The flexible hybrid transparent electrode was prepared by a two-step process: graphene film was firstly grown on Cu foil by modified thermal chemical vapor deposition (CVD) and then transferred onto indium tin oxide (ITO) electrode on the polyethylene terephthalate (PET) substrate. The quality of the graphene is characterized by various analytic techniques, including the AFM, SEM, TEM, and Raman spectroscopy. The gradient flux was found to be beneficial to decrease defect. The thickness, morphology, light transmittance, and electromechanical properties of three conductive electrodes were investigated and compared. The outcomes show that the hybrid electrode could resist mechanical force and the results are better than original ITO electrode. It may be a potential trend to apply the graphene to other conducts in the flexible transparent conductive field. A new generation of flexible devices has been extensively studied for electronics, optoelectronics, and energy harvesting applications –4. The kRecently, graphene becomes a novel kind of two-dimensional (2D) carbon allotrope with a unique band structure, which shows outstanding thermal, mechanical, and electrical properties . BesidesHerein, the graphene/ITO flexible hybrid transparent electrode was prepared by a two-step process: graphene film was grown on Cu foil by modified CVD method and then transferred onto ITO electrode on polyethylene terephthalate (PET) substrate. The transmission electron microscope (TEM) image, atomic force microscopy (AFM) image, and the Raman spectra of graphene films were analyzed firstly. The morphology, light transmittance, and electromechanical properties of the hybrid electrode were investigated. It is found that the gradient flux of carbon source modifies the synthesis of graphene and reduces the defect of surface structure. Moreover, the graphene layer of hybrid electrode enhances the ITO layer with excellent mechanical characteristic, and hence the electromechanical properties are improved compared to the ITO electrode.2 (pressure ~ 150 mTorr) flows. After reaching 1024 °C, the sample was left in H2 for 15 min and then a gradient flux of methane (CH4) was added to the tube for another 15 min . After that, the furnace was turned off to cool down the chamber with both CH4 and H2 presence. The CH4 pump was turned off when the furnace temperature reached 700 °C. Then, the sample cooled down to room temperature in the presence of H2 continually. Meanwhile, another graphene films were prepared by the same process with constant methane flux at annealing process of 1024 °C.The graphene film was synthesized using the CVD method similar to procedures reported previously –23. AccoThe PET substrate with ITO films  were purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. The graphene film was transferred to ITO/PET film by means of reported reference -28. Brie+ laser excitation wavelength of 514.5 nm. The laser beam was focused onto the sample through an ×50 objective lens. The Raman spectra were collected in the frequency range of 1000 to 3000 cm−1 with an acquisition time of 10 s. The morphology of hybrid electrodes of graphene/ITO on PET substrate was analyzed by scanning electron microscope (CARL ZEISS-∑IGMA/VP) and eclipse inverted microscope (Nikon-TE2000U). TEM imaging was carried out by JEM-2100 (HR) with 200-kV acceleration voltage and LaB6 emitter. AFM imaging was conducted with tapping mode on Multimode 8 SPM system (Bruker Inc.) to test the morphology and thickness. The optical transmittance of the samples was investigated by using X-Rite spectrometer (Coloreye 7000A). The sheet resistance and other electrical data were measured by four-probe meter (KDY-1), Hall effect measurement system (Lakeshore 7704A), and LCR meter (Agilent U1731B). The mechanical force in the stretching test was conducted by an electromechanical universal testing machine .The graphene films were characterized by Raman spectroscopy  with an Ar−1 representing the D, G, and G′ modes of representative graphene vibration, respectively. The obvious G peak and sharp G′ peak indicate the formation of graphene. The IG′/IG ratio for two samples is greater than two , which confirms monolayer graphene film. Meanwhile, the G′ bands with the full width at half maximum around 30 cm−1 demonstrate the uniformity of monolayer graphene film [ID/IG ratio, comparing the black curve of 0.11. It means that the gradient flux of methane in the process of graphene growth is beneficial to decrease the defects and improve the quality of graphene.The Raman spectroscopy was used to demonstrate the composition of graphene film. Figure ene film . The weaene film , 19. TheFigure The transferred graphene over the ITO electrode on PET substrate was observed by scanning electron microscope (SEM) image and optical image, as shown in Fig. The sheet resistance of all samples was measured by four-probe meter and Hall effect measurement system. Other measured results were listed in Table To investigate the electromechanical properties of graphene/ITO hybrid electrode, bending and stretching tests were performed. Experiments were repeated three times for each group above. Figure R/R0 to evaluate the trend. Among them, the R0 stands for the original and flat sample resistance with dimension of 1 × 1 cm2 and △R refers to the resistance change compared to R0. Figure R/R0 of the samples as a function of the applied tensile strain. The resistance of samples rises up as tensile strain increases. Before the 5 % tensile strain, the value of the three electrode △R/R0 is nearly square. For clearly distinguishing the difference, the data was shown in a log scale in the inset image. After 10 % tensile strain, the brittle property of ITO is evident. Unlike the ITO electrode, the graphene electrode could resist up to 20 % strain with △R/R0 of ~3.28 compared with that of ~125.91 for ITO at the same strain. Likewise, the hybrid graphene/ITO electrode is well performed with △R/R0 of ~17.78. The result demonstrates the pronounced advantage of graphene in protecting ITO brittle failure to some extent. It could be possible to take use of graphene hybrid conductors in flexible transparent electrode.In real time while observing the electrical change under stretch and bending process, we made lap joints (in electrodes) on the edge of sheet samples. The connector of LCR meter then bit the joint while the mechanical force conducted on samples. We recorded the real-time resistance data and calculated the value of △A comparative experiment for resistance change trend measurements was also performed under stable bending process at different curvature. The bending radius was measured by the scale, which is shown in Fig. In summary, this work demonstrates a feasible two-step process method to employ the CVD graphene in fabricating the graphene/ITO flexible hybrid transparent electrode. With this method, as-fabricated graphene shows lower defect and improves the mechanical stability of single ITO electrode. Such hybrid electrode makes a significant advancement toward traditional transparent flexible film both in electrical and optical properties. The hybrid method meets the criteria of low cost, conductivity, and mechanical stability. The graphene/ITO hybrid film as a flexible transparent electrode has important implications for future devices.


Graphene has been considered as a promising material for opto-electronic devices, because of its tunable and wideband optical properties. In this work, we demonstrate electro-refractive phase modulation in graphene at wavelengths from 1530 to 1570 nm. By integrating a gated graphene layer in a silicon-waveguide based Mach-Zehnder interferometer, the key parameters of a phase modulator like change in effective refractive index, insertion loss and absorption change are extracted. These experimentally obtained values are well reproduced by simulations and design guidelines are provided to make graphene devices competitive to contemporary silicon based phase modulators for on-chip applications. The most widely used approach for realizing phase modulators in integrated Si photonic systems is based on p-n junctions (depletion or injection type) which provides high-speed performance enabling the generation of data rates up to 60 GBit/s4In modern optical high-speed communication systems, phase shift keying is the standard method for data modulation1Graphene, a two dimensional allotrope of carbon, is considered as a promising material for a wide range of photonic applications81011121314In this work, we report on the experimental demonstration of a broad-band electro-refractive phase modulator using graphene as active material. Key parameters of this device such as insertion loss, change in effective refractive index, and change in absorption are extracted from the experiments and simulations have been performed reproducing these values. The results are then compared to the state-of-the-art Si modulators using the typical figure of merits and an outline is given for realizing graphene modulators that can significantly outperform current Si based phase modulators.We use a stack of graphene-oxide-graphene embedded into one arm of a Si waveguide based Mach-Zehnder interferometer (MZI), where graphene is located in the evanescent field of the Si waveguide. The chemical potential of the graphene is changed electro-statically by biasing the two graphene layers with respect to each other. Therefore, the effective refractive index of one MZI arm is changed which causes a shift in the transfer function of the MZI.2O3 were deposited with atomic layer deposition (ALD) at 300 °C using O2 plasma and trimethylaluminium (TMA) as precursors. 2O3. A single layer of CVD grown graphene was transferred to the sample by the PMMA transfer method172O3 at 150 °C using water vapors and TMA as precursors, a second CVD grown single layer graphene, which acts as counter electrode, was transferred, contacted, and patterned using the same methods described for the first layer. In order to passivate the second graphene layer, another 40 nm of Al2O3 were deposited. Finally, vias were etched through the Al2O3 layers wet chemically to access the two nickel electrodes. An optical image of the final device is shown in 16172O3 on the sample (black spectrum), ii) after the first graphene layer was transferred, patterned, contacted, and covered by 90 nm Al2O3 (green spectrum) and iii) the final device (blue spectrum) with two graphene layers. These transmission spectra demonstrate clear interference pattern with a high extinction ratio of >15 dB for each mentioned step.All optical and electro-optical measurements were carried out in air at room temperature using a tunable continuous wave laser (1520–1620 nm) with 1 mW optical output power. To analyze the effect of each fabrication step on the transmission spectrum, the device was characterized at each stage of fabrication by measuring the transmitted optical power as a function of wavelength. 2O3 on top, the extinction ratio reduced from 22.5 dB to 17.5 dB  in between the two graphene layers. −4.where L, d and Δλ are the graphene length (200 μm), the spacing between minima (6.6 nm), and the wavelength shift with voltage V, respectively. 2/H2O redox couples at the graphene/dielectric interface23In an MZI, the change in absorbance Δα in one arm can be determined from the change in extinction ratio. As can be seen in 2O3 are taken from literature182-Si-HSQ-Al2O3-graphene-Al2O3 is considered with refractive indices of 1.44-3.48-1.38-1.64-ng-1.64, respectively  with TE mode propagating along the non-planar waveguide, which is an idealized situation of the stack used in the experiments. In the simulations, the complex optical conductivity of graphene (σ) is expressed as sum of intra-band and inter-band contributions which are determined using Kubo formalism given by27In addition to the experiments, simulations of the optical properties of the waveguide-graphene stack have been performed to get complementary information on the main optical parameters extracted in the experiments  and to explore the parameter space in terms of chemical potential and mobility. The simulations are based on the complex optical conductivity of graphene, which depends on the Fermi energy, the scattering rate and the temperature, and have been carried out using finite difference method in MATLABF) are taken as 300 K, and 0.9 × 106 ms−1, respectively10c, ξ, e, ω, ħ, kB and fd are the carrier scattering rate, chemical potential, energy, electron charge, radian frequency, reduced Planck’s constant, Boltzmann constant and the Fermi-Dirac distribution, respectively. In the simulations, Γ is varied from 5e11 to 1e14 s−1 in order to recognize its effect on the optical properties. These scattering rates correspond to charge carrier mobilities (μ) of 270 to 54000 cm2/Vs at μc = 0.3 eV /(ΓμC )), which are typically found in real devices. Since graphene was found to be p-doped for our device, the simulations have been discussed only for negative electro-chemical potentials here. However, the optical conductivity of graphene is symmetric for positive and negative electro-chemical potentials due to symmetric band structure in graphene1029where temperature (T), and Fermi velocity (vg (and hence refractive index ng) of graphene is related to its optical conductivity byThe dielectric constant εg = 0.33 nm is the thickness of graphene and ε0 is the permittivity of free space. Using finite difference method, values of neff and absorption have been calculated from eigen-solution of Maxwell equationwhere t2-Si-HSQ-Al2O3-graphene-Al2O3 stack. The eigen-solution of above Maxwell equation gives complex eigenvalues, with the real and imaginary parts representing neff and absorption, respectively. The simulated values of neff and absorption are plotted in c = 0 eV, independent on Γ and in good agreement to experimentally obtained value of 0.01 dB/μm. At μc < −0.4 eV a strong dependency of absorption on Γ is observed, as intra-band absorption becomes the dominating process there. In this regime, low Γ, corresponding to high carrier mobility, gives a lower absorption. In contrast to the absorption, neff shows only a dependency on Γ at the maximum value of neff around μc ~ 0.4 eV, and is effectively independent on Γ for higher and lower μc as is clear from where ∈ is dielectric permittivity tensor which takes into account refractive indices of SiOAfter the measured and simulated values of the graphene based phase modulator have been presented, a comparison with silicon based phase modulators can be given using different common figures of merit. We first start to discuss two important intrinsic figures of merit, which do not depend on the exact device layout, but only depend on the doping level and the scattering parameter in graphene, i.e. the insertion loss for a certain phase change and the ratio of change in refractive index to the change in absorption.c| > 0.5 eV, the absorption is significantly reduced due to Pauli-blocking of the inter-band contribution. The insertion loss of a scaled phase modulator, which can perform a phase shift of π, would be only 2 dB at μc = −0.6 eV and Γ= 1.25e13 s−1 (μ = 1080 cm2/Vs at μc = −0.6 eV). Here, lower Γ, i.e. higher carrier mobility, leads to an even lower insertion loss. This would be an improvement compared to Si MZI based phase modulators having an insertion loss of at least 4 dBFor the phase modulator realized in this work, the insertion loss caused by the intrinsic graphene absorption is 2 dB, while a phase shift of π/20 was achieved. This means that for a scaled device, which can perform a phase shift of π, the insertion loss would be ~40 dB, which is not acceptable for practical applications. These values are in good agreement to the simulations. In addition, the simulations suggest that at higher doping levels where |μ−1 (μc is varied from 0 to approximately −0.35 eV in our experiments). These scattering rates correspond to a carrier mobility of 500–1000 cm2/Vs at μc = −0.3 eV, a mobility typically measured in reference devices using the same fabrication process. The low Δn/Δα of 0.1 μm/dB means that for obtaining a phase shift of π, the light intensity is changed by 10 dB, which is unacceptable for most applications demanding constant light intensity. Again our simulations suggests that a significant improvement can be expected either for lower Γ or for higher doping levels where |μc|> 0.5 eV. Under these conditions, Δn/Δα can reach excellent values being larger than 1 μm/dB  are required. Such high doping levels are realizable with molecular doping3314In conclusion, an electro-refractive phase modulator, operating in the wavelength range of 1530-1570 nm, is realized experimentally using graphene as active material. Key parameters of the modulator such as absorption, Δn and Δα have been extracted from the experiment and reproduced with simulations. While the parameters obtained from experiments are far behind state-of-the-art Si based phase modulators, the simulations suggest that outstanding parameters for phase modulation can be achieved using graphene as active material. This requires first an enhanced interaction of the graphene with the waveguide mode and a stronger dielectric coupling between the two graphene layers in order to achieve competitive values of VHow to cite this article: Mohsin, M. et al. Experimental verification of electro-refractive phase modulation in graphene. Sci. Rep. 5, 10967; doi: 10.1038/srep10967 (2015).


The effects of substrate positions and graphene thicknesses on the morphological, structural, and optical properties were found to be very pronounced. By setting the substrate to be inclined at 90°, the growth of ZnO nanostructures, namely, nanoclusters and nanorods, on single-layer (SL) graphene was successfully realized at temperatures of 600°C and 800°C, respectively. For the growth on multilayer (ML) graphene at 600°C with an inclination angle of 90°, the grown structures show extremely thick and continuous cluster structures as compared to the growth with substrate’s inclination angle of 45°. Moreover, the base of nanorod structures grown at 800°C with an inclination angle of 90° also become thicker as compared to 45°, even though their densities and aspect ratios were almost unchanged. Photoluminescence (PL) spectra of the grown ZnO structures were composed of the UV emission (378–386 nm) and the visible emission (517–550 nm), and the intensity ratio of the former emission (IUV) to the latter emission (IVIS) changed, depending on the temperature. The structures grown at a low temperature of 600°C show the highest value of IUV/IVIS of 16.2, which is almost two times higher than the structures grown on SL graphene, indicating fewer structural defects. The possible growth mechanism was proposed and described which considered both the nucleation and oxidation processes. From the results obtained, it can be concluded that temperature below 800°C, substrate position inclined at 90° towards the gas flow, and ML graphene seems to be preferable parameters for the growth of ZnO structures by thermal evaporation because these factors can be used to overcome the problem of graphene’s oxidation that takes place during the growth.A seed/catalyst-free growth of ZnO on graphene by thermal evaporation of Zn in the presence of O Grap103 W/mK -18. Ther103 W/mK ,20. It iSince a decade ago, intensive researches have been focused on fabricating one-dimensional (1D) zinc oxide (ZnO) semiconducting nanostructures because it can provide a variety of important applications due to their unique morphologies, compositions, and chemical/physical properties ,22. For 2) gas [The most common method to grow ZnO on graphene is thermal evaporation due to its simplicity and high growth rates. Recently, we reported the seed/catalyst-free growth of ZnO on multilayer (ML) graphene by thermal evaporation of Zn in the presence of oxygen (O2) gas . The eff2) gas .2)/Si were used as the substrates . Figure 2. The growth was carried out by thermal evaporation technique in dual zone furnace as shown in Figure 2) gas (99.80%) were used as the sources. Prior to the growth process, the substrate was treated with organic cleaning of ethanol, acetone, and deionized (DI) water to remove any unwanted impurity on the substrate. Zn powder of approximately 0.6 g was spread evenly into a ceramic boat. The ceramic boat was placed in the zone 1 of furnace while the substrate was placed with inclination angles of 45° and 90° facing towards the flow of O2 gas in the zone 2 of furnace. The distance between source and substrate was fixed at 23 cm. This is a distance from center-to-center of two zones. Two independent temperatures were applied to the furnace system. Here, T1 denotes to the set temperature (ST) of the source while T2 denotes to the ST of the substrate. Firstly, the temperature of zone 2 was raised to T2  in argon (Ar) environment (Ar flow rate of 200 sccm). Then, the temperature of zone 1 was raised to T1 . The flow of Ar was stopped when the temperature of zone 1 reached 400°C . This was done in order to avoid the transfer of Zn particles to substrate prior to actual growth. The heating of Zn powder was continued until it reached 1,000°C. It was confirmed from several attempts that such high temperature of 1,000°C was needed for continuous and constant evaporation of Zn. After reaching 1,000°C, O2 (400 sccm) was introduced for 1 h of growth time. Finally, the furnace was turned off and the samples were cooled down to room temperature. It is noted here that the above procedures are identical with the procedures reported in ref. [Two kinds of graphene structures, i.e., single-layer (SL) graphene and ML graphene, grown by chemical vapor deposition (CVD) and transferred onto silicon dioxide  was calculated. Figure Figure 2 gas is introduced into the system. This oxidation process seems to be faster than the nucleation of Zn particles when the position of substrate is inclined to 45°. This can be proved where no nucleation of ZnO was obtained for the growth on 45°-inclined SL graphene, but the growth was successfully realized on 90°-inclined SL graphene. It is also speculated that 90°-inclined position seems to help in increasing the nucleation rates. As mentioned in the previous section, the nucleation of Zn on ML graphene is also promoted by the availability of graphene stacking structures which provide graphene edges as the additional nucleation sites [As reported in ref. , the nucon sites , the temon sites .Figure 6In conclusion, the effects of substrate position and graphene thickness on the morphological, structural, and optical properties were found to be very pronounced. Substrate position with an inclination angle of 90° seems to be one of the effective control parameters to increase the nucleation rates which were proven by the ability to grow ZnO nanostructures on SL graphene as well as the growth of thick and continuous ZnO cluster structures and thick base structures of ZnO nanorods on ML graphene. The structures grown at a low temperature of 600°C show fewer structural defects. The possible growth mechanism is proposed and described which considered both the nucleation and oxidation processes. Not only temperature but also substrate position and graphene thickness play significant role in determining the properties of the grown ZnO structures by thermal evaporation because these factors can be exploited to overcome the problem of graphene’s oxidation that takes place during the growth.


Background: Effects of drug abuse on LINE-1 retrotransposition (L1-RTP) in somatic cells are unknown.Results: Methamphetamine and cocaine-induced L1-RTP depends on CREB.Conclusion: Methamphetamine and cocaine activate CREB and L1-RTP in neuronal cell lines.Significance: To our knowledge this is the first study to demonstrate the induction of L1-RTP by drugs of abuse. Long interspersed element 1 (L1) is a retroelement constituting ∼17% of the human genome. A single human cell has 80–100 copies of L1 capable of retrotransposition (L1-RTP), ∼10% of which are “hot L1” copies, meaning they are primed for “jumping” within the genome. Recent studies demonstrated induction of L1 activity by drugs of abuse or low molecular weight compounds, but little is known about the underlying mechanism. The aim of this study was to identify the mechanism and effects of methamphetamine (METH) and cocaine on L1-RTP. Our results revealed that METH and cocaine induced L1-RTP in neuronal cell lines. This effect was found to be reverse transcriptase-dependent. However, METH and cocaine did not induce double-strand breaks. RNA interference experiments combined with add-back of siRNA-resistant cDNAs revealed that the induction of L1-RTP by METH or cocaine depends on the activation of cAMP response element-binding protein (CREB). METH or cocaine recruited the L1-encoded open reading frame 1 (ORF1) to chromatin in a CREB-dependent manner. These data suggest that the cellular cascades underlying METH- and cocaine-induced L1-RTP are different from those behind L1-RTP triggered by DNA damage; CREB is involved in drug-induced L1-RTP. L1-RTP caused by drugs of abuse is a novel type of genomic instability, and analysis of this phenomenon might be a novel approach to studying substance-use disorders. Alu (SINE-VNTR-Alu) NTR-Alu) . A singlefective . Nonetheefective , and ∼10efective , 4, and efective , 7. Lateefective –11, and fective ––15. L1 eective ––, 16, whoective ––, 18, wheective –– and reveective ––. ORF1 anective ––, 16, 21.Recent observations suggest that L1-RTP occurs in somatic cells and that copy numbers of L1 are increased in human brain tissues , 22. In e.g. Ser133 phosphorylation) is mediated by a number of kinases, including phosphorylated extracellular signal-regulated kinase (pERK) (28–The cAMP response element-binding protein (CREB), as the name suggests, binds to cAMP-responsive element (CRE), which is found in most cAMP-regulated promoters –30. CREB–e (pERK) . CREB ispERK) 28–, 33. CREpERK) 28–.Methamphetamine (METH) and cocaine are recreational drugs that cause addiction and are among the most widely used illegal drugs worldwide –38. Thes–42–In this study we found that METH and cocaine induce L1-RTP and that the induction of this activity by METH and cocaine depends on CREB. Biochemical analysis revealed that METH or cocaine recruits ORF1 to chromatin. To the best of our knowledge, this is the first study demonstrating the induction of L1-RTP by drugs of abuse. These data support the existence of CREB-mediated genome shuffling via L1-RTP; in this paper we discuss its possible involvement in psychiatric disorders associated with METH and cocaine use.2 in Eagle's minimum essential medium plus Ham's F-12 medium containing 1% nonessential amino acids and 15% fetal bovine serum . PC12, HeLa, HT1080, and SK-N-SH cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The transfection efficiency was ∼70% for HeLa and HT1080 and ∼30% for PC12, SH-SY5Y, and SK-N-SH cells as determined using fluorescence-activated cell sorting on day 2 after transfection of plasmid DNA encoding enhanced green fluorescent protein . Protease and phosphatase inhibitors were purchased from Roche Diagnostics . METH and cocaine were purchased from Dainippon Sumitomo Pharma Co., Ltd., and Sanseidou (Japan). A license for METH (no. 25-013) and cocaine (no. 1040) was issued by the governor of Hyogo. We prepared a 100 mm stock solution of the RT inhibitor, stavudine (d4T), in DMSO. About 30 min before the addition of METH or cocaine, d4T was added to a final concentration of 50 μm to the cell culture medium.SH-SY5Y (EC94030304), PC12 (EC88022401), HeLa (EC93021013), HT1080 (EC85111505), and SK-N-SH cells (EC86012802) were obtained from DS Pharma Biomedical. SH-SY5Y cells were maintained at 37 °C and 5% CO133, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase  were used as primary antibodies. A rabbit polyclonal antibody against human ORF1 was generated using the peptide MGKKQNRKTGNSKTQSAC as an immunogen  , γ-H2AX (Millipore), CREB , phospho-CREB (Seratories) . As secoR) . Cells were transfected with either pEF06R or pL1-NeoR using Lipofectamine 2000 (Invitrogen). The cells were selected for 2 days with puromycin (0.5 μg/ml) for pEF06R or with hygromycin (50 μg/ml) for pL1-NeoR. Next the cells were incubated for an additional 2 or 3 days with the indicated amounts of chemicals. For the PCR-based assay, genomic DNA was prepared from harvested cells using a DNA extraction system . For the semiquantitative PCR, primers that were designed to cover exons would amplify a product of ∼1040 bp (without L1-RTP), whereas they would generate a product of ∼340 bp (with L1-RTP) after L1-RTP (A). Thus, the occurrence of L1-RTP was determined by evaluating the size of the amplified product. After staining the amplified DNA with SYBR Green I , the signal intensity of the 340-bp bands was measured using a molecular imager  and normalized to β-actin (ACTB) bands, used as an internal control. EFGP was PCR-amplified using 5′-CTGGTAGTGGTCGGCGAGCTG-3′ and 5′-GACCACCCTGACCTACGGCGT-3′ as the forward and reverse primers, respectively. Amplification reactions were performed using the Ex TaqTM polymerase (TaKaRa) and the following cycling conditions: initial step for 15 min at 94 °C followed by 28–32 cycles of 30 s at 94 °C and 15 s at 68 °C. To detect ACTB as the internal control, 5′-GAGGGAAATCGTGCGTGA-3′ and 5′-AGAAGGAAGGCTGGAAAA-3′ were used as the forward and reverse primers, respectively. PCR amplification was performed using the following cycling conditions: initial 5 min at 94 °C, 20 cycles of 30 s at 94 °C, 30 s at 62 °C, and 1 min at 72 °C. The PCR product was analyzed in agarose gels and visualized with SYBR Green I Nucleic Acid Gel Stain.L1-RTP was assayed as described previously –49. For –R) , 51, forr L1-RTP A. Thus, 6 cells transfected with pL1-NeoR and selected with hygromycin (50 μg/ml) were transferred to 100-mm plates at ∼105 per plate. Next, the cells were incubated for 3 days with the selection and further cultured in the presence of G418 (270 μg/ml). After 3–4 weeks, cell aggregates were stained with methylene blue, and the number of colonies was counted. To determine the occurrence of L1-RTP, each of the six plates was incubated with chemicals or a buffer control, and the number of resulting colonies in each plate was counted.In the colony formation assay, ∼2.0 × 10R vector has a modified mneoI indicator cassette with a bacterial/mammalian promoter and the ColE1 origin of replication (R-Asp702Ala (D702A) contains a missense mutation in the reverse transcriptase domain of ORF2. The reverse transcriptase activity of ORF2 is attenuated by the D702A mutation  was used for reverse transcription, and amplification of cDNA was monitored and quantified using SYBR Premix Ex Taq (TaKaRa) on an Applied Biosystems 7900HT Fast Real-time PCR system (Invitrogen) according to the manufacturer's instructions. All data were normalized to ACTB. For the quantification of both endogenous and exogenous ORF2 mRNA, a primer set of L1-EGFP+5653F (5′-CCAAATGTCCAACAATGATAGACTG-3′) and L1-EGFP+5762R (5′-CCATGTCCCTACAAAGGATATGAAC-3′) was used. For the quantification of mature or precursor mRNA of EGFP, primer sets of L1-EGFP+6342F (5′- TAGTGGTTGTCGGGCAGCAG-3′) plus L1-EGFP+7351R (5′-TTCAAGATCCGCCACAACATC-3′) or L1-EGFP+7222F (5′-TGGAAGCTGGGTGTGTAGTTATCTG-3′) plus L1-EGFP+7365R (5′-GGCATCAAGGTGAACTTCAAGATC-3′) were used, respectively. For the amplification of ACTB-derived DNA, a primer set of 5′-GAGGGAAATCGTGCGTGA-3′ plus 5′-AGAAGGAAGGCTGGAAAA-3′ was used. To evaluate the levels of mRNA, each sample was analyzed in triplicate, and statistical analysis was performed based on the relative intensity of the mRNA levels normalized to ACTB levels.qRT-PCR was performed as described previously . Total RE). SH-SY5Y cells were transfected with pEF06R▵3′CMV, and qRT-PCR was performed. The results of 24-h incubation with either METH or cocaine were analyzed.To correctly assess the mature form of L1 mRNA in the case of pEF06R, it was necessary to exclude the effects of the antisense mRNA transcribed from the inverted 3′ CMV promoter . For thiFig. 2m Tris-HCl (pH 7.6), 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40 (radioimmune precipitation assay buffer), and protease inhibitors. After ultrasonication using Bioruptor  for 12.5 min  at the middle level of output (250 W) at 4 °C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific), and cell extracts were subjected to WB analysis. The blots were probed with the primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. The immune complexes were visualized using PierceTM Western blotting Substrate Plus (Thermo Fisher Scientific). WB results were documented and quantified using ImageQuant LAS 4000 mini and Amersham Biosciences Imager 600 .The cells were washed with phosphate-buffered saline (PBS) and resuspended in 50 mm METH or 0.5 mm or 0.25 mm cocaine. The extracts were subjected to WB analysis with α-ORF1 antibody as the primary antibody.SH-SY5Y cells were transfected with pEF06R, and either METH or cocaine was added to the culture medium on day 2. Total cell extracts were prepared 24 h after the addition of 0.13 or 0.06 mm siRNA using Lipofectamine RNAiMAX in SH-SY5Y and SK-N-SH. The nucleotide sequences of each siRNA are shown in R. Two days later, the cells were replated, incubated for 2 days with METH or cocaine, and subjected to analysis. As a control, Silencer Negative Control siRNAs  were used.Two small interfering RNAs (siRNAs) were prepared (Invitrogen), and their functions were evaluated by transfection into cells followed by WB analysis. We transfected 10 nB. To evaluate the roles of the target gene products, cells were transfected with CREB siRNA using Lipofectamine RNAiMAX for each gene on day 3 after the first transfection of pL1-NeoR. On day 4, pSiR-CREB  was isolated using the Subcellular Protein Fractionation kit (Thermo Fisher Scientific) with micrococcal nuclease as described previously (We used the pORF1-TAP (tandem affinity purification) construct , which eeviously , 48, 49.SH-SY5Y cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After incubation with PBS and 5% bovine serum albumin for 30 min, the cells were incubated with α-γ-H2AX antibodies. After 1 h of incubation at 37 °C, secondary antibodies conjugated with Alexa Fluor 555 (Invitrogen) were added for 1 h at 37 °C. Nuclei were stained with Hoechst 33258 (Invitrogen). The slides were mounted in an anti-fade solution and examined under a fluorescence microscope .U test. A p value of <0.05 was assumed to mean statistical significance.Statistical significance was evaluated using the Mann-Whitney R; A and B)  and a rat pheochromocytoma cell line . As shown in Fig. 1C, METH induced L1-RTP in different cell lines, including SH-SY5Y and PC12, but did not induce L1-RTP in a human fibrosarcoma cell line .We initially performed PCR-based and colony formation assays using the filtered drug of abuse and the plasmids pEF06R and pCEP4/L1mneoI/ColE1 (pL1-NeoA and B) , 50, 51.A and B) –49 in whm were detected (data not shown). We then tested if high doses of METH could induce L1-RTP in HT1080 and SH-SY5Y cells and whether low doses of METH could induce L1-RTP in PC12 cells (D). When METH induced L1-RTP, SH-SY5Y cells expressed EGFP (E). Next, we evaluated METH-induced L1-RTP using a colony formation assay in which L1-RTP activated the expression of a functional neomycin resistance gene and supported the growth of cells cultured in the presence of the cytotoxic drug, neomycin. According to the experimental procedures shown in Fig. 1F, HT1080 and SH-SY5Y cells were transfected with pCEP4/L1mneol/ColE1 , selected on hygromycin, and then treated with METH on day 3 after the transfection. Approximately 105 cells were incubated initially with 0.25–0.015 mm METH. After neomycin selection, we observed 50–100 colonies in each plate , indicating that the frequency of L1-RTP induced by METH was ∼1 per 1.0–2.0 × 103 SH-SY5Y cells . METH induced L1-RTP at a low concentration . In contrast, METH did not induce L1-RTP in nonneuronal HT1080 and HeLa cells .No cytotoxic effects of METH at concentrations up to 1 m12 cells D. When Msed EGFP E. Next, m using the colony formation assay in SH-SY5Y cells (A). After neomycin selection, we observed 50–100 colonies on each plate (A and B), indicating that the frequency of L1-RTP induced by cocaine was ∼1 per 1.0–2.0 × 103 in SH-SY5Y cells. On the other hand, ethanol and sodium barbital did not induce L1-RTP in SH-SY5Y cells (B). No cytotoxic effects of cocaine, ethanol, or sodium barbital were observed at the concentrations tested (data not shown). Next, using a mutant vector, we tested whether colonies induced by METH and cocaine were dependent on L1-RTP or if these colonies formed nonspecifically. The mutant pL1-NeoR vector used in this experiment was reverse transcriptase-negative (due to a D702A mutation). The results revealed that the colonies did not form at all (C). We conducted another experiment using an RT inhibitor (d4T). Previous studies reported a suppressive effect of d4T on L1-RTP . Thus, the results suggested that L1-RTP induction by METH and cocaine is a phenomenon dependent on RT.We next tested whether other drugs of abuse induced L1-RTP, such as cocaine, ethanol, and sodium barbital. We evaluated cocaine-induced L1-RTP at 0.13–0.5 m5Y cells A. After ch plate , A and B5Y cells B. No cytm at all C. We conn L1-RTP , 54. TheE, F, and G) or ORF2 mRNA (H) or the expression levels of the ORF1 protein (I).Moreover, we ruled out the possibility that METH and cocaine induce L1-RTP by up-regulating the expression level of L1 mRNA. qRT-PCR analysis revealed that neither METH nor cocaine increased the splicing efficiency of the immature L1 transcript , and we detected no focus formation of γ-H2AX after treatment of cells with METH at 0.13 mm or cocaine at 0.25 mm (C). These observations indicated that induction of L1-RTP by METH and cocaine was attributable to the nongenotoxic effects of these compounds.Farkash et al.  reportedf γ-H2AX , A and B 0.25 mm C. These A). Next we examined the involvement of CREB and found that two CREB siRNAs (CREB-1 and -2) efficiently suppressed the expression of endogenous CREB  and that both siRNAs abolished METH- or cocaine-induced L1-RTP . These data suggested that the siRNA abrogated the activity of endogenous CREB and inhibited L1-RTP. To confirm this hypothesis, we carried out an add-back experiment using siRNA-resistant CREB cDNA. We confirmed that the transfection of a plasmid DNA expressing a siRNA-resistant FLAG-tagged CREB mRNA (pSiR-CREB) could restore protein expression that had been reduced by the siRNA . Subsequently, we found that pSiR-CREB restored the formation of NeoR colonies, which had been suppressed by the siRNA . Moreover, we confirmed these experiments on another cell line. When the experiments with CREB siRNA and added-back siRNA-resistant CREB were performed on the SK-N-SH human neuroblast cell line, the results were the same as with SH-SY5Y cells (data not shown). These data indicated that CREB was involved in METH- or cocaine-induced L1-RTP.To determine the mechanism behind METH- and cocaine-induced L1-RTP, we assessed the involvement of CREB. According to a recent report, METH and cocaine induce the phosphorylation of CREB . To confFig. 4et al. . The METH- or cocaine-induced chromatin recruitment of ORF1 was blocked by both types of CREB siRNA . These data indicated that METH and cocaine separately mobilized the L1 component to chromatin via a mechanism involving the activation of CREB.Goodier et al.  reportedin vitro. In this study the induction of L1-RTP was found to be induced by METH or cocaine in neuronal cell lines such as SH-SY5, PC12, and SK-N-SH but not in nonneuronal cell lines such as HeLa and HT1080. Therefore, it appears that METH or cocaine induce L1-RTP only in neuronal cells. METH induced L1-RTP at low concentrations  and caused fluctuations in the levels of intracellular calcium. When the intracellular calcium concentration increases, calcium interacts with intracellular calcium-binding proteins and produces various physiological effects. Hondebrink et al. (m) stimulate an acetylcholine-evoked calcium increase. Moreover, depolarization-evoked calcium increase is inhibited after exposure to high METH concentrations (1 mm) (C and D).Recently, the incidence of psychiatric disorders associated with abuse of drugs such as alcohol and METH has been increasing worldwide . It is nk et al.  showed ts (1 mm) . The chas (1 mm) . Thus, Ms (1 mm) , C and DWe found that METH- or cocaine-induced L1-RTP are not involved in the mechanisms of DSBs. METH or cocaine did not up-regulate γ-H2AX (H2AX phosphorylated at serine 139) , which iCREB cDNA does not induce L1-RTP (F–I). These findings suggest that CREB functions as the mediator for METH- and cocaine-induced L1-RTP. It is possible that this specific factor induces changes in chromatin structure and relevant transcriptional network, thereby leading to psychiatric disorders.In a variety of species and types of memory, the process of consolidation of a new memory requires gene expression that begins during training. The proteins of the CREB transcription factor family are evolutionarily conserved regulators of this gene expression , 34. CREe L1-RTP . CREB, ae L1-RTP , F–I. Thet al. (Recent evidence demonstrated that neurons actually support high levels of L1-RTP ; for exaet al.  reported


In addition, the extent of PD-1/PD-L1 down-regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD-L1 down-regulation is associated with increased T cell (CD4+ and CD8+) proliferation, increased type 1 effector cytokines (IL-2 and IFN-γ) secretion, and decreased type 2 effector cytokine (IL-10) secretion. Then, PD-L1 up-regulation by the stimulation of IFN-α can significantly convert this representation. Collectively, our report suggest that T(CD4+)/mDCs-associated PD-1/PD-L1 pathway plays a key role in AF immune regulation. PD-1/PD-L1 down-regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.Although immuno-inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD-1 and PD-L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD-1 on CD Atrial fibrillation (AF) is the most common arrhythmia, but the pathophysiological mechanism leading to this condition remains poorly understood. However, increasing evidence suggests the influence of an immune-mediated inflammatory response in AF pathogenesis 4+ and CD8+ T cells, NK cells, B cells and monocytes upon activation. It is engaged primarily by its ligands PD-L1  and PD-L2  4+ and CD8+ T cells Programmed death-1  is a CD28 homolog and costimulatory molecule expressed on CD4+ T cells or mDCs in AF patients, and that the extent of the down-regulation is closely related with the AF burden. Additionally, PD-1/PD-L1 down-regulation significantly enhanced CD4+ and CD8+ T cell proliferation and influenced cytokines secretion in vitro. Our study indicates that the PD-L1 may be induced by IFN-α. We further up-regulated expression of PD-L1 on mDCs by IFN-α treatment and found that mDC-mediated T cell proliferation was suppressed and the pattern of cytokine secretion was converted. This suggests that PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs may modulate immune regulation and play a role in AF pathogenesis.In this study, we examined the expression profiles of PD-1 on T cells and PD-L1 and 2 on T cells, myeloid dendritic cells (mDCs) and macrophages in patients with paroxysmal and persistent AF, to assess their roles in AF pathogenesis. We found that PD-1/PD-L1 expression is substantially down-regulated on CDn = 42) and the persistent AF group (n = 49). The diagnosis of AF was adopted in the ACC/AHA/ESC 2006 guidelines on AF i) episodes of paroxysmal AF lasting less than 6 hrs each and (ii) a sum of paroxysmal AF lasting largely less than 30% of the total time. The frequent paroxysms group (group F) consisted of 15 patients with at least one of the following characteristics: (i) a total time of recurrent paroxysmal AF lasting largely longer than 30% of the total time and (ii) at least one episode of AF lasting more than 6 hrs. The other 10 patients who did not meet the above criteria were excluded. The time of paroxysms was mainly judged by ECG, holter monitor or symptoms of palpitations. This method of grouping can depict the arrhythmia burden and the risk for adverse events Between May 2010 and January 2012, a total of 91 patients with a diagnosis of AF were enrolled in the Department of Cardiology of Tangdu Hospital, Xi'an, China. The AF patients were divided into two groups: the paroxysmal AF group , lymphocytes and white blood cell counting according to routine protocols. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by Ficoll–Hypaque  density gradient centrifugation, and were viably frozen in 80% foetal calf serum , 10% dimethylsulphoxide and 10% RPMI 1640 medium (Gibco) in liquid nitrogen for subsequent analysis.5 cells were counted in each analysis. The results were processed with FCS express V3 software and described with the percentage of positive cells.The following monoclonal antibodies (mAbs) were used: CD3-FITC , CD8-PE , CD4-APC , PD-1-PE-cy7 , PD-L1-PE , PD-L1-PE-cy7 , PD-L2-PE , lineage-1-FITC, HLA-DR-PerCP , CD11c-APC , CD14-APC . All Abs were purchased from BD Biosciences . PD-1 expression on T cells, PD-L1 and PD-L2 expression on mDCs, macrophages and T cells were measured, respectively, using fresh heparinized peripheral blood. In brief, blood samples were stained with a mixture of Abs , as well as isotypecontrol antibodies, for 30 min. at 4°C. The cells were then processed with FACS lysing solution  to remove RBC, fixed and analysed by four-colour flow cytometry on a FACSCalibur (BD Biosciences). A total of 1 × 104+ T cells were positively isolated from PBMCs using the CD4 Positive Isolation Kit . MDCs were obtained from PBMCs by CD19-negative selection, followed by CD1c-positive selection using the CD1c (BDCA-1) Dendritic Cell Isolation Kit (Miltenyi Biotech) with a MidiMACS separator unit. All procedures were performed according to the manufacturer's instructions. CD4+ T cells or mDCs purity exceeded 90%. Unless otherwise stated, freshly isolated cells were carefully collected and suspended in RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. MDCs were treated for 72 hrs in a 96-well plate in RPMI 1640 medium containing 10% FCS only or with saturating doses of IFN-α . Cells were collected at 0, 24, 48 and 72 hrs of in vitro cell culture. The expression of PD-L1 was analysed immediately on a FACSCalibur (BD Biosciences).CD5 cells/200 μl) for 24 hrs. In some cases, IFN-α  was added in the culture medium. On the following day, mDCs were mixed at different concentrations  with purified CD4 or CD8 T cells (1 × 105 cells/200 μl) from a healthy individual in the presence of 2 μg/ml anti-B7-H1  or control antibodies  for 5 days. Cells were pulsed with 1 μCi/well of [3H]thymidine  for 18 hrs before harvest. Before measurement of incorporated [3H]thymidine, part of the culture supernatants was collected for cytokine detection.Purified mDCs cultured with poly(I:C)  were performed in 96-well flat-bottom culture plates (at 1 × 10Cytokine assessment was carried out using ELISA Kit (eBioscience) for three cytokines  of culture supernatants. All procedures were performed according to the manufacturer's instructions. Absorbance was measured on an automatic plate reader. The sensitivity of these ELISA kits were 1 pg/ml for IL-10, 2 pg/ml for IL-2 and 4 pg/ml for IFN-γ.anova was employed for comparing means of multiple groups, followed least significant difference (LSD) for paired comparison. The Mann–Whitney U-non-parametric test was used for differences between non-parametric analyses. To evaluate differences in categorical variables between study groups Pearson's chi-squares test was used. All P-values were two-tailed and values <0.05 were considered statistically significant.All experiments were repeated three times. Quantitative data were expressed as mean ± SD, and categorical variables as absolute and relative frequencies (percentages). The statistic software SPSS 19.0 was employed for statistical processing. Continuous data were tested for normal distribution by the Kolmogorov–Smirnov test and were found to be suitable for parametric analyses. One-way P > 0.05). β-blocker as heart rate-slowing agent was used significantly more in AF patients groups than control group (P < 0.001). AF patients groups had higher CRP levels compared with control group and CRP level in persistent AF group was further higher than paroxysmal AF group .The characteristics of the study participants in this study are presented in Table4+ and CD8+ T cells in paroxysmal AF patients, persistent AF patients and control group. Representative dot plots of PD-1+ distinct subpopulations were shown in Figure4+ T cells in AF patients was significantly decreased (P < 0.001) than healthy controls (12.04 ± 1.95). Compared the two groups, we found persistent AF patients (1.79 ± 0.32) expressed a lower level of PD-1 than paroxysmal AF patients , persistent AF group (3.61 ± 0.54) and control group  than healthy controls (0.95 ± 0.19), and persistent AF group (0.25 ± 0.08) was lower than paroxysmal AF group /mDCs interactions might participate in the pathogenesis of AF.We further detected PD-L1 and PD-L2 expression on the function cells. Representative dot plots of PD-L1+ and PD-L2+ distinct subpopulations were shown in Figuresoups Fig., F and H4+ T cells. To further explore this phenomenon, we examined a paroxysmal AF group with a 3-month follow-up. We divided them into two subgroups (group F and group O) and measured PD-1/PD-L1 expression on CD4+ T cells and mDCs. The results showed that PD-1+ distinct subpopulations of CD4+ T cells and PD-L1+ distinct subpopulations of mDCs in group F  were significantly lower than group O  were more efficient in inducing CD4+ and CD8+ T cell proliferation at all ratios tested compared with mDCs from controls . It suggested that the T cell-stimulating capacity of mDCs from AF patients was enhanced. Then, we examined the effects of mDC-associated PD-L1 up-regulation on allogeneic T cell proliferation in the presence of IFN-α. We found that the upgrade capacity of mDCs from AF patients to stimulate CD4+ and CD8+ T cells proliferations was restored, leading to results similar to that of normal mDCs . Furthermore, we examined the effects on allogeneic MLR in the presence of anti-PD-L1. As expected, we found that the restored capacity of mDCs from AF patients to stimulate CD4+ and CD8+ T cells proliferations was up-regulated again by treated with anti-PD-L1 . These data suggested that the down-regulation of PD-L1 on mDCs appears to transmit stimulating signal to CD4+ and CD8+ T cells in AF patients, resulting in an increase of proliferation and PD-L1 up-regulation on mDCs can reduce the allostimulatory capacity of mDCs from AF patients.In the allogeneic mixed leukocyte reactions (MLR), we cocultured mature mDCs from persistent AF patients and healthy volunteers at different ratios with T cells from a third healthy individual and studied how the decreased PD-L1 expression on mDCs in AF patients influence the T cells proliferation. The results showed that mDCs of AF patients /mDCs-associated PD-1/PD-L1 pathway in immune regulation of AF. Our results suggest that down-regulation of PD-1/PD-L1 on CD4+ T cells or mDCs might, at least in part, take part in AF pathogenesis. Firstly, PD-1 expression on CD4+ T cells and PD-L1 on mDCs is down-regulated in AF patients compared with controls. Secondly, down-regulation of PD-1/PD-L1 was more significant in persistent AF patients, those considered to have a more pronounced inflammatory state compared with paroxysmal AF patients. This suggests that the extent of PD-1/PD-L1 down-regulation may be closely related with AF burden, which is further supported by the subsequent follow-up investigation. Thirdly, allogeneic MLR experiments indicate that low-level mDCs PD-L1 expression promotes T cell proliferation, suggesting that the PD-L1 levels may be linked with the inflammatory state. Cytokine secretion changed with mDC PD-L1 levels, further supporting this assertion. Collectively, PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs may participate in the process of immune regulation and play a role in AF pathogenesis.In this study, we comprehensively investigated the expression of PD-1 and PD-L1 and 2 on key regulatory cells including mDCs, macrophages, and CDRegarding the underlying mechanisms about how PD-1/PD-L1 down-regulation modulates AF pathogenesis, we speculate on the following possibilities. Firstly, PD-1/PD-L1-associated T cell proliferation may contribute to atrial electrical remodelling. T cell proliferation may also increase secretion of cytokines, such as IL-6 and MCP-1, which may affect the contractility and electrical stability of myocytes differently 4+ and CD8+ T cells in AF patients was not statistically different compared with controls, suggesting that PD-1/PD-L1 inhibitory signals between T-T cells may not participate in regulating T cells. Importantly, the expression of PD-L2 on mDCs, macrophages, and CD4+ and CD8+ T cells in AF patients was not statistically different compared with controls. Our study suggests that PD-L2 indeed is expressed on CD4+ T and CD8+ T cells, consistent with earlier studies 4+)/mDCs-associated PD-1/PD-L1-independent mechanism in AF regulation.In this study, we investigated PD-L1 expression on several cell types, and mDCs have been proposed to be responsible for specific T cell regulation in AF. As a subpopulation of DCs, mDCs express elevated levels of MHC and a range of accessory molecules involved in cell migration, adhesion and co-signalling It is important to note that several limitations that need to be addressed regarding this study. First, to supporting the suggestion that the PD-L1 levels may be linked with the inflammatory state, more cytokines except IL-2, IL-10 and IFN-γ need to be examined. In addition, TNF-α as another important inflammatory cytokines may be not relevant to PD-L1 change in MLR (data not shown). The specific mechanisms is unclear and need further research. Second, although the PD-1/PD-L1 pathway appears to be important, additional co-inhibitory pathways such as those involving CTLA-4 and B7 interactions cannot be excluded in AF pathogenesis. Further study will be needed to determine whether the PD-1/PD-L1 pathway is the only mechanism or if it is just one that regulates the immune response in AF. Third, the specific role of the PD-1/PD-L1 pathway in AF is not clear and it remains unclear whether PD-1/PD-L1 down-regulation is a cause of AF, or merely a consequence.4+ T cells and PD-L1 on mDCs in AF patients. The extent of PD-1/PD-L1 down-regulation was closely related with AF burden. Furthermore, PD-1/PD-L1 down-regulation could improve, at least in part, T cell proliferation and cytokine secretion change, which reflect the inflammatory state and contribute to AF pathogenesis. Therefore, PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs participates in the pathogenesis of AF and up-regulating the expression of PD-1/PD-L1 molecules may represent a new therapeutic option for AF.In summary, the present study begins to shed light on the role of immune pathogenesis in AF, showing that PD-1 is down-regulated on CD


Graphene, in single layer or multi-layer forms, holds great promise for future electronics and high-temperature applications. Resistance to oxidation, an important property for high-temperature applications, has not yet been extensively investigated. Controlled thinning of multi-layer graphene (MLG), e.g., by plasma or laser processing is another challenge, since the existing methods produce non-uniform thinning or introduce undesirable defects in the basal plane. We report here that heating to extremely high temperatures (exceeding 2000 K) and controllable layer-by-layer burning (thinning) can be achieved by low-power laser processing of suspended high-quality MLG in air in “cold-wall” reactor configuration. In contrast, localized laser heating of supported samples results in non-uniform graphene burning at much higher rates. Fully atomistic molecular dynamics simulations were also performed to reveal details of oxidation mechanisms leading to uniform layer-by-layer graphene gasification. The extraordinary resistance of MLG to oxidation paves the way to novel high-temperature applications as continuum light source or scaffolding material. Graphene has been considered one of the most promising materials for generating novel technological applications in near future. This exciting material presents exceptional electrical1357Most of these studies have been so far focused on single layer graphene (SLG) structures. However, large-scale production of high quality SLG and its manipulation have remained technologically challenging91112In particular, SLG is a flexible material that can be easily deformed when deposited on rough surfaces and it tends to fold up during processing in liquids, e.g., during transfer to other substrates16171920221920Recently, we have observed26MLG ~ 2000 K) without instantaneous burning out the samples. This strong heating is possible for suspended samples since the main heat losses in this case are due to heat conduction to air (<105 W/m2 K)−5 W. Losses due to continuum radiation emission are much smaller, estimated to be ~10−8 W, so that TMLG up to 2000 K or even higher can be achieved, for laser power of 1 mW and samples with surface area ~10 μm2.Here, we further exploit these ideas comparing exposure of supported (deposited over solid substrates) and suspended MLG samples to localized laser heating in air. It is important that experiments were performed under cold-wall reactor conditions using a low-power (a few mW) laser focused at the sample, where only a small solid sample is heated by the laser, while the surrounding gas is maintained basically at room temperature. Low gas temperature implies extremely low dissociation degreeExperimental configurations for laser heating of suspended and supported nanoplatelets are shown schematically in The observed uniform and gradual thinning of suspended platelets under localized low-power laser heating is in striking contrast to most of reports published so far considering burning of graphitic materials exposed to air or oxygen under high temperatures, as well as to our experiments with supported samples. Extremely high thermal conductivity of high-quality graphitic materials  in graphitic sheets3031193536373839The temperatures estimated from the Raman spectra are surprisingly high, up to 2000 K or even more. On the other hand, we also performed radiation spectra measurements in visible and near-infrared spectral region that confirmed strong increase of the radiation background corresponding to very high MLG surface temperatures see , and theAs can be seen from 19In MD simulations show that the etching process starts when atomic oxygen binds to a graphene layer forming epoxy structures. The presence of the epoxy groups allows carbon atoms to be extracted when more atomic oxygen reacts with the group, forming vacancies. These observations are in excellent agreement with the ones reported by Duin and co-workers3 levels are kept at very small values and tend to zero as temperature reaches even higher values. For the highest temperature values analyzed, CO formation is once again favored causing a drop on the CO2 levels, as the burning process becomes considerably faster as seen in It is important to stress that different gasification regimes are observed at different temperature ranges. Carbon dioxide formation is favored at low temperatures, characterizing a complete burning process. As the temperature reaches higher values, carbon monoxide formation becomes favored and carbon dioxide levels decrease. At this point, the formation of carbon trioxide can also be observed, but COThe reasons for the extraordinary oxidation resistance observed here in the suspended multi-layer graphene and burning under high-temperature laser heating in air are due to differences between our set-up and the ones usually employed in hot-wall reactor experimentsIn summary, we presented a novel approach for processing of suspended high quality multi-layer graphene platelets by irradiation using focused low-power laser in air. For comparison, processing of supported MLG was also studied. The gas was kept at room temperature due to the “cold-wall” reactor set-up. Irradiation by laser was performed using a confocal Raman spectroscopy configuration allowing for simultaneous monitoring of the temperature and quality of samples. We have shown that laser heating results in very high temperatures (up to 2000 K or even more) and the burning process happens in completely different ways for suspended and supported samples. When MLG flakes were supported on a solid (oxidized silicon) substrate, holes through many layers are formed in the heated region. However, for suspended graphene samples, a uniform thinning through selective layer-by-layer etching has been achieved, without significant changes in lateral dimensions of the samples. The analysis of data obtained using Raman spectroscopy during processing of suspended samples indicates also that formation of initial defects within the basal planes exposed to gas is followed immediately by their complete removal through fast in-plane etching. In other words, under the present hot-sample/cold-gas conditions the time required for the formation of initial defects in a basal plane is much larger than that required for burning out of a damaged graphitic layer. Reactive molecular dynamics simulations were used to gain further insights on these processes and are fully consistent with these interpretations.We have shown that with using a simple technique it is possible to controllably burn graphene layer by layer, thus allowing to obtain few layers or even single layer graphene. We would like to remark that using suspended multi-layer graphene samples  as proposed here, two challenging graphene problems can be solved: high quality controlled thinning of multi-layer graphene and achieving high graphene temperatures, even when exposing samples to atmospheric air. The results can provide new ways to achieve even stronger heating of multi-layer graphene in vacuum or in different controlled gas environments. The new methodology presented here can be adapted to the fabrication of few and multi-layer graphene of controlled thickness with remarkable low defect density. At the same time it paves the way for a new series of graphene applications, such as continuous radiation source and/or as scaffolding material stable at extremely high temperatures. We hope the present work will stimulate further investigations along these lines.MLG flakes were produced from high quality natural graphite  using mild ultrasound processing (sonication) in N,N-dimethylformamide (DMF) solutionsThe gradual and uniform thinning of samples was confirmed in a large number (>30) of experiments with different sizes of samples , applied laser power (from 0.1 to 1 mW) and exposure time .−1/K was measured, while for single layer and bi-layer graphene higher values were obtained (−0.016 cm−1/K and −0.015 cm−1/K). No data are available in literature for conversion coefficients at higher temperatures. Maximum G band downshift values observed here were as high as 30 cm−1, corresponding to near 3000 K or 2000 K maximum sample temperatures if −0.011 cm−1/K or −0.016 cm−1/K conversion coefficients  are used. Alternative method of graphene temperature determination is based on comparison of intensities for Stokes and anti-Stokes components of the G bandFrom In heating experiments, we usually kept the laser power below 1 mW. When elevated laser power (>1 mW) was used, samples were observed completely burned out in a few seconds , and we were able to observe the exact moment of the sample disappearance through the backscattered Raman signal. Gradual decrease of the Raman bands was evident for thickness of samples smaller than 10 nm allowing avoiding the complete sample burning.To characterize changes in the shape and thickness of MLG platelets we used high resolution SEM images  taken before and after heating procedure. For MLG flakes deposited onto Si-substrate we also made AFM (atomic force microscopy) measurements (NT MDT) to see profile change at the place where laser beam was focused.We processed a large number of suspended samples, and measurable changes in the thickness and/or lateral size were detected for 24 of them (some of flakes were burnt out completely or fell down as a result of processing). Significant decreases of the surface area and of the thickness during the heating procedure were observed and measured for 18 and 8 flakes, respectively.et al.For the supported flakes, the in-plane etching rates were calculated by estimating the change on the area before and after laser heating from SEM images, normalizing it by the average perimeter of the flake and then dividing this value by the time of laser heating. The cross-plane etching rate was calculated by dividing the thickness change by treatment time. For the supported flakes the cross-plane etching rate was calculated following the approach proposed by Pop 2) exposed to an atomic oxygen atmosphere .


Medicinal products that have been developed and approved for one disease may be the object of additional clinical development in other disease areas or of additional pharmaceutical development for new and different formulations. The newly developed products can be named as repositioned or reformulated products, respectively. Market access of repositioned or reformulated products in Europe and the United States is an interesting object of study as it may provide clarity about which parameters are assessed and considered to bring added value, other than the molecule itself. As such, we aim to evaluate if the added value of repositioned or reformulated medicinal products can be systematically described, quantified, and predicted. As a first step toward investigating the impact of market access on drug research and development trends for repositioned and reformulated products, it is necessary to have consistency in the designations for the case studies evaluated in this project. In an attempt to achieve that consistency, the current study aims to propose harmonized definitions for the repositioning and reformulation strategies and to propose a taxonomy for the medicinal products derived thereof.A systematic literature review was conducted to collect information on existing cases of repositioning or reformulation. A search strategy was developed by defining the search objectives, targeted data sources, search keywords, and inclusion/exclusion criteria for the retrieved documents.A total of 505 publications were retrieved through a search of the main data sources. The screenings and the ad hoc search led to a total of 56 publications to be used for the case study data extraction. In total, 87 repositioning and/or reformulation cases were found described in the literature, 23 of which presented different definitions and/or classifications by different authors.Given the disparity and inconsistency of terminologies and classifications in the literature, a harmonized nomenclature for drug repositioning, reformulation, and combination cases will allow for a robust analysis of the added value and market access conditions attributed for each strategy and case type as assessed by regulators and payors in Europe and the United States. After evaluation of the existing terminologies and given the absence of clear and consistent definitions for drug reformulation and repositioning in the literature, we propose a global terminology and taxonomy in order to cover all of the previously unclear definitions and classifications for repositioned and reformulated products. De novo drug development is costly ($1.3 billion) and time-consuming (10–17 years) (ostly $1. billion 7 years) .As a result of such market forces, pharmaceutical companies are looking for cost-effective and reduced-risk strategies for developing drug products and protecting existing products from competition as well as extending their patent protection time . Develop‘Reformulation’ is the development of different formulations for the same pharmaceutical drug , whereasThe study of the added value attributed by regulators and payors to potential new development directions for existing drugs is of interest to developers who wish to maximize the potential of their products and sustain their pipelines. As such, identifying opportunities and a rationale for market access of ‘repositioned’ and ‘reformulated’ drugs is a subject of high importance. In addition, market access of repositioned and reformulated products can help to illustrate the parameters that are assessed and considered to bring added value, other than the molecule itself. The regulatory strategies and regulatory pathway trends selected for repurposed drugs might vary considerably and also impact the market access conditions attributted for such products. However, the lack of consensus regarding a harmonized taxonomy for repositioned and reformulated products makes it difficult to identify and review existing cases.Part I: Reassessment of nomenclaturePart II: Regulatory pathPart III: Market access implicationsTherefore, we propose a three-part publication with the ultimate goal of understanding the rationale and eventually predicting the impact on market access and conditions attributed to drug reformulation and repositioning in the pharmaceutical industry in Europe and the United States:As a first step toward investigating the market access association with drug research and development trends for repositioned and reformulated products, it is necessary to have consistency in the designations for the case studies that are underlying this project. In an attempt to achieve that consistency, the current study aims to propose clear definitions and classifications for repositioning and reformulation strategies and to propose a harmonized taxonomy for the medicinal products derived thereof.Collect and describe potential cases of drug repositioning and reformulation mentioned in the literature.Describe and evaluate the circumstances that lead to drug repositioning and reformulation.Describe and evaluate the classification and nomenclature used for drug repositioning and reformulation in the literature.Evaluate the possible implications of existing regulations and guidelines for the development and market approval of drug reformulation/repositioning.Describe the success criteria to be achieved for drug repositioning and reformulation, from a payor perspective.Evaluate the impact of pricing and reimbursement requirements, regulations, and evaluations for the market access of repositioned drugs and reformulated drugs, and if these can be forecasted.Evaluate the potential lost benefit for patients and the society as a whole in cases where pricing or reimbursement cannot be achieved.A systematic literature review was conducted to collect information on the identified repositioning and reformulation cases. In line with the project's main goal, which is to research reformulation and repositioning cases in order to evaluate the impact on pricing and reimbursement decisions in Europe and the United States, the following objectives were set for the literature research:The present study focuses on objectives 1, 2, and 3 in order to evaluate the consistency of nomenclature of drug reformulation and repositioning cases.ad hoc search facilitated the retrieval of publications covering some of the topics of interest. In line with the objectives of the project, a list of keywords and a search algorithm were established  were considered for the search. For the initial selection, it was deliberately decided not to have a time limit regarding the time of publication. Any literature that mentioned only potential candidates for drug repositioning or reformulation were excluded given the impossibility to assess the impact on market access for these products.All the sourced references were imported to a Data Manager file. The list of titles and abstracts were screened (1st selection process) and once the full publications were collected, according to the process previously described, they were thoroughly screened (2nd selection process).ad hoc search allowed for the inclusion of eight more pertinent articles and four proprietary reports, which resulted in a total of 56 publications for data extraction.The search in the Embase database sourced 505 publications. After screening for duplicates and analyzing titles and abstracts, 87 publications were selected. The reasons for rejection were recorded . From thThe selected documents were published between 1995 and 2012. As shown in Our results included 125 cases of reformulation and repositioning. By using basic definitions for reformulation, ‘a new formulation of existing product’, and for repositioning, ‘a new indication for a known drug’, the number of cases was reduced to 87 after eliminating duplicates. In particular, some cases of repositioning such as sildenafil, thalidomide, and imatinib were frequently found. Timing of launch of the new productRepositioning or reformulation can be done with drugs that were never commercialized for their originally developed goals. This was the case with sildenafil for its first repositioning from angina to erectile dysfunction. Several cases were found for marketed products, including specific situations of discontinuations. A notorious example of that situation is that of thalidomide, which was repositioned twice after being tragically withdrawn from the market , 5, 7.Availability of generics of the original product when the change occurred® were already available when the new product was launched. It is common that line-extension drugs are introduced prior to the introduction of generics (®) was reformulated 1.5 years prior to patent expiry  and Paxil® (paroxetine), respectively  and minoxidil , 7, 18.In contrast, a rational approach relies on the understanding of the disease physiopathology and/or the drug mechanism. Imatinib is an illustrative example: the understanding of the role of KIT tyrosine kinase was the trigger for initiating the preclinical studies for gastrointestinal stromal tumors , 18, 23.Pharmacological target compared to the original productSeveral drugs, including duloxetine, sunitinib, and everolimus, were found to be effective in another disease via the same pathway or protein interaction. This is what some authors refer to as ‘on-target repositioning’ , 5.‘Off-target repositioning’ is defined as a new therapeutic use based on a newly discovered (or previously unexplored) additional pharmacological mechanism for a known drug . This waTherapeutic area compared to the original productIn many cases, the new and original indications are within the same therapeutic area [trastuzumab, sunitinib , and pacThe same drug product can be found effective in a different therapeutic area. There are many examples of on-target repositioning that fit in this category, including that of finasteride , 17, 18.The events and processes leading to the repositioning of a product are diverse. Repositioned products are classified based on three main criteria.Modified release formulations®)  technology for mesalazine tablets (Mezavant XL®) .Change in pharmaceutical form and/or change in administration routeChanges in the pharmaceutical form can be varied and can include no change in the administration route, such as the microemulsion form of cyclosporine A and the syrup form of valproic acid, both for oral use , 16. A cExcipients change without impact on pharmacokinetic parameters®) when reformulated to Astepro®   can be modified to form a new formulation. This modification ranges from a ‘simple’ chiral switch, such as escitalopram and esomeprazole , 12, 26,®) .The change of specific ingredient and structural change of the active compound may involve the change of chemical, physical, or clinical parameters as illustrated in Cases of reformulation are particularly frequent in the realm of psychiatry because non-adherence to treatment is a key issue in the treatment of mental illnesses , 12, 25.®) (®)   or in th®) (®) .Many definitions were available in the literature for drug repositioning and reformulation. There was an unclear definition and limitation on what is to be considered ‘true’ repositioning or reformulation, if these can include or exclude ‘simple’ line extensions and indicate how to define and denominate the cases when the drug is simultaneously developed through a repositioning and a reformulation process. Additionally, it wasn’t clear in the literature in which classification category can drug combinations be included. This was also the situation for the cases with structural changes of the active ingredient such as chiral switch, prodrugs or use of active metabolites.This lack of consistency was also observed in the classification criteria of the cases. Some authors based their classification on the change of pharmacokinetic properties, while others used a classification based on the timing of the original product's lifecycle. Furthermore, other authors based their classification on the differences in the approach used for discovery of indication or on the commercial success of the repositioned product.Our research findings confirmed the non-availability of a complete and homogeneous definition for both drug repositioning and reformulation. Thus, different authors have been developing and using different, and sometimes, even contradictory definitions , 28, 29.One of the common points in the different definitions used by different authors was that drug repositioning is defined as the process of finding a new use for an already known drug. However, definitions diverged in several parameters, such as the exact meaning of ‘new use’ and ‘known drug’. Some authors consider a new indication within the same/similar therapeutic use as repositioning, while others exclude this category from the repositioning definition. Another case was the so-called ‘geographic repositioning’, which is considered by some authors as a repositioning category while this particular case does not meet the definition criteria of discovery of a new use.Some authors considered reformulation as part of drug repositioning efforts. Also, differences regarding the classification of types of reformulation were found in the literature.Drug repurposingThe term repurposing includes all the re-development strategies based on the same chemical structure of the therapeutically active ingredient as in the original product. More precisely, under this term, we regroup drug repositioning, reformulation, and combination strategies.Drug repositioningRepositioning is the process of finding a new indication for a drug or compound. By this, we assume that the new indication is distinct from the already approved/intended indication of the original product, where ‘distinct’ implies an anatomical and/or therapeutically distinct indication referring to the 10th version of the International Classification of Diseases (ICD-10). The situation where the new indication involves a different pharmacological target (off-target repositioning) is the only exception where a new use in a similar indication will be covered by the actual definition.The original product candidate to drug repositioning should fall into one of the following categories:Drugs that were never commercializedDrugs in clinical developmentDrugs with finalized but failed/negative clinical development due to poor efficacy profile and/or less frequency due to efficacy issuesDrugs that were not completely developed, particularly from academic institutions and public sector laboratoriesDrugs that are/were commercializedDrugs that have been marketed but discontinued for commercial reasonsDrugs that have been marketed but discontinued for safety/public health reasonsMarketed drugs for which IP rights are still in placeMarketed drugs for which patents are already expired or when generic versions are already available in the market.Some authors also consider drugs that have been discovered, developed, and marketed in small or emerging markets but not widely launched nor launched in larger pharmaceutical markets, especially in the United States and Europe, as potential candidates for drug repositioning , 7, 33. ReformulationsReformulation is, by the simple definition of the term, making a particular change in the formulation of the original drug. This can be achieved by exploiting advances in formulation technology to change the release of the active substance, pharmaceutical forms, and/or route of administration but it can also concern some excipients with no impact on the pharmacokinetic parameters. No change should be incurred in the structure of the API except when it is a chiral switch. By this, we assume that approaches modifying the chemical structure of the API, such as prodrugs or active metabolites, are not included in our definition. Cases where the development of a new product does not include a change in the original formulation  should also be excluded.Repositioning aided by reformulationRepositioning aided by reformulation is a particular situation wherein a new formulation  is developed for a new therapeutic use of a candidate for repositioning. To be considered as a repositioned and reformulated product, the change incurred to the original product should adhere to both aforementioned definitions.Drug combinationRegarding the specific case of drug combinations, we concluded that drug combinations can be considered a separate strategy, and where criteria of definitions are met, they can fall under the classification of reformulation and/or repositioning. By this, combinations can be classified as cases of reformulation or repositioning provided there was a change in formulation or indication, respectively.The previously described absence of a common, clear, and concise definition for repositioning and reformulation of drugs prompted us to propose a global definition that attempts to cover all of the previously unclear and sometimes contradictory criteria for the definitions and classification of cases of repositioning and reformulation.Based on the proposed definitions and from our understanding of actual classifications, we propose a complete classification algorithm  for drugThe chosen parameters for classification reflect our evaluation of the attributes having a direct or indirect impact on the development of products, particularly on their market access conditions. The combination of factors such as the commercialization and genericization status of the original product at the time of launching the new product, being developed by the same or a different company, under the same or a different brand name as for the original product, leads to a large set of development scenarios.More specifically, cases of repositioning  and 4 arGroup 0: Chiral switch, excipient change without pharmacokinetic impact and cases where none of the other classification of groups  is applicable;Group 1: Same pharmaceutical form, same administration route, and different pharmacokinetic parameters ;Group 2: Different pharmaceutical form, same or similar administration route, and same pharmacokinetic parameters;Group 3: Different pharmaceutical form and different administration route.Concerning drug reformulation  and 5, iFinally, the classification of drug combinations  and 6 isAlthough our proposal aims to be universal yet precise in its content, some cases may be beyond its purview. Due to the ultimate goal of analyzing the association of pricing and reimbursement regulations on development of repurposed drugs, we have excluded in-development or non-approved cases and cases for which data could not be retrieved. As such, we cannot extrapolate the utilization of our proposed nomenclature to all possible existing cases of repositioning or reformulation. For all new cases not included due to date limitation inherent to the current literature review, we suggest that these can be analyzed and categorized for their nomenclature as soon as they are known and eventually the proposed nomenclature be re-evaluated in light of these future findings, if necessary.Given the disparity and inconsistency of terminologies and classifications found in the literature, a harmonized nomenclature for repositioning, reformulation, and combinations will allow a robust and consistent analysis of the added value and market access conditions attributed for each strategy and case type as assessed by regulators and payors in Europe and the United States.Click here for additional data file.Click here for additional data file.