nominal threshold of TDP-43 neuronal and glial inclusions were present in 547 of 1108 (49.4%) participants. TDP-43 cytoplasmic inclusions in entorhinal and neocortical regions tended to be more frequent in the second layer than in deeper cortical layers. Most inclusions were compact Fig.  while grIn the TDP-43 positive cases, the most frequent neocortical area showing TDP-43 inclusions was the ATPC 41.9%) followed by the midtemporal cortex, the OFC and inclusions were least common in the midfrontal cortex (Table .9% follon = 547), showed inclusions in the amygdala and in 206 of the 547 cases (37.7%), the inclusions were confined to the amygdala (stage 1) , a combination of HS and LB disease (n = 4) and 1 case had a chronic macroinfarct in the caudate. Phospho-PHF-tau and FUS immunostaining done in these cases was negative excluding a diagnosis of FTLD.All participants having TDP-43 pathology  1) Fig. , Table 1 Fig. , T18 of the 1160 (1.5%) cases failed to show the proposed regional progression of TDP-43 pathology due to one skipped stage despite examination of an additional section of the regions without identified TDP-43 pathology. Of the 18 cases, eight showed no TDP-43 pathology in the amygdala which showed no degenerative changes on microscopy. In 5 cases the hippocampus/entorhinal cortex was skipped, in another 3 cases the ATPC was skipped and in 2 cases the midtemporal cortex was skipped. The clinical diagnoses of these 18 cases were probable AD (6 cases), mild cognitive impairment (7 cases) and no cognitive impairment (5 cases). Fourteen of the 18 cases met criteria for pathologic AD while the remaining 4 cases did not, however, numerous macroinfarcts/microinfarcts with significant tissue loss was present in these cases. These18 cases were not included in the statistical analyses.Overall, the frequency of a pathologic diagnosis of AD was higher in those having TDP-43 pathology (73.5%) as compared to those negative for TDP-43 56.2%). The proportion of cases with a pathologic diagnosis of AD increased across the TDP-43 stages to 80% in Stage 3 and 85% in each of stages 4 or 5 (Table %. The prp < 0.001). Post hoc pairwise comparisons between the age of stage 2 and 3 cases, stage 3 and 4 cases and stage 4 and 5 cases showed no statistical difference . Frequency of females was slightly higher in those having TDP-43 pathology while education did not differ by TDP-43 status. Of the cases having no TDP-43 pathology, 67% had no dementia and the percentage of participants with no dementia decreased across the TDP-43 stages with only 15% showing no dementia in stage 5  MMSE scores in the stage 5 cases. Of the cases without TDP-43 pathology, one-third had dementia. The percentage frequency of dementia increased across the TDP-43 stages with 65% of stage 3, and 85% of stage 5 cases having dementia.The mean MMSE score, proximate to death was 22.8 (SD 8.2) in the group without TDP-43 pathology while the mean MMSE score was significantly lower in those having TDP-43 pathology being 18.5 (SD 9.8). The MMSE scores were progressively lower across TDP-43 stages 2 to 5 (Table p < 0.001), 4  and 5 cases  as compared to those without TDP-43 pathology . In those with TDP-43 pathology impairment of specific cognitive domains was varied by the TDP-43 stage (Table p = 0.003) in the stage 4 cases while impairment of semantic memory was greater (p = 0.002) in stage 5 cases as compared to stage 4 cases.Overall impairment of cognitive domains was greater in those with TDP-43 pathology as compared with those without TDP-43 pathology  and the later stages with more widespread neocortical TDP-43 distribution and that involvement of the ATPC is an important pathologic marker of the transition to dementia. While episodic memory is impaired in stages 2-5, significantly lower MMSE scores, impairment of semantic memory and perceptual speed only occurs when TDP-43 protein involves the midfrontal cortex.Although the ATPC is known to be degenerated in FTLD , 9, and Eighteen of the 1160 cases (1.5%) failed to show the proposed regional progression of TDP-43 pathology due to one skipped stage. Since additional sections of these skipped regions were examined and significant neuronal loss was not detected, lack of TDP-43 pathology in these regions was ascribed to the biological variation of TDP-43 distribution. Skipped regions were also reported in other studies of TDP-43 staging , 17.TDP-43 pathology in the ATPC was associated with a higher percentage of cases with pathologic AD and slightly higher percentage of cases with HS pathology compared to TDP-43 involvement limited to the hippocampus or entorhinal cortex (stage 2). These observations support the ATPC being an intermediate pathologic stage of age-related TDP-43 proteinopathy. The observation that the frequency of AD was significantly higher in cases as the TDP-43 stage increases beyond the amygdala was reported previously , 14. AlsTDP-43 pathology stages 3 through 5 were associated with higher odds of dementia which was independent of coexisting pathologic diagnoses of AD, LB disease or HS, suggesting that the progression of TDP-43 from the mesial temporal lobe to the ATPC represents an important transition and likely marks the onset of more severe functional changes.Impaired episodic memory, a significant finding in AD was present across all the TDP-43 stages except stage 1. The independent association of TDP-43 pathology with impaired episodic memory was also observed in our previous studies of mixed AD and non-AD cases , 26 and Strengths of this study include detailed data on multiple neuropathologies on a large number of participants obtained in a blinded manner and the availability of detailed clinical data on diagnosis and neuropsychological testing of these participants performed proximate to death. These studies also have high follow-up and autopsy rates that provide internal validity of findings.A perceived limitation could be the lack of TDP-43 data from the basal ganglia and brainstem. Although, the staging presented in this study is contracted as compared to a previous study of TDP-43 staging in AD , the higPathological and clinical data justified expansion of our previous 3 stage TDP-43 pathology distribution scheme to a 5 stage distribution scheme. Of the neocortical areas studied, ATPC was the earliest neocortical region showing TDP-43 pathology therefore TDP-43 pathology involves the temporal cortex earlier than the frontal cortex. Cases with TDP-43 in the ATPC had significantly increased odds of dementia justifying separation of ATPC to a distinct stage. Impairment of episodic memory was present in stages 2 to 5 but impairment of semantic memory and perceptual speed was only observed when TDP-43 pathology spread from the temporal cortex to involve the midfrontal cortex.


PD-L1 expression in de-novo and recurrent glioblastoma multiforme and to explore associated genetic alterations and clinical traits. We show that PD-L1 expression was reduced in recurrent GBM in comparison to de-novo GBM. Additionally, patients who received an extended dose of temozolomide (TMZ) chemotherapy showed a significantly reduced level of PD-L1 expression in the recurrence stage compared to the corresponding de-novo tumour. Our findings may provide an explanation for potentially lower response to immunotherapy in the recurrent stage due to the reduced expression of the therapeutic target PD-L1.The biology of recurrent glioblastoma multiforme (GBM) is a dynamic process influenced by selection pressure induced by different antitumoural therapies. The poor clinical outcome of tumours in the recurrent stage necessitates the development of effective therapeutic strategies. Checkpoint-inhibition (PD1/PD-L1 Inhibition) is a hallmark of immunotherapy being investigated in ongoing clinical trials. The purpose of this study was to analyse the  Glioblastoma multiforme (GBM) is the most common type of malignant brain tumour, which is characterised by poor clinical outcome and short survival time, rarely longer than 14 months , 3. UntiPD-L1) on the tumour cell surface or the programmed death-ligand protein 1 (PD-1) receptor on the T-Cell in attempt to support antitumoural immune response.Recently, a novel class of immunotherapies - the immune checkpoint inhibitors - have successfully influenced the treatment of a large variety of different solid cancer types . These iRecurrent GBM show an increased number of genetic alterations, which are potentially induced by radio- and chemotherapy  and may The purpose of this study was to analyse PD-L1 expression on mRNA and protein-level in patients with de-novo and recurrent glioblastoma multiforme. First, we investigated the PD-L1 expression in GBM at different stages (64 cases) (de-novo tumour (n=64), first (n=38), second (n=18) and third recurrence (n=10)). Secondly, de-novo and recurrent samples from the same patients (38 cases) were analysed and the change of PD-L1 expression over time within the same tumour was examined. Finally, the laboratory results were correlated with clinical data and confirmed by taking the results of 874 patients of 6 independent cohorts from different publicly available databases into consideration.nd recurrence and 5 (7.81%) patients received a surgery of the 3rd recurrence.Tissue samples of 876 patients were screened for paired samples of de-novo and recurrent tumours, which were acquired during surgical resection between 2011-2016. 102 patients with matched samples (de-novo and recurrent specimens) were identified. Of these, 64 patients achieved all quality criteria and were included in the study  was analysed by qRT-PCR. De-novo tumours from all patients (n=64) showed highly variable expression values  IQR 2.72-8.89 NE). Samples of the first recurrence  showed a decreased median expression of PD-L1 of 54.04% in comparison to de-novo GBM  was further reduced (3.38 %) in comparison to the fist recurrence . Tumours of the 3rd recurrence (2.63 NE IQR 1.35-3.57) showed a significant difference in their PD-L1 expression . Additionally, immunohistochemistry (IHC) of PD-L1 was performed and showed a reduction of 66.71% (p=0.0045) in the first recurrence, Figure PD-L1 of 6 different public-available datasets, Figure M Figure . This diIn line with reported results , IHC andPD-L1 expression-level in the recurrent stage was predicted. Additionally, the age of the patients in the de-novo stage (Odds ratio 1 CI (0.7-1.2) p>0.05), IDH1/2 mutation status (Odds ratio 0.8 CI (0.3-1.2) p>0.05) was not associated with a lower PD-L1 expression in the recurrent tumour. Leuco- and thrombocytopenia regularly occurred in patients who underwent chemotherapy. However, our model could not predict any association with the expression-level of PD-L1 and cytopenia complications. Finally, we analysed patients who received an extended temozolomide therapy  beyond the classical STUPP-protocol (6 cycles). Interestingly, a significant down-regulation of PD-L1 in the recurrent tumour was found in those patients (Odds ratio 2.3 CI (1.2-5.5) p=0.02), Figure We identified 38 patients with matched de-novo and recurrent GBM and analysed associated clinical features, which promote the recurrent down-regulation of PD-L1 Figure . First, The purpose of this study was to analyse the occurrence of PD-L1 expression in recurrent glioblastoma. Being a “key-player “ of the immune escape mechanism of glioblastoma multiforme, PD-L1 is one of the most promising targets for future immune therapy . In de-nUp to today, little is known about recurrent GBM and the abundance of tumour-specific alterations has so far only partially been investigated. Since recurrent GBM are difficult to treat and show a high therapeutic resistance, there is variation in treatment strategies. Most of the recurrent tumours received several therapies (TMZ and radiation) and contained an increased number of mutations. Due to the increase of potential neoantigens, it is assumed that recurrent tumours should be more accessible for immune system recognition and attack. In contrast to the hypothesis that PD-L1 expression would increase as a reaction of the tumour to protect itself form a stronger immune response (immune-escape mechanism) in recurrent tumours, we found the opposite effect. PD-L1 mRNA expression and protein level were significantly reduced in recurrent tumours, which was confirmed by an additional analysis of 6 independent cohorts. Berghoff et al., (n=18 patients) reported a reduction of PD-L1 in 22%, a stable protein-level in 66.7% and only in 5.5% an increased PD-L1 within the recurrent tumour. PD-L1 expression was measured using IHC only, and staining was very heterogeneous within one sample especially in recurrent tumours. In line with the findings by Berghoff et al., the results from an analysis of CD8 and CD3 infiltration in de-novo and recurrent tumours showed reduced levels in recurrent tumours. However, Miyazaki and colleagues did not confirm the reduction of PD-L1 in a recurrent GBM in a small n=16) cohort. Additionally, the cohort partially achieved immunotherapy, which biased the findings and hinder a clear interpretation of the results [ cohort. Finally, a regression model of PD-L1 expression and clinical data showed one significant parameter, which was associated with a down-regulation of PD-L1. The extended TMZ therapy (>6 cycles) might have a negative influence on the immune system activity, which then results in less immune infiltration within the tumor and consecutively leads to a decreased need for PD-L1 expression as immune-escape mechanism within the tumour. Other parameters for immune system malfunction like leuco- and thrombocytopenia  did not showed a significant connection to PD-L1 expression. The immune modulation function of TMZ in the context of immunotherapy was observed in different studies but is not well understood and needs to be explored in the future .This study was limited by a relatively small number of de-novo and recurrent pairs, but achieved a number of 38 cases. With regard to the limited number of patients, a statistical power of 0.8 was achieved for all tests, which reflects the robustness of statistical testing. In conclusion, this study reported a reduction of PD-L1 expression in recurrent GBM, which might be caused by the immune modulating effect of TMZ therapy. The effect of PD-L1/PD-1 immune therapy of recurrent tumours is still unclear and needs accurate evaluation. As known from other cancer entities, PD-L1 occurrence and the therapeutic effect of PD-L1/PD-1 therapy are not necessarily connected. The exact mechanisms of PD-L1 regulation in de-novo and especially recurrent tumours will need more examination in future studies.https://cancergenome.nih.gov/) data were used for analysis. Data were downloaded at the UCSC Cancer Genome Browser. Expression analysis was performed based on Agilent array data (TCGA GBM G4502A) for high-grade glioma. Additional datasets of recurrent GBM were downloaded from publicly available platforms (http://gliovis.bioinfo.cnio.es) [Publicly available Level 3 TCGA (cnio.es) .Tumor tissue was sampled from contrast enhancing regions identified by intraoperative neuronavigation  during tumor resection. The tissue was snap-frozen in liquid nitrogen immediately after resection and processed for further analysis. The local ethics committee of the University of Freiburg approved data evaluation and experimental design (protocol 100020/09 and 5565/15). The methods were carried out in accordance with the approved guidelines. Written informed consent was obtained from all patients. Descriptive statistics of the patient cohort was given in Table Tissue samples were fixed using 4% phosphate buffered formaldehyde and paraffin-embedded according to standard procedures. H&E staining was performed on 4 μm paraffin sections using standard protocols. Immunohistochemistry was applied using an autostainer (Dako) after heat-induced epitope retrieval in citrate buffer. IDH1 mutation was assessed by immunohistochemistry using an anti-IDH1-R132H antibody . Immunohistochemistry was performed on 3 μm paraffin-embedded tissue sections after deparaffinization and heat-induced epitope retrieval in citrate buffer by using the SignalStain Kit by Cell Signaling according to the manufacturer’s instructions. As primary antibody, Anti-PD-L1-antibody  was applied to the tissue in a concentration of 1:200 and incubated overnight at 4°C. The next day, after application of SignalStain® Boost Solution and Secondary Antibody Solution, counterstaining with Meyer’s haemalaun solution was performed. The samples were then mounted and analyzed with an Olympus microscope. PD-L1 positive cells were counted in 6 high-fields (40x magnification) per slide and compared to the total number of cells in each field. From this data, the mean percentage of PD-L1 positive cells was calculated.The following antibodies were used for Imm-munofluorescence: PD-L1  and CD8 . Primary antibodies were used at the concentration indicated by the manufacturers. Anti-Mouse and anti-Rabbit Alexa488- or Alexa594-conjugated (Life Technologies) were used as secondary antibodies. Alexa594/Alexa488-conjugated antibodies were used at 1:100 dilution. Pictures were acquired using a fluorescence microscope . Image quantification was performed by ImageJ and analyzed by R-software.PD-L1 (for (TGGCATTTGCTGAACGCATTT), rev (TGCAGCCAGGTCTAATTGTTTT)) and S18 (for (TTTGCGAGTACTCAACACCA), rev (CC-ACACCCC-TTAATGGC))RNA was prepared using the All Prep DNA/RNA Protein Mini Kit (Qiagen) and used for first strand cDNA synthesis using random primers and SuperscriptIII Reverse Transcriptase (Invitrogen). RNA and cDNA concentration was controlled using the NanoDrop(Thermo Fisher) spectrophotometre. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Green PCR Master Kit (Applied Biosystems). Primersequence were: st recurrence) samples we used the paired Wilcoxon signed-rank test (IHC and mRNA expression). The validation cohort was normally distributed (Shapiro-Wilk p=0.3) and differences were tested by unpaired student’s t-test with an alpha-level of 5%. A binominal generalized linear model was used to find associated clinical parameters to up/down regulation of PD-L1 in the recurrence stage. Significance was calculated by chi-square with an alpha-level of 5%. All statistical analysis was performed with R-software.The primary endpoint was to determined the difference of mRNA expression between de-novo GBM and recurrence. Distribution and variances of all data was tested by Shapiro-Wilk test (p>0.05) to confirm normality. We tested the difference between de-novo and recurrent GBM by Wilcoxon signed-rank test (unpaired) and determined a 5% alpha-level. For the paired (de-novo/1


The human papillomavirus (HPV) vaccines consist of major capsid protein (L1) virus-like particles (VLP) and are highly efficacious against the development of cervical cancer precursors attributable to oncogenic genotypes, HPV16 and HPV18. A degree of vaccine-induced cross-protection has also been demonstrated against genetically-related genotypes in the Alpha-7 (HPV18-like) and Alpha-9 (HPV16-like) species groups which is coincident with the detection of L1 cross-neutralising antibodies. In this study the L1 domains recognised by inter-genotype cross-neutralising antibodies were delineated. L1 crystallographic homology models predicted a degree of structural diversity between the L1 loops of HPV16 and the non-vaccine Alpha-9 genotypes. These structural predictions informed the design of chimeric pseudovirions with inter-genotype loop swaps which demonstrated that the L1 domains recognised by inter-genotype cross-neutralising antibodies comprise residues within the DE loop and the late region of the FG loop. These data contribute to our understanding of the L1 domains recognised by vaccine-induced cross-neutralising antibodies. Such specificities may play a critical role in vaccine-induced cross-protection. Clinical trials have demonstrated that both vaccines are highly efficacious against the development of cervical cancer precursors and other anogenital diseases attributable to vaccine genotypes HPV16 and HPV1818®9, has recently been licensed for use following successful clinical trialsL1 virus-like particles (VLP) precipitated on an aluminium salt adjuvant are the basis of the prophylactic vaccines, CervarixVaccine type L1 neutralising antibodies can be detected in both the serum and cervicovaginal secretions of vaccine recipients222423Pseudovirions (PsV) are used as surrogates for authentic virions for the measurement of antibody-mediated neutralisation in a range of viral systems including HIV, influenza virus, SARS coronavirus and HPV282930222722272333That cross-neutralising antibodies are generated in response to the L1 VLP-based HPV vaccines indicates that the L1 proteins of the vaccine genotypes harbour immunogenic domains that share sequence and/or structural similarity with the L1 proteins of non-vaccine genotypes. Within this present study L1 crystallographic homology models were used to predict structural differences in the loops between HPV16 and the non-vaccine Alpha-9 genotypes. These data informed the design and generation of chimeric PsV with inter-genotype loop swaps for the delineation of specific L1 domains recognised by HPV vaccine-induced cross-neutralising antibodies. The identification of such L1 antigenic domains will contribute to our understanding of virus-host interactions induced in response to vaccination.The crystal structure of the HPV16 L1 capsomer (Protein Data Bank [PDB] Code: 2R5H) was used as the template to which the L1 amino acid sequences representing the Alpha-9 PsV were modelled. Three amino acid residues differ between the L1 sequences of the HPV16 crystal and PsV: two positons in the EF loop (Q177N and Q181N) and one position in the FG loop (A266T). These sequence differences did not appear to adversely impact upon the quality of the HPV16 L1 homology model which had a Global Model Quality Estimation (GMQE) score of 1.00. The L1 homology models representing the remaining Alpha-9 genotypes produced GMQE scores of 0.99 (HPV31 and HPV35) and 0.98 .The crystal structure of the HPV35 L1 capsomer (PDB Code: 2R5J) was used as a control to evaluate the structural accuracy of the HPV35 PsV L1 homology model based upon the HPV16 crystal structure. The pairwise comparison of the homology model on the HPV35 crystal structure by superimposition demonstrated a root mean square (RMS) deviation of 0.68 Å.–f) with the distance between altered loop structures summarised , HPV33 (Ala58), HPV52  and HPV58 (Asn58) in comparison to HPV16. No structural changes in the DE loop were predicted between the L1 homology models representing the non-vaccine Alpha-9 genotypes compared to HPV16  or insertions (HPV52: Asn284 and Ser285) in comparison with HPV16 which appeared to influence these predicted structural changes. The FG amino acid sequence of HPV31, HPV33 and HPV58 contained no residue deletions or insertions in comparison with HPV16, and no structural changes in the FG loop were predicted for these non-vaccine genotypes compared to HPV16. The HI loops of HPV33, HPV52 and HPV58 have an amino acid residue deletion which corresponds to Thr350 within the HI loop of HPV16. The three L1 homology models representing these non-vaccine genotypes were predicted to have structural changes compared to HPV16 homology model within the HI loop, in comparison to HPV31 and HPV35 for which no structural changes were predicted , double  or triple (DEFGHI) loop swaps  generate® n = 19; Gardasil® n = 17) were tested against the chimeric L1L2 PsV. The cross-neutralising titres generated against the chimeric L1L2 PsV with a HPV31 backbone were compared against the HPV31 PsV whilst the titres generated against the chimeric L1L2 PsV with a HPV35 backbone were compared against the HPV35 PsV (Thirty-six HPV vaccinee sera (CervarixPV35 PsV .p < 0.001). The largest impact of any single or combination loop swap was observed with the HPV35 FG loop alone which resulted in a median decrease in cross-neutralising antibody recognition of 19.0-fold  compared to the HPV31 PsV.It was reasoned that the introduction of HPV35 loops into a HPV31 backbone would result in the reduction of cross-neutralising antibody recognition of HPV31. The incorporation of the HPV35 HI loop alone did not result in a reduction in cross-neutralising antibody recognition; however, the incorporation of the HPV35 DE loop  reduced p = 0.637) and 22 , respectively, compared to the HPV35 PsV titre of 22  (p < 0.001) compared to the HPV35 PsV.The transfer of cross-neutralising antibody recognition to HPV35 was not supported by either the introduction of the HPV31 DE or HI loop into the HPV35 backbone, both of which had no significant effect on cross-neutralising antibody recognition of HPV35 producing geometric mean titres (GMT) of 25  . Conversp < 0.001) and 20.1-fold  decrease relative to the HPV31 PsV for Cervarix® and Gardasil® sera, respectively. The replacement of the DE and FG loops within the HPV35 L1 backbone with those of HPV31 significantly increased recognition of the HPV35 PsV  by both Cervarix®  and Gardasil® sera .The cross-neutralising antibody recognition of the chimeric L1L2 PsV was independent of the HPV vaccine received. For example, the replacement of the FG loop within the HPV31 L1 backbone with that of HPV35 resulted in a median 17.8-fold  and FG loops . These residues were selected to undergo site-directed mutagenesis to determine their contribution to the epitope footprint recognised by cross-neutralising antibodies, alongside additional sites where the residue differed between HPV31 and HPV35  (In order to predict the residues within the DE and FG loops which may be involved in the epitope footprint(s) recognised by vaccine-induced cross-neutralising antibodies, the DE and FG loop amino acid sequences of HPV16, HPV31 and HPV35 were aligned . Amino a Thr274) .A panel of seven chimeric L1L2 PsV with a HPV35 backbone and HPV31 DE and FG loop swaps were generated. These chimeric PsV harboured single or double amino acid residue switches incorporated in either the DE or FG loop whereby the amino acids present in HPV31 were swapped for the amino acids present at those positions in HPV35 . The PsV® n = 12; Gardasil® n = 12), from the original panel of thirty-six, were tested against the seven new chimeric L1L2 PsV and the cross-neutralising titres generated were compared against the chimeric L1L2 PsV with a HPV35 backbone and HPV31 DE and FG loops (p < 0.001).A subset of twenty-four HPV vaccinee sera (CervarixFG loops . Within 264 to Lys278) and the late region (Gly279 to Ser288) of the loop. The dual amino acid switches within the late region  resulted in a significant reduction in cross-neutralising antibody recognition; however, the most dramatic impact was observed by the dual amino acid deletion at positions S281Del and G282Del which produced a cross-neutralising titre [GMT of 17 ] lower than the titre observed against the HPV35 PsV [40 ] (The amino acid switches within the FG were separated into the early region (HPV16 numbering: Ala to 47)] . In contMapping of the mutagenised residues within the DE and FG loops on to the HPV31 L1 homology model demonstrated that these positions are all surface-exposed on the capsid  and thatThis study attempted to delineate the L1 domains recognised by HPV vaccine-induced Alpha-9 cross-neutralising antibodies. We designed and generated chimeric L1L2 PsV with inter-genotype loop swaps which were tested for their sensitivity to cross-neutralisation by antibodies elicited by HPV vaccines.The cross-neutralising antibodies detected following immunisation with HPV16 VLP recognise L1L2 PsV representing a number of non-vaccine Alpha-9 genotypes2227The surface-exposed L1 loop regions of the HPV capsid demonstrate a higher degree of sequence heterogeneity than the core regions as evident from the higher levels of intra- and inter-genotype amino acid variability located to the loops116The analysis of L1 loop structure within this study has to be interpreted with the caveat that they are structural predictions derived from homology models representing the Alpha-9 PsV L1 amino acid sequence rather than the resolved L1 crystal structures. Nevertheless homology modelling is a standard approach used for H1N1 pandemic influenzaThe L1 loops of HPV16 have been investigated extensively using L1 VLP as target antigens for antibody recognition. The manipulation of individual L1 loops by the insertion of foreign B-cell epitopes from HIV and HBV identified the FG and HI loops as immunogenic regions of the L1 capsid42431045The limited use of chimeric L1L2 PsV as antigenic targets may reflect the reduced tolerance of these complex particles to manipulation. Within this present study, the chimeric L1L2 PsV consisted of inter-genotype loop swaps between two genotypes from within the same species group (Alpha-9: HPV31 and HPV35) an approach which resulted in the successful formation of functional chimeric L1L2 PsV particles. Chimeric PsV have been utilised as antigens in other virus systems including HIVThe chimeric L1L2 PsV demonstrated that the FG loop is necessary and sufficient for the epitope footprints of a significant proportion of cross-neutralising antibody specificities and that the DE loop appears to enhance this recognition. The HI loop appeared not to contribute towards cross-neutralising antibody recognition. Two or more L1 loops appear to contribute towards the majority of identified L1 MAb epitopes, although there are instances where a single loop has supported an epitope footprint, for example the BC loop alone is targeted by some HPV6, HPV33 and HPV16 L1 MAbs455014Fine-mapping of the epitope footprint recognised by cross-neutralising antibodies was undertaken, whereby residues at DE and FG loop amino acid positions of HPV16 and HPV31 which differed from HPV35 were subjected to site-directed mutagenesis alongside sites where the residues were variable between all three genotypes. Cross-neutralising antibodies appeared to primarily target amino acid residues located in the late region of the FG loop particularly the amino acids at positions 281 and 282, the deletion of which resulted in almost complete loss of recognition by cross-neutralising antibodies. In contrast, the mutation of two amino acid positions in the early region of the FG loop had no effect upon cross-neutralising antibody recognition, indicating that the early region is not a target for such antibody specificities.278 (HPV16 numbering) which mediates primary host attachmentin vivo by vaccine induced L1 antibodies13164551The FG loop contains a LysCross-neutralising antibodies appear to recognise a single non-vaccine Alpha-9 genotype or multiple non-vaccine genotypes, in addition to HPV16We propose that the L1 domain recognised by inter-genotype cross-neutralising antibodies elicited by the HPV vaccines comprise residues within the late region of the FG loop possibly stabilised by one or more residues within the DE loop. The cross-neutralising antibodies which target this L1 domain may play a role in HPV vaccine-induced cross-protection.http://home.ccr.cancer.gov/LCO/packaging.htm) using SWISS MODEL (http://swissmodel.expasy.org/)56L1 homology models were created from the L1 amino acid sequences of L1L2 PsV representing HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58 (sequences available from DeepView Swiss-Pdb viewer v4.0® (Thermo Fisher Scientific) and were applicable site-directed mutagenesis was carried out using the QuikChange kit (Stratagene). L1 genes were subcloned into the p31sheLL or p35sheLL plasmids in conjunction with the Rapid DNA Dephos & Ligation kit (Roche). HPV31, HPV35 and chimeric L1L2 PsV carrying a luciferase reporter were expressed and purified as previously described50) was estimated for each PsV preparation using the Spearman-Karber equations and a standardised input of 300 TCID50 was used for all PsV7 particles per ng L1 protein (http://home.ccr.cancer.gov/lco/production.asp), with the ratio being normalized for the input volume and the TCID50.The L1 genes with HPV31 or HPV35 backbones and reciprocal inter-genotype loop swaps were synthesised by GeneArt® or Gardasil® as part of a Phase IV clinical trial comparing HPV vaccine immunogenicity . A subset of samples  taken 1 month after receiving the final dose, were selected for analysis based upon HPV31 cross-neutralisation antibody titresSerum samples were available from 12–15 year old girls randomised to receive three doses of CervarixSamples were subjected to 5 serial dilutions, with the antibody titre resulting in an 80% reduction in the luciferase signal produced by control wells containing PsV and cells only estimated by interpolation. HPV antibody control reagents were included in each assay runThe Wilcoxon paired signed-rank test was used for the comparison of cross-neutralising antibody titres between different L1L2 PsV targets. The test was performed using the statistical package, Stata 12.1 (StataCorp LP).How to cite this article: Bissett, S. L. et al. The DE and FG loops of the HPV major capsid protein contribute to the epitopes of vaccine-induced cross-neutralising antibodies. Sci. Rep.6, 39730; doi: 10.1038/srep39730 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We previously showed increased growth associated protein 43 (GAP-43) expression in brain samples resected from patients with cortical dysplasia (CD), which was correlated with duration of epilepsy. Here, we used a rat model of CD to examine the regulation of GAP-43 in the brain and serum over the course of epileptogenesis. Baseline GAP-43 expression was higher in CD animals compared to control non-CD rats. An acute seizure increased GAP-43 expression in both CD and control rats. However, GAP-43 expression decreased by day 15 post-seizure in control rats, which did not develop spontaneous seizures. In contrast, GAP-43 remained up-regulated in CD rats, and over 50% developed chronic epilepsy with increased GAP-43 levels in their serum. GAP-43 protein was primarily located in excitatory neurons, suggesting its functional significance in epileptogenesis. Inhibition of GAP-43 expression by shRNA significantly reduced seizure duration and severity in CD rats after acute seizures with subsequent reduction in interictal spiking. Serum GAP-43 levels were significantly higher in CD rats that developed spontaneous seizures. Together, these results suggest GAP-43 as a key factor promoting epileptogenesis, a possible therapeutic target for treatment of progressive epilepsy and a potential biomarker for epilepsy progression in CD. About 30–40% of patients are not responsive to pharmacological treatment and only ~47% of patients will remain seizure-free 10 years after surgical resection4. Seizure freedom after surgery is negatively correlated with duration of epilepsy, such that the longer the disease duration, the worse the outcome is after surgery5. In order to develop new treatments for the prevention and cure of epilepsy, it is important to understand mechanisms of disease development and progression.Epilepsy is the fourth most common neurological disorder in the United States11. The first seizure is typically followed by a latent period, leading to seizure recurrence and the development of drug resistance in some patients. These observations suggest that epilepsy is a non-static disease, and the epileptic brain region may continue to undergo progressive cellular and molecular changes over time.Congenital focal brain malformations known as cortical dysplasia (CD) are common pathological substrates of medically intractable epilepsies. Patients with CD may not develop epilepsy until the occurrence of the first seizure in the setting of a “trigger”; a phenomenon we refer to as a “second hit” 13. We have recently shown that the duration of epilepsy in patients with CD is correlated with increased expression of growth-associated protein 43 (GAP-43), a marker of axonal and synaptic growth14. GAP-43 is upregulated following stroke and traumatic brain injury19. Our results suggest the interesting possibility that GAP-43 is upregulated after a second hit (e.g. seizure), leading to the formation of highly interconnected, synchronized epileptic neural networks. However, studying surgically resected human brain specimen has a significant limitation for the investigation of the dynamic changes of GAP-43 during epileptogenesis. Therefore, we investigated the role of GAP-43 expression in epileptogenesis using a well-characterized animal model of CD.There is increasing evidence that cellular and neural networks at the local epileptic area extend beyond the lesion, which may explain why some surgical resections classically failOur study shows that GAP-43 expression is located predominantly within excitatory neurons, and its expression is increased in brains after an acute seizure. GAP-43 in serum is also increased in CD rats that developed spontaneous epilepsy. Knockdown of GAP-43 significantly reduced epileptogenesis and epileptogenicity in CD rats. The findings presented in this study provide valuable insight into some possible molecular mechanisms of epileptogenesis and identify GAP-43 as a potential new target protein for the treatment and prevention of epilepsy in cortical dysplasia.24. Only implanted rats  were used for EEG analyses , displaying generalized tonic-clonic behaviors, while seizures in control rats were less severe, and equally distributed across all stages of behavioral severity  type Ises Fig. . All CD 23.All CD + PTZ rats exhibited increasing interictal spiking after the initial acute seizure induced with PTZ Fig. . After 114. Bilateral somatosensory cortex images (A/P = −2.0 ± 0.2 mm) were taken and analyzed . Each field of view contained 53.5 cells (SEM = 4.17). There was no difference in the number of cells imaged in CD and non-CD brains regardless of the architectural differences. The IHC staining intensity in the cortex was given a grade 2–8 based on neuronal rim staining (cell surface) and tubular punctate expression (intracellular) by two blinded investigators (AN and ZY). As previously reported, the scores were calculated based on the sum of qualitative grading of Rim and Tubular Punctate GAP-43 expression14. Rim Staining Value: 4 = more than 50% of neurons in the image positive for GAP-43 stained rim, 3 = more than 10% but less than 50%, 2 = about 5–10%, 1 = less than 5% of neurons; Tubular Punctate Staining Value: 4 = intensely dark, 3 = moderately dark, 2 = some punctate staining, 1 = no staining.There was no significant effect from surgical implantation of EEG electrodes on GAP-43 expression, therefore rats were pooled for GAP-43 analyses . Sections stained with immunohistochemistry (IHC) for GAP-43 protein were randomized and qualitatively graded as previously describedA mixed models regression analysis was conducted on GAP-43 immunohistochemistry staining with Tukey-Kramer adjustments. There was no change in GAP-43 expression over time  within CD (p = 0.381) and non-CD (p = 0.252) groups, suggesting that normal brain maturity did not affect GAP-43 expression. Therefore, data were pooled into CD and non-CD saline groups. CD rats treated with saline had significantly higher GAP-43 expression than non-CD rats with saline, showing that CD pathology alone is associated with a baseline increase in GAP-43 levels , suggesting a possible relationship between the appearance and recurrence of spikes and GAP-43 upregulation.Quantified results of ELISA and NIR western blotting were analyzed with mixed models regression analyses testing measured response against group, day, and group-day interaction. Tukey-Kramer adjustments were made for all pairwise comparisons. Similar to the IHC observations, NIR western blotting results showed an increase in GAP-43 protein level at day 30 in CD rats as compared to non-CD rats Fig. . CD rats2 = 0.04, n.s. p = 0.63). Interestingly, serum GAP-43 levels were correlated with interictal spikes at days 100 and 120 . Although further research is necessary to determine the mechanisms of serum GAP-43 upregulation with seizures, GAP-43 protein was found localized to capillary lumen in the CD + SS rat brain, suggesting that it may be released into the blood after synapse formation. These results support the notions of a seizure-induced breakdown of the blood brain barrier27. Further research is necessary to determine the mechanisms and timing of GAP-43 increase in the serum throughout the development of SS.Remarkably, serum GAP-43 levels were significantly higher in CD rats after SS (Day 120) as compared to CD rats without SS (p = 0.028), and non-CD rats (p = 0.015) , and a significantly higher pixel intensity in GAP-43 stained sections (p = 0.01) than non-CD brains. There was no significant difference in VGLUT1 pixel intensity (n.s. p = 0.07). Double-labeled images showed a stronger co-localization between GAP-43 and VGLUT1 in both CD and non-CD brains (p = 0.005). The subcellular localization of GAP-43 within axons containing spherical vesicles was confirmed by immuno-EM staining  knockdown. Animals which received a LTV vector containing shRNA against GAP-43 (shGAP43) injected into the right somatosensory cortex, showed ~50% reduction in local GAP-43 RNA  in rats with CD leads to long-lasting (up to 120 days) upregulation of new synapse protein formation (GAP-43) in predominantly excitatory synapses. Persistent GAP-43 upregulation is correlated with the early development of epileptogenicity  and ictogenesis (spontaneous epileptic seizures). Conversely, the knockdown of GAP-43 in CD brains decreased interictal spike frequency, further suggesting a direct role of GAP-43 in cortical dysplasia and its upregulation in epileptogenesis.We recently demonstrated that GAP-43 proteins are higher in the dysplastic and epileptic cortex compared to normal appearing cortex in human patients with medically intractable epilepsy who underwent surgical resections30. It is activated through phosphorylation at ser41 via protein kinase C (PKC) leading to actin polymerization31. GAP-43 expression is crucial in neurodevelopment, yet it is significantly reduced in multiple brain areas (including sensorimotor cortex) after brain maturation. It remains abundant in plastic areas of the adult brain such as the limbic system, the hippocampus and associative areas of the brain, but is low in primary sensory and motor areas33. As GAP-43 is elevated during normal neurodevelopment, it is possible that the “first hit” (e.g. in utero irradiation model), causes GAP-43 expression to remain high in dysplastic cortex16. This could allow for the development of a highly plastic brain prone to synaptic outgrowth, potentially creating an imbalance between excitatory and inhibitory connections. GAP-43 protein expression is higher in the sensorimotor area of CD rats (as compared to control brains) with sustained upregulation up to older ages . Animals with CD express high baseline GAP-43 levels and show a lower threshold for the development of longer and more severe PTZ-induced seizure than control rats with lower baseline GAP-43 proteins. Moreover, the abundant localization of GAP-43 proteins in excitatory synapses may suggest that baseline axon outgrowth is involved in decreased seizure threshold, as observed in CD rats.GAP-43 is an intracellular protein located at branching points, growth cones, and axon terminals involved in axonal elongation, neurotransmitter release and synaptic vesicle recycling36. GAP-43 proteins were significantly higher in CD rats in the acute period following the “second hit” and the upregulation only persisted beyond the acute period in CD rats (as compared to control rats). We have also previously observed an increased expression of GAP-43 in other seizure models, including focal intracortical penicillin injection, kainic acid, and traumatic brain injury37. Acute, non-sustained GAP-43 upregulation may be a non-specific response to an insult to the normal brain. However, persistent GAP-43 up-regulation may trigger abnormal synaptogenesis in a dysplastic brain, and lead to the development and progression of chronic epilepsy.Our findings demonstrate a relationship between epileptogenesis and upregulation of GAP-43 following a second hit in a model of cortical dysplasia. Acute seizures lead to an early and short-term upregulation of GAP-43 in both control and dysplastic brains. These results are consistent with previous studies showing the implications of GAP-43 protein regulation following stroke and traumatic brain injury, as well as neurodevelopment, learning and memory; thus, suggesting a role of GAP-43 in plastic events, even in adulthood40. The results of our confocal imaging show that GAP-43 positive synapses are primarily glutamatergic. These results could support the notion that there is a change in the balance of excitation and inhibition throughout epileptogenesis, and further suggests that GAP-43 upregulation is involved in this process. This data is consistent with research by Console-Bram et al. showing GAP-43 upregulation following exposure to glutamate, but a decrease with exposure to GABA41. With an increase in excitatory neurotransmission through glutamate receptors, GAP-43 may be further upregulated, creating more excitatory synapses and a potential positive feedback loop in epilepsy-prone brains. This pathway may begin with an excitatory stimulus, or heightened expression of an excitatory receptor such as NMDA, leading to an increase in GAP-43 with new excitatory synapses and a pro-epileptic network. Previous studies showed that NMDA receptors play a role in the regulation of GAP-43, and may serve as a potential up-stream target43. Additionally, upregulation of the NMDA receptor complex in human dysplastic neurons, contributes to the formation of local epileptic networks48. We have observed an increase in NMDA receptor 2 A/B subunits in CD brains as compared to non-CD, with GAP-43 positive axons surrounding the NR2A/B positive areas on the neuronal cell bodies37. Previous reports also showed that blocking NMDA receptors prevents the increased expression of GAP-43; making NMDA receptors an attractive upstream candidate for GAP-43 regulation49.The localization of GAP-43 protein in excitatory synapses, in addition to heightened epileptic activity with increased GAP-43 expression suggest that axon outgrowth plays a role in the epileptogenesis in CD following a “second hit”. The sustained increase in GAP-43 aligns at least in part with results of surgical outcome studies51.The potential role of GAP-43 in epileptogenesis after a second hit is further highlighted in CD animals following GAP-43 knockdown. The knockdown of GAP-43 reduced the peak seizure severity after PTZ induced acute seizures. Moreover, the reduction in interictal spike frequency following the acute seizure, or “second hit”, provides further evidence for a role of GAP-43 in epileptogenesis. Excitatory post-synaptic protein PSD-95 was significantly reduced with GAP-43 knockdown. This may contribute to the observed inhibition of chronic epileptogenesis. Further research is warranted to investigate the downstream targets that may play a role in modulation of a pro-epileptic neuronal network. The mechanisms involved in the increased expression of GAP-43 protein are likely multifactorial. For example, an intrinsic program-driven transcription of GAP-43 expression, supported by previously published results showing increased GAP-43 mRNAs in dysmorphic neurons27.Levels of GAP-43 in the serum were elevated after spontaneous seizures, but not prior to their onset. The abrupt increase in GAP-43 levels in the serum is observed at day 120, but not day 100. Similarly, while interictal spikes continuously increase up to 120 days, we did not observe spontaneous seizures until 120 days after PTZ. There was also a correlation between serum GAP-43 levels and interictal spikes at days 100 and 120. This phenomenon must be further explored, and future studies should focus on protein changes at day 120 and later. We also observed (using immuno-EM) aggregated GAP-43 protein in the capillary lumen of CD + SS rats, but did not observe this phenomenon in other groups. These observations suggest that the post-ictal GAP-43 serum level increase could be due to leakage secondary to a seizure-induced breakdown of the blood brain barrierIn summary, our results indicate that the sustained increase of GAP-43 in dysplastic, but not in control brains, is involved in epileptogenesis and epileptogenicity. This rather specific expression of GAP-43 may point to a fundamental pathophysiological mechanism for expression of epileptogenicity and disease progression in epilepsy due to CD. We postulate that during the latent period, molecular/cellular changes, such as aberrant axonal sprouting, occur in the CD brain resulting in the development and growth of local, highly excitable and electrophysiologically synchronized epileptic network(s).A total of twenty timed-pregnant Sprague-Dawley rats and their 113 male pups were used in this study . The care and use of the animals were approved by the Animal Research Committee of Cleveland Clinic Foundation. All methods were performed in accordance with the approved guidelines by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of Cleveland Clinic. All rats included in the study were housed in individual cages, under 12-hour dark-light cycles, and had free access to food and water. Animals were housed in the Cleveland Clinic Biological Resources Unit accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.22. An additional ten timed-pregnant rats served as age-matched control animals. All pups were grown to adulthood of post-natal day 55 (PND55) prior to experimental procedures, which is when non-CD and CD animals weigh roughly the same weight, therefore reducing possible differences in drug metabolism.Ten timed-pregnant rats were irradiated with 145 cGy of cesium-137 on E17 for a model of multifocal cortical dysplasia as previously describedstatus epilepticus in the vast majority of CD rats23. Fifteen  animals were injected with saline and used for normal and CD controls. All animals were monitored visually by video and given a behavioral score for seizure severity25. Rats were then sacrificed at post-PTZ induced seizure day 2, day 15, day 30, or day 120 and their brains were harvested for western blot and ELISA or fixed in paraformaldehyde solution for immunohistochemistry.In order to account for potential molecular changes caused by surgical implantation of EEG electrodes, parallel groups of rats were studied with and without EEG. In the first set of experiments, 50 rats  without surgical manipulations were injected with pentylenetetrazole (PTZ) at a sub-convulsive dose . This subconvulsive dose only induces seizures in a small portion of normal/non-irradiated rats, but it leads to et al., 2016. Electrodes were implanted bilaterally over the frontal  and parietal cortices . Additionally, two reference electrodes were placed REF1: above the frontal sinus +3.0 mm anterior and 1.5 mm lateral to bregma and REF2: above the cerebellum −2.0 mm posterior and 2.5 mm lateral to lambda for a back−up reference electrode. After 72 hours of recovery, rats were placed in EEG recording chambers. They were injected with either PTZ or saline, in the same manner as described above. Rats were monitored in 12-hours per day, three to four days per week, for up to 120 days . Four rats were monitored simultaneously during each session at an acquisition sampling rate set at 200 Hz, a low-frequency filter setting of 7 Hz and high-frequency filter of 70 Hz. Animals had free access to food and water during the recording sessions.In the second set of experiments, 28 rats  were surgically implanted with EEG electrodes for monitoring of epileptic activity over time. These animals were also evaluated for GAP-43 expression, but since GAP-43 may increase due to the acute damage involved in surgery, the initial experiments were also necessary to confirm that the surgical procedures were not the cause of the changes in expression. Under isoflurane anesthesia, rats were placed in a stereotactic frame and implanted with six epidural EEG electrodes as described in Nemes 52 were purchased from Sigma-Aldrich, Inc.  at a titer of 109 TU/ml. Lentivirus injection was conducted in a biosafety level 2 laboratory within a biosafety cabinet. Rats were injected during surgical implantation of EEG electrodes under isoflurane anesthesia. Using stereotactic technique as described above, a burr hole was created at A/P: −2.0 mm L: +3.0 mm D: 1.0 mm. A Hamilton syringe  was loaded with 2 µl of lentiviral suspension and placed into the cortex. After 2 minutes, infusion of the lentivirus began, with a rate of 0.1 µl per minute. After 2 µl of the lentivirus was injected, the needle remained in place for 5 minutes to ensure proper infusion. Afterwards, it was slowly removed and the burr hole was closed with dental acrylic.In a third experiment, 20 rats  received an intracortical injection of a lentiviral vector (LTV) containing shRNA against GAP-43 , or a control vector containing a scrambled sequence of the GAP-43 shRNA . Lentiviral vectors25.Six days after LTV injection, rats began EEG monitoring with a baseline recording of 24 hours. At day 7 post-LTV, rats were given 10 mg/kg, i.p., of pentylenetetrazole (PTZ) every 10 minutes until an EEG seizure was observed to determine their seizure threshold. Seizure severity was graded based on duration and clinical semiology using Racine’s behavioral scale22. Seizure and spike data were analyzed based on latency from PTZ injection, frequency, and severity.Digitized EEG was reviewed using NeuroWorkbench software . EEG signals were reformatted in referential and bipolar montages. All EEG and video was visually inspected for the presence of seizures, and 25% of EEG was sampled for manual quantification of interictal spikes (3 hours per 12 hour recording). Analyses were performed by two blinded investigators . The EEG seizures were defined as repetitive and evolving spikes with interruption of the background activity for 10 seconds or longer. As previously reported, epileptiform/interictal spikes were defined as paroxysmal electrical sharp activities lasting 20–150 ms, with an amplitude that was at least five times the background EEG activity23.Once animals completed their monitoring periods, they were deeply anesthetized with ketamine  and xylazine  and perfused transcardially with either phosphate-buffered saline (PBS) or 4% paraformaldehyde (PFA). Their brains were then harvested and either flash-frozen in liquid nitrogen, or stored in 4% PFA for 24 hours and then transferred into 30% sucrose for at least 48 hours before processing. The brains were serially sectioned on the coronal plane in 30 µm slices using a cryostat. The slices were stained with Cresyl Violet (CV) for histological examination. Slides were also used to confirm the CD as previously described53. Slices were incubated overnight at 4 °C in the following primary antibodies: anti-GAP43  or anti-GAP43  combined with anti-Vesicular Glutamate Transporter 1  or anti-Vesicular GABA Transporter . IHC staining was developed using VECTASTAIN Elite ABC Kit (Rabbit IgG)  according to manufacturer’s instructions. IF staining was developed using Alexa Fluor® 488 goat anti-mouse and 568 goat anti-rabbit secondary antibodies according to manufacturer’s instructions . As a negative control used to confirm the absence of non-specific staining, the method was also completed without primary antibodies. In addition, antibody specificity was confirmed using pure GAP-43 protein with western blotting and ELISA. Positive signals were observed with purified protein, and western blotting bands were located only at 43 kDa.Immunohistochemistry (IHC) and immunofluorescence (IF) staining was done on free-floating sections55. A Pearson’s correlation was used to compare the relationships of each antibody pair. Image stacks were also analyzed for pixel intensity by each color and data was analyzed using Students t-tests. For immuno-electron microscopy (EM), brains were processed as described by Fujioka et al., 2011 for pre-embedding immuno-EM. Thin sections were blocked using bovine serum albumin and incubated in anti-GAP43  for 12 hours at 4 °C. Sections were washed and incubated for 1 hour in 10-nm gold conjugated goat anti-rabbit IgG  and fixed to stabilize gold particles for imaging with electron microscopes .Slides were imaged using a confocal laser scanning microscope  at 1024 × 1024 with a 40x lens. Z stacks were gathered from each sample with 1 µm step sizes throughout a total thickness of 100 µm. Two slides per animal were analyzed with one image taken from each hemisphere of the somatosensory cortex . Once the settings for laser intensity, signal offset and amplifier gain were calibrated, they were left the same for all slides. Background was subtracted and monochromatic images were analyzed for co-localization using the Intensity Correlation Analysis plugin (ImageJ v1.37a) and positive product of the differences from the mean (PDM) as described56. Total RNA was isolated from LTV brain samples using TriZol reagent. Reverse transcription was done with the High-Capacity cDNA Reverse Transcription Kit, and quantitative real-time PCR was completed with a 7500 Fast Real-Time PCR System instrument using Taqman Fast Universal PCR Master Mix (2X), No AmpEraseUNG . Probes and primers were from Taqman gene expression assay reagents: GAP-43 (Hs00967138_m1). Data were normalized to GAPDH (Hs02758991_g1) RNA amplification and calculated relative to the expression of the target gene in the brains of LTV rats.Brains were homogenized and prepared for RNA and protein quantification as described®-FL polyvinylidene fluoride (PVDF) membrane . Membranes were blocked with Odyssey Blocking Buffer (TBS) and incubated with anti-GAP43 , anti-GAPDH , PSD-95  or Gephyrin  overnight at 4 °C. Membranes were washed thoroughly in TBS-t and incubated in secondary antibodies for 1 hour . After drying overnight, membranes were scanned on the Odyssey® Classic Imager . Signals were quantified and normalized using methods to eliminate background as well as determine relative signal over total protein, and then expressed as fold changes over the signal of the control group58.For increased sensitivity and quantification of protein expression, NIR western blotting technique was used. Protein concentrations adjusted to exactly 0.5 μg were electrophoresed and transblotted onto Millipore Immobilon59. ELISA plates were coated with monoclonal anti-GAP-43 antibody  at the concentration of 1 µg/ml in ELISA coating buffer (Bicarbonate/carbonate buffer) overnight. Plates were then blocked with 3% BSA for two hours at room temperature and washed with PBS +0.05% tween. Diluted brain homogenates (100 ng/mL), serum (1:100 dilution) or purified GAP 43  at varying concentrations (1–1000 ng/ml) as standards were added overnight at 4 °C. Following washes, biotin conjugated polyclonal anti GAP43 antibody  was added (1 µg/ml) and plates were incubated for one hour at room temperature. Plates were then washed and incubated with streptavidin HRP for one hour at room temperature, and developed using ABTS peroxidase substrate system. Plates were read on an ELISA reader at 410 nm. Using standards, known concentrations, their optical density (OD), and the optical density of samples, linear regression curves were constructed and the concentration of GAP43 was calculated in the samples using the equations from the linear regression line.A sandwich ELISA method was used as previously described to measure the concentration of GAP43 in either the brain homogenates or serum25 and compared with a mixed models regression analyses with Tukey-Kramer adjustments. Severity was also evaluated by percentage of time spent in each stage over the total seizure duration. GAP-43 protein expression from IHC was graded as previously described14. Mixed models regression analyses were run on all numeric data comparing CD and Non-CD across groups of control, day 2, day 15, day 30 and day 120 time points. Students t-tests were used to evaluate independent groups when appropriate. Results were considered statistically significant with p-values < 0.05.Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22.0  and R Statistical Software . Comparisons were made between groups based on seizure prevalence, latency and duration. Seizure severity was graded behaviorally using a standard scaleSupplementary Raw Image for Figure 8Supplementary Raw Image for Figure 3