This use of a chemical waste product as a solid carbon source instead of commonly used explosive hydrocarbon gas sources for graphene synthesis has the dual benefits of converting the waste to a valuable product, and reducing pollution.We have developed a simple, scalable, transfer-free, ecologically sustainable, value-added method to convert inexpensive coal tar pitch to patterned graphene films directly on device substrates. The method, which does not require an additional transfer process, enables direct growth of graphene films on device substrates in large area. To demonstrate the practical applications of the graphene films, we used the patterned graphene grown on a dielectric substrate directly as electrodes of bottom-contact pentacene field-effect transistors (max. field effect mobility ~0.36 cm The CTP film was passivated with a Ni capping layer, then annealed under high temperature, low vacuum and a reducing atmosphere. The method produces multi-layer graphene on the top and bottom surfaces of the Ni layer. This result suggests that carbon atoms are released during pyrolysis of the CTP, then diffuse into the Ni layer during annealing, and precipitate to form graphene on both sides of the Ni layer upon cooling4344454643452/Si substrate in an electronic device. We demonstrated an organic field-effect transistor (OFET) as the first example of organic electronic devices using graphene electrodes based on direct patterned growth from solid carbon source on the device substrate without using any additional graphene-transfer process; the Ni capping layer was removed by wet etching, thereby leaving the graphene films on SiO2/Si substrate.Here we introduce a simple, scalable, transfer-free, ecologically sustainable, value-added method to synthesize patterned graphene electrodes for high-performance electronic devices by recycling coal tar pitch (CTP) as an inexpensive carbon feedstock. CTP as an industrial by-product of steel production and is mainly composed of a complex mixture of aromatic hydrocarbons and heterocyclics2 (500 nm)/Si substrates (2 cm × 2 cm) were treated with UV/ozone for 30 min to establish good wetting between CTP solution and substrate, and then a 20-nm thickness of CTP film was deposited on SiO2/Si substrates uniformly by spin coating . The thickness of CTP film was measured using ellipsometry and the film uniformity was confirmed using atomic force microscopy (AFM) (2 (10 sccm) gas, and low vacuum (~330 mTorr) at 900–1100 °C for 1–4 min to find the optimal conditions for graphene synthesis. After annealing, the samples were cooled to room temperature under the same Ar/H2 flow. A Raman spectroscopy system with excitation of 532 nm was used to evaluate whether CTP films had turned into graphene films512/Si substrate in various growth conditions, the Ni layer and the graphene films grown on top of the Ni layer were removed by simple immersion in aqueous FeCl3 solution for 1 min; the underlying graphene films remained on the SiO2/Si substrate. This process enabled direct growth of graphene films on dielectric substrates, and avoided damage to the graphene that could occur if the graphene were transferred using additional transfer process. In the average Raman spectrum (n = 2500 points), the D peak (~1350 cm−1) corresponds to defects in the graphene films. At annealing time of 4 min, the D peak intensity decreased as annealing temperature increased in the range of 900–1100 °C  . A 200-n–1100 °C . When an–1100 °C . The temDc is diffusion coefficient, Dco is the maximum diffusion coefficient at infinite temperature and QID is the activation enthalpy for the diffusion, R is the gas constant and T is absolute temperature. This equation is Fick’s first law of diffusion expressed in terms of an Arrhenius type. Based on this equation, increase in annealing temperature increases the amount of carbon that can dissolve in the Ni film, and therefore increases the amount of carbon precipitated to the Ni surface upon cooling, and increase in annealing time gives sufficient supply of carbon atom to form high-quality graphene at high temperaturewhere The effects of various softening points (SPs) and concentrations of CTP solution were investigated, because these factors also affect the graphene quality. As the SP of CTP increased (due to an increase in the portion of large molecules in CTP solution), the film thickness increased at the same concentration (8 wt%) and spin coating condition  . The num2/Si substrates to measure the average Raman spectrum. We used a poly[methyl methacrylate] (PMMA)-layer-supported graphene transfer process with metal etching solution (FeCl3) described elsewhereFurthermore, CTP-derived films on top of the Ni layer were transferred onto SiO2 gas. Graphene films formed even under low-vacuum condition with Ar atmosphere (~3.8 mTorr), although the average Raman spectrum had higher D peak intensity  indicate that of mono-layer to few-layer graphene had formed. This synthesis method does not use any explosive gases (CH4 and H2), and can be used to mass-produce graphene easily and safely.We also obtained graphene films from the growth process without using Hntensity  than did2/Si substrates at 1100 °C for 4 min were prepared for use in further characterization. This annealing condition was chosen based on the results of preliminary trials. In the Raman scanning over a large area , pronounced peaks occurred at ~1580 cm−1 (G peak) and ~2700 cm−1 (2D peak), in addition to the D peak51D to G peak intensity IG ~0.1)  , then transferred to the TEM grids. TEM images at the folded edge of graphene films show that the multilayer graphene consisted of mono-layer to few-layer graphene with regular interlayer spacing (~0.34 nm)  in agreement with our Raman analysis data 26CTP-derived graphene films grown on the SiOIG ~0.1) . The qua1) (D/IG . On most(I2D/IG) . Raman mportions , and thasis data . Hexagonsis data  and elecsis data , inset26The graphene growth using Cu catalyst was also investigated. The Ni capping layer was replaced by a Cu capping layer (200 nm) and the samples were annealed at same growth conditions but at 1000 °C for 4 min because the melting point of Cu (1085 °C) is lower than that of Ni (1455 °C). After annealing, the Raman spectrum of Cu surface did not show any peaks related to carbon . After Cu etching, the Raman spectrum of the film that remained on the substrate had a large D and G peak without 2D peak; this spectrum is associated with amorphous carbon . Because the Cu is a surface catalyst due to the low solubility of carbon in it, the Cu layer did not act as an efficient catalyst to form a graphene when our metal capping layer structure was used432 gas, 0.2 Torr, 10 s) to remove the CTP film from regions not covered by the Ni pattern. During this process, the patterned Ni layer functions as a passivation mask that protects the CTP film underneath the Ni pattern from RIE treatment. After applying this method, Raman spectrum of the gap between the Ni patterns did not reveal any peaks related to carbon materials ; this means that RIE treatment removed the unprotected CTP film effectively. After etching the Ni patterns, the patterned graphene source/drain electrodes (gaps ~100 μm) can be obtained directly on the 500 nm SiO2/Si substrate. Finally, a pentacene layer (50 nm thick) was deposited on the patterned graphene electrodes at 50 °C through a shadow mask to complete OFET device fabrication.We used the multi-layer graphene films obtained from our transfer-free method to fabricate bottom-contact OFETs  based oninset) were characterized by measuring their output and transfer characteristics . We calculated RC of Gr-P and Au-P FETs by using the transfer line method with channel lengths of 30, 50, 80, and 100 μm . As gate voltage varied from −60 to −150 V, RC of the graphene electrode, normalized by channel width (1500 μm), decreased from 0.14 MΩ·cm to 0.043 MΩ·cm (545556μFET in the saturation regime was an order of magnitude higher in Gr-P FETs (0.05–0.13 cm2·V−1·s−1) than in Au-P FETs (0.011–0.017 cm2·V−1·s−1). The transfer curve of the Gr-P FETs showed a high on/off current ratio (1.1 × 107) with small hysteresis . In this case, the fabrication process was the same as used to fabricate the Gr-P FETs, except for the use of the large area substrate. The distribution of the μFET of the Gr-P FET large-area arrays showed ~95% operation and maximum μFET 0.13 cm2·V−1·s−1 (average μFET ~0.07 cm2·V−1·s−1)  , inset weristics . For comt levels . The Gr-43 MΩ·cm , which icm 545556. Calculasteresis , so theysteresis , inset. V−1·s−1) .RC in Gr-P FETs. RC between the electrode and the organic channel is primarily affected by the magnitude of the carrier injection barrier at the interface54WF of each electrode and electrode-pentacene interface. The calculated hole injection barrier of graphene electrode was 0.43 eV, which is 0.23 eV lower than that of Au electrode (0.66 eV), even though the WF of the graphene electrode (4.32 eV) was lower than that of Au electrodes (4.60 eV)  . We attr2/Si substrates that had graphene and Au electrodes patterns were immersed in an OTS SAM solution (0.1% in toluene) for 3 min, then dried under blowing using N2 gas. The OTS-treated Gr-FETs showed increased drain current and μFET (0.25–0.36 cm2·V−1·s−1), and the OTS-treated Au-FETs were also affected (μFET 0.04–0.07 cm2·V−1·s−1)  .μFET, ~0.36 cm2·V−1·s−1) in a bottom-contact geometry than did Au-electrode pentacene FETs. Using the same method, we also fabricated large-area graphene-electrode pentacene FET arrays (144 devices on a 4-inch wafer) to demonstrate transfer-free fabrication of graphene-based electronic devices. Our results provide a promising way to use graphene films converted from the carbon waste (CTP) as a reliable conducting material and as an alternative electrode for electronic devices. Furthermore, using this industrial waste product of steelworks to produce graphene will contribute to a clean environment and sustainable graphene growth.In conclusion, we demonstrated a simple, scalable, low-cost, ecologically sustainable, value-added process to synthesize graphene films from inexpensive CTP films under a Ni capping layer without using explosive hydrocarbon gas sources and physical transfer process. We synthesized multi-layer graphene films underneath the patterned Ni layer in a tube furnace and then achieved patterned graphene films as source/drain electrodes directly on the OFET device substrate without physical transfer process after removing the patterned Ni layer. The pentacene FETs using graphene source/drain electrodes synthesized using our transfer-free patterned growth method showed higher carrier mobility (max. 2 (500 nm)/Si substrates (NAMKANG HI-TECH). After baking at 240 °C for 30 min, the Ni layer (200 nm) was deposited  on a coal tar pitch film at room temperature. Then the sample was annealed using thermal CVD  with flowing 50 sccm Ar and 10 sccm H2 at a total pressure of ~0.3 Torr, then cooled to room temperature by removing the tube from the furnace while maintaining the same Ar/H2 flow. The Ni layer was etched away by dipping the sample into FeCl3 solution (Iron(III) chloride solution 45°Be’, JUNSEI Chemical) for 1 min and the sample was rinsed with DI water. Raman spectra and mapping images of graphene films were investigated using a Raman spectroscopy (WITEC Alpha 300R Raman spectroscope equipped with a 532 nm diode laser). The CTP-derived graphene was further characterized by HR-TEM .The coal tar pitch  was diluted with quinoline solvent  to a concentration of 8 wt%. The Coal Tar Pitch solution was spin coated  on a SiOA 50-nm-thick pentacene (Sigma Aldrich) layer was deposited on the electrodes through a shadow mask at 50 °C. The electrical properties of the graphene and Au-electrode pentacene FETs were characterized using a parameter analyzer (Keithley 4200). The work function of each electrode and electrode-pentacene interface was measured using ultraviolet photoelectron spectroscopy (UPS) .How to cite this article: Seo, H.-K. et al. Value-added Synthesis of Graphene: Recycling Industrial Carbon Waste into Electrodes for High-Performance Electronic Devices. Sci. Rep.5, 16710; doi: 10.1038/srep16710 (2015).


The intriguing structures have several advantages for straightforward fabrication on patterned substrates, high-performance graphene sensors without post-annealing process.We introduce a high-performance molecular sensor using self-corrugated chemically modified graphene as a three dimensional (3D) structure that indicates anisotropic charge distribution. This is capable of room-temperature operation, and, in particular, exhibiting high sensitivity and reversible fast response with equilibrium region. The morphology consists of periodic, “cratered” arrays that can be formed by condensation and evaporation of graphene oxide (GO) solution on interdigitated electrodes. Subsequent hydrazine reduction, the corrugated edge area of the graphene layers have a high electric potential compared with flat graphene films. This local accumulation of electrons interacts with a large number of gas molecules. The sensitivity of 3D-graphene sensors significantly increases in the atmosphere of NO Specifically, graphene-based gas sensors can detect individual molecular levels of gas adsorption3437891114151617Molecular detection using graphene is a promising method for use in gas sensors and biosensors because of its outstanding electrical properties and large specific surface area12 gas molecules. Moreover, the sensitivity of the gas sensor increased significantly at various NO2 gas concentrations.Thus, the goal here is to develop an easily fabricated, high-performance rGO molecular sensor capable of room-temperature operation, and, in particular, exhibiting high sensitivity and reversible fast response with saturation region. For this purpose, we introduce self-corrugated, chemically modified graphene as a 3D structure with anisotropic charge distribution. The accumulated electrons on the corrugated-edge area of the graphene layers exhibit a high electric potential compared with two-dimensional rGO (2DrGO) films. This local accumulation of electrons interacts with a large number of gas molecules, resulting in a reversible faster response and higher sensitivity than with conventional rGO sensors. The density of states of the self-corrugated graphene surface was confirmed by Scanning Kelvin Force Microscopy (SKFM), and the gradient of electric potential as a function of wall height on the 3DrGO was modeled by computational simulation as a function of anisotropic charge distribution. The field-enhancement factor is five times higher than that of 2D graphene film, and a fast response with saturation of resistance change was confirmed for the adsorption and desorption of NO2 thickness of 300 nm for the fabrication of a resistive-type gas sensor. In order to produce a well-aligned, corrugated rGO structure, the GO film was placed in a prepared chamber with a relative humidity of 80%. The rapid evaporation of toluene reduces the temperature at the air–solution interface because the toluene is highly volatile and immiscible with water vapor. Thus, condensation and nucleation of micro-sized water droplets results on the substrate. Rapid evaporation and airflow across the surface of the ODA-functionalized GO solution led to convective flows in the solution; the water droplets then created regular corrugation by packing of GO nanosheets. The GO nanosheets encapsulate the water droplets and precipitate at the water–solution interface. Following the evaporation of water from the GO nanosheets, self-corrugated regular vertical GO structures were produced on their surfaces. It has been established that solvents with a low viscosity have a low surface energy and their droplets have a tendency to adopt a spherical shape on the surface of high-viscosity solvents to minimize surface energy2H4) at 80 °C overnight. The both materials are deposited on the same interdigitated structure. This process constitutes a simple means for the fabrication of resistive-type rGO-based devices with various flexible substrates. 2 at thickness of 300 nm. The length and width of interdigitated electrodes are 50 mm, respectively. The −1, with lower intensity ratio of I2D/IG than single layered graphene, confirming heterogeneous structureD/IG ratio of 3DrGO reveals a large number of structural changes or defects in the 3DrGO. However, the I2D/IG of 3DrGO is lower than that of 2DrGO in 2, ~284.6 eV), C–C/C–N , C–O , C = O , and O–C = O  bonds. With subsequent reduction by fuming hydrazine, the presence of oxygen functional groups was dramatically reduced. These results suggest that the structural changes of rGO were not affected by the functionality of oxygen moieties. ds–Vds of the interdigitated rGO electrodes for both 2DrGO and 3DrGO. The resistance of 3DrGO (37 Ω) is slightly higher than that of 2DrGO (29 Ω), which agrees with the Raman analysis of structural changes of rGO. Although the morphology is significantly different, the resistance is only slightly altered because of the similar quantities of oxygen functional groups as insulating sites and the presence of nearly flat rGO films on the bottom layer of 3DrGO, as shown in Structural changes, such as mechanical deformation, wrinkles, doping, and the number of layers of rGO, can be described by Raman spectra, as shown in 2 substrate with respective Cr and Au source and drain electrodes of 30 mm length and width. ds–Vgs characteristics for both samples. It is clear that the strain and mechanical defects lower the on/off ratio due to the delocalization of electrons. In addition, the threshold voltage is similar for both samples. However, the on current of 2DrGO is higher than that of the 3DrGO, while the on/off ratio is lower. This is caused by mechanical deformation of graphene surfaces such as corrugation, breakage, and wrinkles29There are several issues involved in producing high-performance molecular sensors. A large surface area with corrugated surfaces facilitates the adsorption of large amounts of gas molecules and accelerated charge accumulation, which can improve gas detection in terms of sensitivity and fast-response time. In particular, the charge transfer induces significant changes in resistance due to the relation between electrostatic potential and charge density which can be described by Coulomb interaction. Also, the corrugated surfaces induce an inhomogeneous charge distribution. For instance, electron charge density increases on the rippled area of graphene surface2 gas concentration of 20 ppm. The gas sensitivity (S) is defined as S = ΔR/R0, where R0 is the initial resistance when exposed to pure N2 gas, and ΔR is the resistance change after exposure to the N2 and NO2 mixture. After exposure to NO2 gas at 20 ppm for 300 s, the resistance decreases due to the p-type property of rGO and the electron acceptor of the NO2 molecules0 × 100 (%). The sensitivity of 3DrGO (65%) is roughly three times greater than that of 2DrGO (20%). 2 gas concentration for 20, 50, and 100 ppm; the sensitivity increases linearly with increasing gas concentration. An extrapolation to lower gas concentrations predicts a sensitivity of about 0.2 at 1 ppm. This value is three times higher than that predicted for 2DrGO. The observed sensitivity values indicate that resistance increases with NO2 adsorption. Since the NO2 molecule is electrophilic, charges are expected to transfer from rGO to the physisorbed NO2, which confirms the p-type characteristic of rGO shown in 2 gas exposure can be separated into three regions, namely, (i) a sharp initial in gas response (fast-response region), (ii) a nearly linear intermediate region (slow-response region), and (iii) a region in which the sensor completely saturates (saturation region), as shown in 2 gas exposure, the first and second cycle are stable. The base-line is saturated as shown in 2 carbon and gas molecules, while the slow response is due to mechanical defects or oxygen functional groups, as described earlier2 sites. Conversely, the 3DrGO exhibits a reversible response with the saturation region (iii). This indicates that a certain limit adsorption of gas molecules over the surface could be present. The excess NO2 molecules that did not reach the surface active sites of the sensor could then be expected to remain in the surrounding region. This establishes a constant gas response. These results demonstrate that a gas molecules interacting with high-energy binding sites during are adsorbed continuously during gas exposure. Meanwhile, the adsorption of gas molecules were saturated by accumulated gas molecules with fast response owing to high electric potential on the graphene.The response curves for NOWe introduce self-corrugated, chemically modified graphene as a 3D structure that governs anisotropic charge distribution. This morphology can be formed by condensation and evaporation of water on a GO solution using the “breath figure” technique. In particular, the accumulated electrons on the edge sites of the corrugated graphene layers exhibit a strong electric field compared to flat graphene films. The sensitivity, stability, and fast response of 3DrGO were significantly improved in terms of gas-molecule detection. This method is a promising candidate among practical molecular sensors and flexible devices using chemically modified graphene without post-annealing process.2SO4 are mixed in a flask. Subsequently, 60 g of KMnO4 is slowly added for 1 h. For safety, stirring was carried out for 2 hours in ice bath. Again, the mixture is stirred vigorously for 24 hours at room temperature. DI (deionized) water is added and stirred for 10 min. A volume of 50 mL H2O2 (30 wt% aqueous solution) was then added, and the mixture was stirred for 2 h at room temperature. The mixture was precipitated and filtered to obtain the graphite oxide powder. This was then exfoliated into GO nanosheets in deionized water by homogenizer for 1 h2 substrate with thickness of 300 nm. The interdigitated electrode was fabricated by simple shadow mask with deposition of metal source such as Cr and Au. The channel width and length of metal electrodes were 50 μm. The Cr/Au electrodes were deposited using thermal evaporator at 2 × 10−6 torr with a thickness about 20 nm and 100 nm respectively. In order to produce the regular structure of 3DrGO, humid air which has a relative humidity of 80% and a flow rate of 0.1 L/min was supplied from a nozzle affixed to a funnel. The vertical distance between the substrate and the end of the funnel was kept at 6 mm inside a desiccator. After complete evaporation of the solvents, vertically aligned patterns of the ODA-functionalized GO were obtained. Subsequently, the film was transferred to a furnace to eliminate the ODA from the GO. Pyrolysis was carried out at 250 °C for 1 h under atmospheric pressure. The self-corrugated rGO with uniform array structures were achieved after fume reduction using hydrazine monohydrate (N2H4) at 200 °C for 24 h. The 3DrGO gas sensor could be fabricated with high uniformity as shown in Graphenen Oxide (GO) was obtained by natural graphite  according to a modified Hummers method−1 for Stokes shift. To confirm the alteration in the atomic ratio of carbon to oxygen with presence of functional group with respect to the 2DrGO and 3DrGO samples after fuming reduction by hydrazine, the X-ray Photoelectron Spectroscopy (XPS: Thermo VG Scientific Inc. MultiLab2000) analyses were carried out on an X-Ray photoluminescence spectrometer with an Al cathode as the X-ray source with power of 150 W. The fitted peaks of XPS spectra were determined by considering a combination of Gaussian and Lorenzian distribution. The Ids-Vds and Ids-Vgs characteristics were measured using a two-probe method (Keithley 4200-SCS). The total resistance with respect to 2DrGO and 3DrGO devices with interdigitated electrodes was measured by Ids-Vds. In order to measure the gating effect as depending of structures in 2 gas was mixed with N2 gas. The concentration of NO2 gas with respect to 20, 50, 100 ppm was controlled by Keithley 2700 multimeter with solenoid valve system. The resistances of sensors were measured by Keithley 2000 multimeter, recording electrical resistance as a function of time for gas exposure. Two-probe DC resistance measurements was used by Au wires for current and voltage leads. The measured values were carried out under atmospheric pressure.The morphologies of rGO were obtained by field emission scanning electron microscopy . The optical image of interdigitated electrodes for gas sensor was also revealed by optical microscope (Nikon Eclipse LV100). The structural and chemical characteristics in the 2DrGO and 3DrGO sheets were investigated by confocal Raman spectrometer  with an excitation wavelength of 532 nm and Rayleigh line injection filter with a spectral range of 100–3600 cmHow to cite this article: Yol Jeong, S. et al. Enhanced response and sensitivity of self-corrugated graphene sensors with anisotropic charge distribution. Sci. Rep.5, 11216; doi: 10.1038/srep11216 (2015).


Drosophila, a single Sin3A gene encodes different isoforms of SIN3, of which SIN3 187 and SIN3 220 are the major isoforms. Previous studies have demonstrated functional non-redundancy of SIN3 isoforms. The role of SIN3 isoforms in regulating distinct biological processes, however, is not well characterized.The multisubunit SIN3 complex is a global transcriptional regulator. In Drosophila S2 cell culture model system in which cells predominantly express either SIN3 187 or SIN3 220. To identify genomic targets of SIN3 isoforms, we performed chromatin immunoprecipitation followed by deep sequencing. Our data demonstrate that upon overexpression of SIN3 187, the level of SIN3 220 decreased and the large majority of genomic sites bound by SIN3 220 were instead bound by SIN3 187. We used RNA-seq to identify genes regulated by the expression of one isoform or the other. In S2 cells, which predominantly express SIN3 220, we found that SIN3 220 directly regulates genes involved in metabolism and cell proliferation. We also determined that SIN3 187 regulates a unique set of genes and likely modulates expression of many genes also regulated by SIN3 220. Interestingly, biological pathways enriched for genes specifically regulated by SIN3 187 strongly suggest that this isoform plays an important role during the transition from the embryonic to the larval stage of development.We established a These data establish the role of SIN3 isoforms in regulating distinct biological processes. This study substantially contributes to our understanding of the complexity of gene regulation by SIN3.The online version of this article (doi:10.1186/s12864-016-2428-5) contains supplementary material, which is available to authorized users. Nucleosomes are comprised of DNA wrapped around histone proteins to form a stable chromatin structure. Post translational modifications (PTMs) of histones influence chromatin structure and the transcriptional state of genes . HistoneDrosophila S2 cells compromises the G2/M phase transition during cell cycle progression [Sin3A causes curved wings in adult flies [mSin3A impacts both G1 and G2/M phases of the cell cycle [sin3 in budding yeast show an accumulation of asynchronous cell population in the G2 phase [Drosophila cell lines in which Sin3A was knocked down [. Several investigators have shown that the two highly similar mouse Sin3 proteins play distinct roles during development. For example, mSin3A is essential for early embryonic development, survival and growth of cultured cells [mSin3B plays a regulatory role during the late gestation period of embryogenesis, suggesting the functional non-redundancy of the mSIN3 genes [SIN3 activity is linked to several cellular processes throughout metazoan development. SIN3 has been shown to be involved in protein stability, oncogenic transformations, senescence and cell survival . SIN3 isgression . Additiolt flies . Rescue lt flies . In mousll cycle , 18. NulG2 phase . SIN3 haked down . In addiked down , 21. In ked down . Severaled cells , 18, whiN3 genes . DespiteDrosophila, splicing of a single gene results in the expression of multiple isoforms of SIN3 [Drosophila, Saccharomyces cerevisiae express a single Sin3 protein [The intricate mechanism of gene regulation by SIN3 is augmented by the presence of distinct SIN3 isoforms or discrete SIN3 HDAC complexes in multiple eukaryotic model systems . In Dros of SIN3 . Additio of SIN3 . Unlike  protein . Althoug protein –31. The  protein . Further protein , 33. TheDrosophila [. SIN3 187 and SIN3 220 are the most prevalent isoforms of SIN3 expressed during fly development [Drosophila S2 cells and larval imaginal disc cells, whereas SIN3 187 is the prevalent isoform during the latter stages of embryonic development and in adults [Drosophila.Previously, we demonstrated the existence of distinct SIN3 HDAC complexes in osophila . SIN3 18elopment . SIN3 22n adults . Addition adults . Differen adults , which iDrosophila cultured cells that predominantly express one or the other major isoform. To our knowledge, it is the first time the binding sites of SIN3 isoforms across the Drosophila genome have been mapped. Interestingly, we found that overexpression of SIN3 187 led to replacement of SIN3 220 at a majority of the genomic sites, indicating that the binding sites of the SIN3 isoforms overlap with each other. RNA-seq analysis, however, demonstrate that SIN3 187 plays unique gene regulatory roles, in addition to having some functions in common with SIN3 220.Genome-wide binding sites of SIN3 have been previously published by several groups , 37, 38,, we established two S2 cultured cell lines in which cells express either of the two major SIN3 isoforms with a tag for immunoprecipitation ), Cytochrome c1 (Cyt-c1), Pyruvate kinase (Pyk), twine (twe), CG9548 but not at control regions dachsous (ds) or CG31819  analysis [Through investigation of the chromosomal distribution of SIN3 isoforms, we found that they are enriched over the euchromatic regions of the analysis . Specifianalysis . We founThe enrichment patterns of SIN3 187 and SIN3 220 at genomic features  are similar to each other Fig. . AdditioDrosophila genome) and 6905 (approximately 40 % of the Drosophila genome) genes bound by SIN3 187 and SIN3 220, respectively, supporting the idea of a global transcriptional role of SIN3 isoforms. Additionally, the ChIP-qPCR data showing localization of the SIN3 isoforms at putative gene targets  and 46 % (162/349) of the genes repressed and activated by SIN3 220, respectively, are direct targets  to verify gene expression changes resulting from Sin3A knockdown. We selected S-adenosyl methionine synthetase (Sam-S) and tumbleweed (tum), which were found to be altered upon Sin3A knockdown, and bound by SIN3 220 as well  analysis on the genes that were directly repressed and activated by SIN3 220 using the DAVID gene annotation module Fig. . 52 % 1. AdditioDrosophila embryogenesis, the levels of SIN3 isoforms are comparable [Our findings demonstrate that the ectopic expression of SIN3 187 in S2 cells led to a reduction in the level of SIN3 220 Fig. . In addimparable . During mparable . Based or) or activated (Aa) by SIN3 220, B. Genes directly repressed (Br) or activated (Ba) by SIN3 220 as well as SIN3 187, C. Genes directly repressed (Cr) or activated (Ca) by SIN3 187 alone and D. Genes that are directly regulated in opposing directions by SIN3 187 and SIN3 220  and Glutamate-cysteine ligase modifier subunit (Gclm), which are shown to be repressed and activated specifically by the SIN3 187 isoform, respectively. In addition, these genes are bound by SIN3 187 at the promoter region as determined by the ChIP-seq data . Our results demonstrate that reduction of Sin3A (SIN3 220) did not impact the expression of these genes  knockdown  knockdown. For example, biological processes such as neuron development, cell morphogenesis and aging are overrepresented by both SIN3 187 regulated genes and genes that show a change in expression due to reduction of Sin3A (SIN3 220). Collectively, these data indicate that SIN3 187 specifically modulates biological processes involved in differentiation and development, in addition to regulating processes in common with the SIN3 220 isoform.Our previous research demonstrated that during the late stages of  reduced . These rIn this research, we have described the differential role of the SIN3 isoforms in regulating gene activity, summarized in Fig. Drosophila cells, ectopic expression of SIN3 187 led to a reduced level of SIN3 220  + L-glutamine supplemented with 10 % heat-inactivated fetal bovine serum (Invitrogen). Gentamycin (50 mg/ml) was added to S2 cells. S2 cell lines stably transfected with expression constructs of HA-tagged SIN3 187 and SIN3 220 were grown in the media with 0.1 mg/ml of penicillin/streptomycin and 0.1 mg/ml Geneticin. Cells were maintained at 27 °C.The 6 cells were harvested by centrifugation and lysed in Laemmli Sample buffer (Bio-Rad). Proteins (15 to 20 μg) were resolved on an 8 % SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride (PDVF) membrane . Membranes were incubated with primary antibody: HA-HRP , SIN3 PAN (1:2000 [Western blot was carried out following standard protocols . To prep (1:2000 ), SIN3 2 (1:2000 ), α-Tubu7 exponentially growing S2 cells were subject to cross-linking by the addition of formaldehyde to a final concentration of 1 % for 10 min at room temperature. Excess formaldehyde was quenched by addition of glycine to a final concentration of 125 mM. The cross-linked cells were washed with 1X phosphate buffered saline and resuspended in lysis buffer ), followed by incubation on ice for 15 min. These cells were disrupted by dounce homogenizer  followed by low speed centrifugation (170 x g for 10 min). The pellet was resuspended in 200 μl MNase digest buffer  and subjected to micrococcal nuclease digestion by addition of 20 units of MNase  for 10 min at room temperature. 10 mM EDTA was added to quench the MNase activity. Following MNase digestion, samples were made up to 1.2 ml with resuspension buffer  and sonicated using a probe sonicator (Fisher Scientific) to extract cross-linked chromatin in the solution.Approximately 4x10Approximately 75 μg of crude chromatin was used to prepare input for the ChIP experiment. Briefly, samples were treated with 0.05 μg/μl of RNase A (Sigma) and incubated overnight with 200 mM NaCl at 65 °C. Samples were treated with 0.04 μM Proteinase K (Fisher Scientific), 10 μM EDTA and 20 μM Tris (pH 8) and incubated at 45 °C for 1.5 h. DNA was purified by standard phenol chloroform extraction. This method leads to the formation of mono, di, tri-nucleosomal DNA fragments as determined by agarose gel electrophoresis analysis of purified DNA.3) at 65 °C for 30 min. Elution was done twice in a total volume of 500 μl. Cross-links were reversed in the eluted samples as for the ChIP input DNA preparation. Purified DNA samples from HA-tag ChIP were quantified by real-time PCR using 2-ΔΔCT method of analysis [For HA-tag ChIP, we used monoclonal anti-HA-agarose antibody (Sigma). Anti-HA-agarose bead slurry was washed with RIPA buffer. 75 μg of chromatin was incubated with 30 μl washed anti-HA-agarose for 4 h at 4 °C. Chromatin prepared from wild type S2 cells was used as control. Chromatin bound HA-agarose was then washed, once with 1 ml of modified 2X RIPA buffer  for 8 min at room temperature, once with 1 ml of high salt buffer  at 4 °C for 10 min, once with 1 ml of low salt buffer  at 4 °C for 10 min and once with 1 ml of Tris-EDTA (pH 8). To perform SIN3 PAN ChIP, we incubated chromatin with 10 μl polyclonal SIN3 antibody  overnighanalysis . All genWe prepared sequencing libraries using the method previously described by Ford et al., 2014 . In brieDrosophila genome. Reference Drosophila melanogaster sequence (dm3) was downloaded from the UCSC table browser and raw reads were aligned to the reference genome. Only uniquely mapped tags were subsequently used for further analysis. Input DNA tags were used to normalize ChIP DNA tags. Protein bound regions were called using Model-based analysis of ChIP-seq (Macs 2.1.0) [To map the binding sites of SIN3 isoforms, we used Bowtie2  to aligns 2.1.0)  using ths 2.1.0) –59(AdditPeaks were assigned to UCSC Refseq genes (dm3). Using the bedtools  windows Sin3A knockdown was carried out based on a published protocol [6 cells were plated in 4 ml of Drosophila Schneider’s medium. After 3 h, FBS-containing media was carefully removed and supplemented with 2 ml of serum free media. 50 μg of dsRNA was added to plates and mixed by swirling. After 30 min, 4 ml of complete Drosophila media was added. Cells were incubated for 4 days. Construction of dsRNA targeting Sin3A was previously described [GFP sequence. The primer pairs to amplify GFP containing sequence are GAA TTA ATA CGA CTC ACT ATA GGG AGA TGC CAT CTT CCT TGA AGT CA (forward primer) and GAA TTA ATA CGA CTC ACT ATA GGG AGA TGA TGT TAA CGG CCA CAA GTT (reverse primer). Efficient knockdown of Sin3A compared to GFP RNAi was verified by western blot analysis.RNAi mediated protocol  with sliescribed . dsRNA a4. Ectopic expression of SIN3 187HA was verified by western blotting.S2 cells carrying stably transfected HA-tagged SIN3 187 transgene under the metallothionein promoter was induced by addition of 0.07 M CuSOSin3A was compared to GFP RNAi S2 cells. The primers used for RT-qPCR are listed in Additional file Taf1 gene was used to normalize the mRNA level. At least three independent biological replicates were performed. Results are represented as the mean of the fold difference between the experimental and control samples.Gene expression analysis by RT-qPCR was carried out as previously described . The chaSin3A knockdown versus GFP RNAi was retrieved through GEO under GSE68775 .Procedures from RNA isolation to next generation sequencing were performed at the Applied Genomics Technology Center, Wayne State University. Briefly, total RNA was extracted using the EZ1® RNA Universal Tissue Kit (Qiagen). Cell lysis and homogenization was carried out by bead-milling on the TissueLyser® II (Qiagen) in 750 μl QIAzol™ lysis reagent. Extracted RNA was purified on the EZ1® Advanced (Qiagen). Additional DNase treatment was done to remove any residual DNA. Quantification and quality assessment of purified total RNA was done using the DropSense96® Microplate Spectrophotometer (Trinean) and microfluidics using the RNA R6K assay for the Agilent 2200 TapeStation, respectively. cDNA was generated by reverse transcription of the purified mRNA. The TruSeq RNA Sample Preparation Kit (Illumina) was used to prepare sequencing libraries following the manufacturer’s protocol. Single indexed samples were multiplexed and sequenced on an Illumina HiSeq 2500 sequencing system in paired end mode with a read length of 2 x 50 bp. A total of three independent biological replicates were performed. In the current study, we report the RNA-seq analysis done on total RNA isolated from cells overexpressing SIN3 187HA. Wild type S2 cells treated in the same way as the SIN3 187HA cell line was used as control. Correlation among replicates is shown in Additional file Sin3A knockdown sample and FDR ≤ 0.001 for the SIN3 187HA overexpression sample. The R statistics environment was used to visualize the data. The volcano and scatter plots were generated using the ggplot2 package.The Tophat pipeline was utilized for the analysis . Reads oP-value <0.05 were pooled.Genes bound by SIN3 isoforms and that also showed change in expression upon alteration in the level of SIN3 isoforms were included in the GO analysis. GO analysis, performed separately on genes repressed or activated by SIN3 isoforms, was done utilizing the online tool DAVID . Utilizit-test.Statistical calculations were done using the online statistical tool Graphpad and significant values were calculated using the unpaired Student’s www.ncbi.nlm.nih.gov/geo/). ChIP-seq datasets are deposited under accession number GSE72171 and RNA-seq data for SIN3 187 overexpression are deposited under accession number GSE72172. These two datasets are included in a superseries GSE72173. RNA-seq datasets for Sin3A knockdown samples can be retrieved from GSE68775.ChIP-seq and RNA-seq data can be accessed from the GEO database (


The ability to control the charge-potential landscape at solid-liquid interfaces is pivotal to engineer novel devices for applications in sensing, catalysis and energy conversion. The isoelectric point (pI)/point of zero charge (pzc) of graphene plays a key role in a number of physico-chemical phenomena occurring at the graphene-liquid interface. Supported by theory, we present here a methodology to identify the pI/pzc of  graphene, which also allows for estimating the nature and extent of ion adsorption. The pI of bare graphene -grown) is found to be less than 3.3, which we can continuously modify up to 7.5 by non-covalent electrochemical attachment of aromatic amino groups, preserving the favorable electronic properties of graphene throughout. Modelling all the observed results with detailed theory, we also show that specific adsorption of ions and the substrate play only an ancillary role in our capability to tune the pI of graphene. Miniaturized devices with nanostructures as active elements are highly promising to realize the dream of complete chemical analysis on a single chip2467911514One of the fundamental challenges in such a scenario is the need to have control over the charge-potential landscape of the graphene-liquid interface in addition to providing for the right chemical functionalities and biological receptors for the application of interest. The charge density on graphene has been found to be largely dependent on the nature and density of chemical groups available on the surface171967σOHP) can be modelled using the Gouy-Chapman-Stern theory asFirst we present a theoretical formulation of the charge-potential relationships at the graphene-liquid interface as shown in 22ψOHP is the potential at the OHP, β = 1/kT, κ−1 is the Debye screening length given by cI is the total ionic strength of the solution, ε0 and εr are permittivity of free space and relative permittivity and e is the electronic charge. On the graphene side, we model the presence of functionalities using a layer of a definite thickness (tFL) with a dielectric constant (ε−FLr) different from that of water (εr−H2O = 80). The functionalities present ionizable groups on the graphene surface, whose charge distribution can be modeled by a Langmuir-Freundlich type of an isotherm1617Ka/Kb or pKa/pKb) of the ionizable groups dictate the magnitude and sign of the charge distribution (σ0) and the potential (ψ0) at the interface between functional layer and the Stern layer. This dissociation or protonation of the groups can be schematically generalized in a manner similar to that of amino acids aswhere An example for the former case are carboxyl groups which are dissociated at high pH, while the latter case can be thought of as hydroxyl or amine groups which are protonated at low pH. The fractions of dissociated and protonated groups are respectively given by+], which corresponds to the bulk proton concentrationσmax) at maximum dissociation or protonation, (see Notes/N1 in SI)where the square brackets indicate the surface concentration of the respective species, except for [Hi or j refers to different kinds of negatively or positively chargeable functional groups respectively each with a corresponding Ka and m specifies the degree of heterogeneity in the distribution of ionizable groups as specified by modified Langmuir-Freundlich theorywhere the superscript index σoff is a charge density offset which is constant over a range of pH and ionic strengthwhere sign[CStern is the specific capacitance of the Stern layer (thickness tStern) given by ε0εr−Stern/tStern.where VecG) applied through a reference electrode leads to an electrified interface, whose effect on the charge carrier density in graphene is given byIn order to arrive at the charge carrier distribution in graphene, we need to consider that in an experiment an external voltageψgr referring to the graphene electrostatic potential, CFL = ε0εr−FL/tFL and ψoff is a cumulative offset potential that is independent of pH or ionic strength of the solution. This includes measurement offsets, offset voltage arising due to the choice of the reference electrode and offsets due to the varying filling levels of graphene from impurities or the underlying substrate (the part which is only in contact with graphene and not with the liquid)ψOHP by ensuring charge neutrality using the transcendental equationwith ψOHP at every triple {pH, cI, VecG}, the charge-potential distribution at all planes of the graphene-liquid interface can be self-consistently computed. From the experimental perspective, we can extract the gate voltage at the Dirac point (cI of the liquid can be varied and the Dirac point recorded as a function of pH and cI. The Dirac point corresponds to the point of minimum charge carrier density10cI by solving for σgr(ψOHP) = 0. The minus sign occurs because the applied gate voltage in an experiment occurs at the reference electrode, whereas in the model it occurs at the graphene plane. Since we restrict here only to the situation at the Dirac point, the gate dependent variation in charge carrier density occurring due to the linear energy dispersion in graphene and the effect of quantum capacitanceUsing the value of Ka of 7 and hence we can talk of an isoelectric point of the surface to be 7. It is apparent that the Dirac point variation at higher pH is compressed at higher ionic strength due to the smaller thickness of the electrical double layer, consistent with previous measurements on grapheneψoff is taken as −0.1 V here (ψoff = 0), although this may vary from sample to sample due to the spatially varying chemistry of CVD-grown graphene, size of the flake and local charging effects from the underlying substrate. We can exclude this parameter by comparing the pH-Dirac point behavior at 1 M ionic strength to the behavior at the other IS values. difference Dirac curve (M-I) correlates directly with the sign of the net surface charge. We have used the data at 1 M ionic strength as the baseline due to this reason. This is due to the fact that the compressed double layer at high IS leads to an effective interface potential whose absolute magnitude is smaller than the value at low IS. Now, in order to determine the pI experimentally, we measure the Dirac point as a function of pH and IS and deduce difference Dirac curves such as the one in σ0 = 0, i.e. the net charge of the surface is found to be 02023Before going to the experimental details, it is worth clarifying the definitions of pI/pzc312 chips and patterning them using photolithography (See Methods for details)Rgr) at a frequency of 1 kHz continuously in buffer solutions of varying pH and ionic strength (see I and 1000 mM: M). From every cycle of field-effect scan, as shown in M-I difference curve representative of the sign of surface charge on graphene.Graphene devices were fabricated by transferring CVD-graphene on to Si/SiO−1L. This corresponds to an average charge density of around 1 μC/cm2 close to the values reported for homogenized alkane emulsions in water (around 5 μC/cm2)412 substrate. The capacitance due to trapped charges in SiO2 is much weaker than the double layer capacitance at the graphene-liquid interface45From 193638404-aminobenzylamine (ABA) and aniline (ANI)Ka of ABA is 8.5, in contrast to that of ANI, which is 4.253Now we turn towards the effect of attaching functional groups on the isoelectric point of graphene surface. For this purpose we choose an electrochemical route since it allows for the versatile attachment of a broad range of chemical moieties by simple variation of experimental parameters4849M-I difference Dirac curve (blue) obtained from this measurement along with the difference Dirac curve before modification (black curve). The curve before modification does not have a zero crossing consistent with the discussion that the pI/pzc of graphene is less than 3.3. However, after modification the difference curve exhibits a clear zero crossing at around a pH of 6, which is attributed to be the approximate pI of the functionalized surface. In order to obtain support for this claim, we have used the model to simulate the presence of pABA by introducing an additional type of ionizable group with a charge density that is around 1.2 times that of graphene (Ka of 8.5. The effect of the silanol groups at (15%) and that of specific ion adsorption (7%) remain the same as for bare graphene. The fitted curves are presented as solid lines in M-I difference curve occurs at 5.92. Further support for this estimation is obtained from Ka of the type of ionizable group to 4.2 (instead of 8.5 as done before). The results collected in M-I zero crossing occurs at 4.05. Further details of the model parameters are discussed in M-I difference Dirac curve directly as an approximate estimate of the surface pI.Ka (or pI) of bare (unmodified) graphene, the attached moieties and to some extent the underlying substrate. The weighting factor is then determined by the density of ionizable sites (see Ka is presented). It is worth mentioning that we are modifying the acid-base equilibrium of the graphene-liquid interface through electrochemical functionalization and hence the pI is modified in this process. The change in pzc is a consequence of this modification and sets in only when we have specific adsorption of ions. The immediate question that comes up is to see the effect of increasing thickness of the functional layers on the isoelectric point of functionalized graphene. For this purpose, we have repeated the above experiment for varying thickness of the polymer layer (see Methods) in many different devices. We first measure the pH-IS-behaviour of unmodified graphene at 4 different IS values, perform the electrochemical functionalization and repeat the pH-IS-measurement followed by an estimation of the layer thickness using AFM. The most important conclusion from the foregoing discussion is that the functionalized graphene surface assumes an effective pI that is a weighted balance between the p2 on the value of pI of the functional interface. For this purpose, we have performed a similar set of measurements on another kind of substrate (referred to as Set 2), namely an oxide prepared using a wet etching procedure (the previous devices were measured on dry oxide). The wet oxide results in a comparatively higher density of silanol groups55Another important aspect concerns the role of SiOFinally, we demonstrate that we can continuously tune the value of pI of the functionalized graphene surface by exploiting the versatility of the electrochemical modification protocol. For this purpose, we perform the characterization of pH-IS-behaviour at the initial stage and after every consecutive electrochemical modification. With every modification the density of functional groups on the graphene surface increases resulting in a shift of the isoelectric point towards higher pH values. We perform this series of experiments continuously without drying the sample in order to ensure that the functional groups remain intact during the entire measurement series. In conclusion, we have presented a strategy to estimate the isoelectric point of bare (unmodified/as-prepared) and functionalized graphene. The surface charge behavior is determined by the weighted average of acid-base properties of the ionizable groups on unmodified or functionalized graphene. While as-prepared CVD-graphene exhibits a pI below 3.3, we could tune this value to higher levels by a judicious attachment of a controlled density of ionizable groups on the graphene surface. In this manner the graphene surface can be rendered positively or negatively charged with the same type of chemical functionality at a given pH. Using a theoretical model we could also show that specific adsorption of ions indeed occurs at the graphene-liquid interface albeit with a minor effect on the capability to tune the net surface charge density of functionalized graphene. The presented theory and the methodology can be used to identify changes in pI/pzc using other chemical functionalization strategies and thereby serves as a versatile platform for engineering the interfacial properties of graphene for a targeted application.2 wafers using photolithography. Two kinds of wafers have been used – one with a dry oxide of 575 nm thickness obtained by thermal oxidation (Set 1) and the other obtained by wet chemical etching with an oxide thickness of 500 nm. CVD-graphene obtained either commercially (Graphene Supermarket Inc.) or by a peel-off process Ref. 2O2) leading to removal of copper in less than 10 minutes. Then, graphene was transferred to Si/SiO2 chips with the prepatterned Ti/Pt electrode lines and baked in the oven at 95 °C for 20 minutes before removal of PS using toluene. The patterning of the flakes (of size 2 μm × 2.5 μm) was also performed by photolithography. For this purpose, 10 nm of copper was first evaporated on graphene to be used as a sacrificial protective layer in order that the photoresist does not come in direct contact with the graphene surface. Following this, the structures are patterned using a positive process using the resist S1805 (Microposit). After exposure and development the unprotected regions are removed using mild oxygen plasma etching. After this, the resist and the remaining copper are removed in N-ethylpyrrolidone and HCl/H2O2 respectively. Subsequently we do another round of lithography to deposit SiO2 by thermal evaporation in order to passivate all electrode lines using an insulating layer. This ensures that graphene is exclusively in contact with the solution. Following this an electrochemical etching procedure was carried out in HCl in order to remove trace metal impuritiesFirst, pre-patterned electrode lines with 4 micron gaps are prepared on 4” Si/SiOThe field-effect measurements in liquid were carried out in a PDMS (poly(dimethylsiloxane)) channel placed on the graphene device to hold the liquid (volume is approx. 200 μL). The gate voltage was applied using a Ag/AgCl reference electrode (WPI Inc.). 0 mV vs Ag/AgCl corresponds to 50 ± 5 mV versus SCE measured in 0.1 M KCl. The impedance of the device was measured using an LCR Meter (Agilent) at 1 kHz. The gate voltage was continuously swept in a fixed range and the real part of the impedance recorded to obtain the data as in 4 was introduced into the microwell and the voltage at graphene was swept from −0.2 to +0.7 V against the Ag/AgCl reference for 1 cycle. For Gr/pANI, a mixture of 10 mM ANI and 100 mM LiClO4 was used, and the voltage was swept from −0.2 to +0.8 V for 4 cycles. For tuning the pI, a series of electrochemical polymerization runs were carried out, using ABA as precursor. For the first 3 modifications, 0.1 mM, 1 mM and 10 mM ABA were used at a voltage of 0.7 V for 1 cycle. The subsequent modifications were also carried out at 0.7 V and 10 mM ABA but increasing the number of cycles from 3 up to 12.The electrochemical modification (ECM) to deposit the functional layers was carried out via oxidative polymerization. The same PDMS channel was filled up with the precursor solution and an Ag/AgCl electrode was used as the reference electrode. The same electrical setup mentioned above was used to perform the modification2 chips were measured using an NT-MDT NTEGRA system or a S&I MonoVista system, with a laser excitation of 632.8 nm at a power of 2.7 mW or 1 mW respectively. This system was equipped with a 520 mm monochromator and a 600 l/mm grating. The acquisition time was 5s. AFM images were obtained using a Digital Instruments Veeco Dimension III in tapping mode. The acquired AFM images were processed using Gwyddion software.Raman spectra of graphene samples on Si/SiOAll equations are modelled symbolically and solved numerically in Mathematica 10. The fitting of model parameters to the data was performed in a semi-automatic manner by programmatically varying the parameters in a given range and minimizing the deviation between the measured and simulated curves.How to cite this article: Zuccaro, L. et al. Tuning the isoelectric point of graphene by electrochemical functionalization. Sci. Rep.5, 11794; doi: 10.1038/srep11794 (2015).


This indicates that SHI induces annealing effects at lower fluences. We also observe that the number of graphene layers is reduced at fluences higher than 1 × 1013 ions/cm2. Using inelastic thermal spike model calculations, we estimate a radius of 2.6 nm for ion track core surrounded by a halo extending up to 11.6 nm. The transient temperature above the melting point in the track core results in damage, whereas lower temperature in the track halo is responsible for annealing. The results suggest that SHI irradiation fluence may be used as one of the tools for defect annealing and manipulation of the number of graphene layers.Studies on interaction of graphene with radiation are important because of nanolithographic processes in graphene-based electronic devices and for space applications. Since the electronic properties of graphene are highly sensitive to the defects and number of layers in graphene sample, it is desirable to develop tools to engineer these two parameters. We report swift heavy ion (SHI) irradiation-induced annealing and purification effects in graphene films, similar to that observed in our studies on fullerenes and carbon nanotubes (CNTs). Raman studies after irradiation with 100-MeV Ag ions (fluences from 3 × 1060.80.x; 81.05.ue Graphene is a new member of the carbon family that has two-dimensional honeycomb lattice structure ,2. It is+ ions has shown that the disorder produced in graphene leads to two competing processes resulting in an initial increase in the formation of defects as depicted by the increase in ID/IG ratio followed by its decrease at higher fluence [+ ions [The graphene properties are sensitive to defects. For example, it has been reported that metal to insulator transition occurs with small introduction of defects in graphene perfect lattice . Hence,  fluence . The def [+ ions  and 2-Me [+ ions  show tha [+ ions . Structu [+ ions . Graphen [+ ions . Zhao et [+ ions . Yang et [+ ions .As mentioned above, most of the irradiation studies on graphene are focused on damage by electron and low-energy ions, and there are only few reports available on the ion irradiation studies of graphene at high energy . The stu2/Ni substrate from Graphene Laboratories Inc., USA were used. Though mechanically cleaved graphene samples generally provide better quality single-layer graphene, we have used commercially available CVD-grown samples which are available in larger size, with industrial applications in mind. Irradiation is carried out with 100-MeV Ag7+ ions using 15-MV Pelletron accelerator at IUAC, New Delhi, India, at fluences of 3 × 1010, 1 × 1011, 3 × 1011, 1 × 1012, 3 × 1012, 1 × 1013, 3 × 1013 and 1 × 1014 ions/cm2. The pressure in the irradiation chamber was of the order of 10−6 mbar, and the average ion current was 1 particle nanoampere . The sample was cut into pieces of 1 × 1 cm2 size for irradiation. Half of the area of each sample was masked with thin aluminium foil during irradiation to keep half the portion unirradiated for comparing the results.In the present work, graphene flakes grown with chemical vapour deposition (CVD) technique on Si/SiOThe surface topography was studied using Digital Instruments Nanoscope IIIa atomic force microscope  in the tapping mode. Raman measurements were carried out using Renishaw inVia microRaman set-up . Initially, we performed Raman measurements on pristine sample at several spots with different laser powers to check the self-heating effect. We took 10% (2 mW) of the laser power, which is below the threshold value for laser-induced heating, for measurement on all the samples. Therefore, self-annealing effect during Raman measurement is ruled out.Raman spectra of pristine (unirradiated) as well as irradiated portions of each sample were recorded at four different spots (flakes) using Ar ion laser with excitation wavelength of 514.5 nm and averaged out for further analysis. The size of the laser spot is approximately 2 μm with × 50 optical magnification of the attached optical microscope. The peak intensities have been obtained by fitting the peaks assuming Lorentzian distribution.A typical AFM topographic image of the sample is shown in Figure h ~ 0.35n + t0, where h is the measured height, t0 is the approximate distance between the first graphene layer and the substrate and n is the number of layers. Substrate to the first layer height t0 is approximately 0.6 to 0.7 nm [The film thickness shows a linear relation of −1), G peak  and 2D peak . The G peak is the characteristic peak of graphitic material, and its origin is due to Eg2 symmetry mode near the k = 0 point in the first Brillouin zone. It is the first-order process and a stretching vibrational mode of the carbon atom both in the rings and in the chain [Ag1 symmetry phonons near the K-zone boundary. In the absence of defect, these phonons are Raman inactive due to the momentum conservation in the scattering [The evolution of Raman spectra of pristine as well as irradiated part is shown in Figure he chain . Any chaattering . They beattering . So, theattering .y-axis for clarity. It has been observed that D peak areal intensity first decreases up to a fluence of 3 × 1011 ions/cm2 and then starts to increase with increasing fluences up to 1 × 1014 ions/cm2.The Raman spectra of unirradiated and irradiated graphene at different fluences with 100-MeV Ag ions are shown in Figure α defined by ID/IG ratio, where the ID and IG are the intensity of D and G peaks, respectively, in the Raman spectrum. It measures the disorder present in the sample which may be due to defects or misalignment in the graphene sample. Samples of lower disorder have low value of α, and a high value of α indicates larger amount of disorder. Since there are variations in different parts of the sample, we have normalized the value of ID/IG of irradiated part with the value of ID/IG of pristine part, and these normalized ratios have been studied for further analysis. The variation of α for different fluences is plotted in Figure The defects induced in carbon-based materials are studied using disorder parameter α is observed up to the fluence of 3 × 1011 ions/cm2, and with further increase in the fluence, there is an increase in its value. This initial decrease signifies ordering or purification of graphene at low fluences and is induced by energy dissipated by the incident energetic ion. To the best of our knowledge, this is the first kind of report on the ordering or purification of graphene. Electron beam irradiation studies [It is clear from Figure  studies ,38-40 haI2D/IG at a lower fluence also confirms the crystal quality improvement as shown in Figure 13 ions/cm2, the ratio I2D/IG decreases sharply due to higher defect density.The small increment in intensity ratio 2-bonded structure [g as compared to graphite (g = 3 × 1013 W/cm3/K) and subsequently result in a larger calculated value of core and halo radius. Figure In the present case, as the energetic ion passes through the film, a large fraction of its energy is transferred to the electronic subsystem of the target material by inelastic collisions. According to the thermal spike model, this results in high transient local energy density along the ion path. The deposited energy is transferred to the atomic system by electron-phonon coupling which generates a localized heating of the atomic system in the ion track of nanometric dimension. This results in the transient thermal spike lasting for a few picoseconds. Moreover, the temperature shows a gradual radial decay as the radial distance from the ion track increases . The spatructure . The temtructure . Hence, The temporal distribution of lattice temperature as projected from the 3D plot for radial distances of 0.0 nm (centre of the track core), 2.6 nm, 11.6 nm and 15.0 nm are plotted in Figure The temperature higher than the melting temperature is reached in a radius of 2.6 nm, thus creating a damaged molten zone along the ion trajectory, referred as the track core. Similarly, temperatures above 800 K, essential for graphene annealing , are rea12 and 3.4 × 1011 ions/cm2, respectively. Hence, we can expect that annealing will increase up to a fluence of 3.4 × 1011 ions/cm2 when halos cover the sample. Beyond this fluence, the region damaged by the track core starts to dominate. Kumar et al. have observed similar behaviour in carbon nanotubes due to ion irradiation [The damage and annealing occurs simultaneously. However, because the ratio of the core and halo radii is approximately 1:4  × 10−12 cm2, 4.3 ± 0.2 and (6.7 ± 0.8) × 10−14 cm2 respectively, with chi-square value of 0.04. The simulated curve with the above parameters is shown in Figure In Equation 1, the first term represents exponential decay due to the annealing effect of irradiation in an annular region and the second term represents exponential increase due to damage effects within the ion track core. Constants  6.7 ± 0. × 10−14 σ1 and σ2, respectively, represent the cross-sectional area of cylindrical regions which are annealed and damaged by an incident ion. The annular halo and core radii estimated from the value of σ1 and σ2 are 5.7 and 1.4 nm, respectively. The overlapping fluence calculated with this core radius is 1.6 × 1013 ions/cm2. The halo and core radii observed for graphene are found to be smaller in comparison to radii 11.6 and 2.6 nm obtained from thermal spike calculations. The smaller core and halo radii can be explained in terms of difference in the strength of electron-phonon coupling  of graphene and graphite. Larger thermal conductivity and mean free path may lead to a lower value of g in the case of graphene as compared to graphite (g = 3 × 1013 W/cm3/K) and subsequent estimated lower value for core and halo radii.−1 at fluence of 1 × 1014 ions/cm2  there is more coverage of one- and two-layer graphene instead of three to four layers observed before irradiation. The ion irradiation-induced ablation in graphene can be used for controlling the number of layers with spatial selectivity. Similar reduction in the number of graphene layers with laser ablation above a given threshold laser energy density has been demonstrated by Dhar et al. [Atomic force microscopy imaging Figure  also conr et al. . This sh11 ions/cm2 after which a rapid increase in disorder is observed. The effect is similar to that shown earlier in fullerenes and CNTs. It is concluded that graphene can be purified by the use of SHI irradiation which is of particular importance for using CVD-grown graphene in device applications. Our results suggest that swift heavy ion with selected ranges of fluence may be used as one of the tools for defect annealing and manipulation of the number of graphene layers.In summary, the annealing effect of swift heavy ion irradiation on graphene in low-fluence regime has been observed. Raman studies show that 100-MeV Ag ion irradiation induced annealing up to a fluence of 3 × 10SHI: swift heavy ion; CNTs: carbon nanotubes; EBL: electron beam lithography; GFET: graphene FET; SEM: scanning electron microscope; MWCNT: multiwalled carbon nanotubes; CVD: chemical vapour deposition; pnA: particle nanoampere; AFM: atomic force microscope; FWHM: full width at half maxima; i-TS: inelastic thermal spike.The authors declare that they have no competing interests.SK carried out the experiments and data analysis and drafted the manuscript. AT and DKA contributed in the design and discussion of this work and in the revision of the manuscript, SAK carried out the theoretical simulations and fitting of curve. FS and VB carried out the Raman characterization and helped in the analysis of data. All authors read and approved the final manuscript.


As single agents, durvalumab (MEDI4736), a human IgG1 anti-PD-L1 antibody, and tremelimumab, a human IgG2 anti-CTLA-4 antibody, have shown acceptable safety profiles and antitumor activity. Similar to other anti-PD-L1/anti-PD-1 monotherapies, durvalumab has shown greater objective tumor response rates in PD-L1-positive patients compared with PD-L1-negative patients. Anti-CTLA4 therapies activate T-cells and may increase immune infiltrate and PD-L1 expression in tumor cells and tumor infiltrating immune cells. Thus, combination therapy with durvalumab and tremelimumab could be active in NSCLC regardless of baseline PD-L1 expression.This is a phase 1, open-label, dose-escalation/expansion study (NCT02000947) of D+T in patients with Stage III/IV NSCLC . The primary endpoint is safety and tolerability; secondary endpoints include investigator-reported RECIST v1.1 response. PD-L1 expression was tested retrospectively using an immunohistochemical assay (Ventana).As of 1 June 2015, 102 patients received treatment in the dose escalation phase; combinations of durvalumab [3 mg/kg (D3) to 20 mg/kg (D20) every 2 (q2w) or 4 weeks (q4w)] and tremelimumab [1 mg/kg (T1) to 3 mg/kg (T3)] q4w, plus a D15 + T10 combination, were explored. Across all cohorts, 80% and 42% of patients had ≥1 treatment-related AE ; 28% discontinued treatment due to a related AE. A greater frequency of AEs, without a corresponding increase in tumor response, was seen with increasing T dose. In the combined T1 cohort (D10–D20), 73% and 30% of patients had ≥1 related AE ; 16% discontinued treatment due to a related AE. There were 3 treatment-related deaths .84 patients  were evaluable for response Table . The oveD+T at selected phase 3 dose D20, T1) has a manageable tolerability profile and anti-tumor activity in NSCLC. Unlike anti- PD-1/PD-L1 monotherapies, the combination of D+T appears to be active regardless of PD-L1 status, including even in patients with no tumor cell membrane PD-L1 staining, a setting where patients would not be expected to derive significant benefit from anti-PD-1/PD-L1 monotherapy over current standard of care [ has a ma


Legal problems are more numbered even in Neonatology.Hospital policy should establish strategies to limit systematic, environmental and human errors, including solutions to optimize organization and increase knowledge .Staff reSimulation works through mechanisms that are proper of our brain for decision-making (i.e. simulated mind) . The capThe aim of this lecture is to focus on methods of simulation on neonatal stabilization and transport .Simulation retraining for medical (or nurse) staff should be based on the andragogic approach: adults are conscious of their own educational needs and focus their own attention on specific interests (related to daily practice) . This meA precious additive methodological element is fun: the serious medical game is the approach to optimize technical memories trough emotions .In our experience, materials for simulation are daily clinical devices and innovative stuff developed by a multidisciplinary collaboration ;we discuAs regards neonatal stabilization and transport, we perform: i) annual retraining sessions for all the operators in our Unit, ii) low and medium fidelity simulation sessions for nearby hospitals, iii) high fidelity sessions for colleagues working at geographically uncomfortable hospitals (i.e. island) and so needing for transport by helicopter, as a kind of “full scale CRM (Crisis Resources Management)”.In our opinion the traditional approach to teaching is inadequate for retraining of adult professionals. We propose simulation as the method to deepen knowledge, strengthen abilities and so optimize performances. Recording sessions and analysing them to discuss behaviours is a main instruments for debriefing. Every kind of support  is admitted and should be creatively promoted.


Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells  themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation  Endothelial cell (EC) proliferation is majorly regulated by soluble factors, such as vascular endothelial growth factor (VEGF), which is required for angiogenesis. However, mechanical forces, generated by pulsatile blood flow (shear and strain), also control EC proliferation, possibly via modulation of EC responses to soluble growth factors124572+ influx in EC through ion channels, including members of the transient receptor potential (TRP) family12in vitro. However, the signaling mechanism(s) upstream of this abnormal TEC phenotype are not yet known. We recently found that TEC express reduced levels of TRPV4 compared to normal endothelial cells (NEC), and exhibit abnormal Rho-dependent mechanosensing and angiogenesis, which were restored upon overexpression or pharmacological activation of TRPV4While calcium has not been widely pursued as a potential anti-angiogenic target, intracellular calcium has been demonstrated to regulate angiogenesis, including the processes of cell proliferation, survival, and migration10In the present study, we investigated the role of TRPV4 in TEC proliferation, including the potential underlying molecular mechanism(s).We investigated if TEC proliferation levels are altered compared to NEC, which may contribute to the abnormal angiogenesis and structure of tumor vessels. We found that TEC exhibited significantly (p ≤ 0.05) increased proliferation compared to NEC, as measured by XTT and cell viability assays . HoweverSince TEC express low levels of TRPV4 ;15 and pTo further confirm the specificity of GSK on TEC proliferation, we investigated if GSK treatment inhibits Lewis Lung Carcinoma (LLC) cell proliferation. While LLC cells express TRPV4 . OverallWe next investigated the molecular mechanism(s) by which TRPV4 mediates the above effects. Since members of the mitogen-activated protein kinase (MAPK/ERK) family are critical regulators of cell proliferation, and mechanical signaling is known to stimulate ERK1/216171820Activation of the ERK signaling pathway, as well as the extracellular matrix (ECM)/integrin/receptor tyrosine kinase signaling pathway, has been demonstrated to induce cyclin D1 mRNA expression, allowing entry of cells into S-phasein vivo, we induced LLC tumors in wild-type (WT) mice, as previously describedFinally, to determine the functional significance of TRPV4 in the regulation of TEC proliferation in vitro. Further, we found a correlation between TRPV4 expression, abnormal TEC proliferation, and increased ERK1/2 phosphorylation, which was attenuated by pharmacological activation or overexpression of TRPV4. Finally, we demonstrate that EC proliferation in tumors is significantly inhibited by treatment with GSK in mice subcutaneously implanted with LLC tumors.In the present study, we demonstrate that reduced expression of the mechanosensitive ion channel TRPV4 in TEC results in increased proliferation, which may contribute to abnormal tumor angiogenesis. We show that the pharmacological activation of TRPV4, with a small molecule activator GSK, decreases TEC proliferation Several studies have indicated different TRP channels to influence the angiogenic process through multiple mechanisms1223Previously, TRPV4 dysfunction has been found to increase renal cystogenesis, and pharmacological activation of TRPV4 reduces proliferation of renal duct cells as well as cholangiocytes2514in vitro, and significantly reduced TEC proliferation in vivo, revealing a correlation between TRPV4-dependent ERK1/2 activity and TEC proliferation. We have previously shown that TRPV4 expression is lower in TEC compared to NEC and treatment with TRPV4 agonist, GSK, up-regulates TRPV4 expression in TEC but not in NEC. Further, GSK treatment normalized abnormal angiogenesis exhibited by TEC in vitro. Similarly, increased expression of TRPV4 by GSK may inhibit TEC proliferation via inhibition of ERK1/2 phosphorylation. Based on these findings, we hypothesize that TRPV4 expression (and/or activity) should be at a threshold level to maintain EC mechanosensitivity and proliferation, and beyond this threshold TRPV4 expression may not have any additional effects, as seen in NEC.Indeed, our results have shown that TEC exhibit increased expression of cell cycle genes, S phase protein, PCNA, and proliferation. Importantly, in untreated animals (−GSK), we found increased co-localization of the proliferative marker ki-67 and endothelial marker CD31, confirming that there is increased proliferation of TEC in tumors. However, pharmacological activation of TRPV4 with GSK normalized ERK1/2 phosphorylation, TEC proliferation Taken together, our data supports a significant role for the mechanosensitive TRPV4 channel in regulating TEC proliferation via modulation of the ERK pathway. We have demonstrated that pharmacological activation of TRPV4 induces vascular normalization in tumors by modulating Rho activity2 incubator, split at ~90–95% confluence, and used between passages 11–22, as previously described9Normal (NEC) and tumor EC (TEC) were extensively characterized and cultured on fibronectin or gelatin coated tissue culture dishes and grown in a defined medium in a 37 °C, 5%COCells were treated with (GSK: GSK1016790A 100 nM) or vehicle (DMSO), washed with PBS, and lysed in RIPA buffer or Triton X-100 with protease and phosphatase inhibitors (Boston Bioproducts). Lysates were loaded and separated by electrophoresis on 8%, 10%, or 12% SDS acrylamide gels. Proteins were transferred onto a PVDF membrane and the membrane was blocked in 5% milk in TBS with 0.01% Tween-20 (TBS-T) for 1 h. The blots were incubated with primary antibodies: phospho-ERK1/2 (1:1000) ; total ERK1/2 (1:1000) ; phospho AKT (1:1000) , total AKT (1:1000) , anti-PCNA (1:1000) (Abcam) and tubulin (1:5000) (Abcam) overnight, rinsed three times with TBS-T, and incubated with the appropriate secondary antibody goat anti-mouse  or goat anti-rabbit  conjugated with horseradish peroxidase (Jackson Laboratories). Signals were detected using chemiluminescent substrates (Thermo Scientific) and developed with a FluorChem M Simple Imager (Protein Simple).RNA isolation of cells was performed using an RNeasy Mini Kit (Qiagen) and measured using the NanoDrop 2000 UV-Vis Spectrophotometer. cDNA synthesis was performed using qscript cDNA SuperMix (Quanta Biosciences) and qPCR analysis was performed using the Fast SYBR green master mix (Applied Biosystems) on the Fast Real-Time PCR system (Applied Biosystems). Real time PCR was performed using real time cyclin D1, cyclin A, cyclin E1, CDK1, CDK2, CDK4, CDK6, TRPV4, and GAPDH primers obtained from IDT technologies. Gene expression was first made relative to GAPDH and ΔΔCT values were expressed as fold change compared to NEC.Cells were transfected with TRPV4-EGFP or EGFP constructs using targefect (Targeting Systems)15156) were subcutaneously injected in the flank region of C57BL/6 mice. Once the tumors were palpable (after 7 days), mice either received daily intraperitoneal (i.p.) injections of TRPV4 agonist GSK1016790A (10 μg/kg) (+GSK) or saline (−GSK), until day 21, as previously describedIn vivo proliferation assays were performed using frozen sections  fixed and permeabilized in ice cold acetone (20 min), washed with Tris buffered saline (TBS), and incubated with rat-anti-CD31 (Invitrogen) (1:50) and anti-ki-67 (Abcam) (1:200) overnight. Sections were then washed with TBS (3x–5 min) and incubated for 1 h with appropriate secondary antibodies coupled to Alexa Fluor-488 or Alexa Fluor-594 (Invitrogen) and mounted with DAPI (Vector labs). Images were acquired with Olympus Epifluorescence Microscope (IX71) using QCapture Pro (QImaging) and quantified using ImageJ. Total number of endothelial cells (CD31+ stained vessels) as well as the number of EC that co-localized with ki-67 were counted and expressed them as % of proliferating EC (CD31+-ki-67+/CD31+). The rationale for this is to differentiate proliferating EC (vessels) from non-proliferating EC.All animal experiments were performed according to an approved protocol by Northeast Ohio Medical University, IACUC. Mouse Lewis Lung Carcinoma (LLC) cells  for 2 h. The cells were fixed with 4% PFA, washed with PBS containing 0.1% Triton X-100 (PBST) (3x–5 min), and incubated for 10 min with 1N HCL on ice. Fixed cells were then incubated with 2N HCL at room temperature (RT) for 10 min and moved to the incubator (37 °C) for 20 min. Borate buffer (0.1 M) was used to buffer the cells for 12 min at RT. Cells were washed with PBST (3x–5 min), blocked with PBST solution containing 1M glycine and 5% fetal bovine serum (FBS) for 1 h, and incubated with anti-BrdU primary antibody (1:100) (Abcam) overnight at RT. Following incubation, cells were washed with PBST (3x−5 min), incubated with secondary antibody, Alexa Fluor-488 (Invitrogen), and mounted with DAPI (Vector labs). Images were captured using an Olympus IX71-fluorescence microscope, and analyzed with ImageJ. Quantitative analysis was performed by measuring BrdU positive cells in NEC and TEC  and expressed as fold change over respective control conditions (NEC in the case of NEC vs TEC or TEC in the case of TEC vs TEC+GSK) (p ≤ 0.05).Cell proliferation was determined using XTT kit (Biotium) and cell viability kit (Enzo biosciences). Cultured EC were trypsinized, counted, and plated in a 96-well plate (1000–2000 cells/well). 24–48 h post plating, media was removed and cells were treated with GSK1016790A (GSK) (10–500 nM) in serum free media for overnight. Cells were washed once with PBS; and XTT or Calcein dye was added to the cells and incubated for 30 min-24 h. Wells containing no cells with the dye served as controls to determine the level of the background signal. Absorbance was read at 485–535 nm or 450–500 nm, respectively for Calcein and XTT.t test, analysis of variance (ANOVA) and Tukey’s post-hoc analysis with significance set at p ≤ 0.05.All the data shown is mean ± SEM from at least three independent experiments. Significance was determined using Student’s How to cite this article: Thoppil, R. J. et al. TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation. Sci. Rep.5, 14257; doi: 10.1038/srep14257 (2015).


Background: Prior cognitive research in posttraumatic stress disorder (PTSD) has focused on automatic responses to negative affective stimuli, including attentional facilitation or disengagement and avoidance action tendencies. More recent research suggests PTSD may also relate to differences in reward processing, which has lead to theories of PTSD relating to approach-avoidance imbalances. The current pilot study assessed how combat-PTSD symptoms relate to automatic behavioral tendencies to both positive and negative affective stimuli.Method: Twenty male combat veterans completed the approach-avoidance task (AAT), Clinician Administered PTSD Scale, Beck Depression Inventory-II, and State-Trait Anger Expression Inventory-II. During the AAT, subjects pulled (approach) or pushed (avoid) a joystick in response to neutral, happy, disgust, and angry faces based on border color. Bias scores were calculated for each emotion type (avoid-approach response latency differences). Main and interaction effects for psychological symptom severity and emotion type on bias score were assessed using linear mixed models.Results: There was a significant interaction between PTSD symptoms and emotion type, driven primarily by worse symptoms relating to a greater bias to avoid happy faces. Post hoc tests revealed that veterans with worse PTSD symptoms were slower to approach as well as quicker to avoid happy faces. Neither depressive nor anger symptoms related to avoid or approach tendencies of emotional stimuli.Conclusion: Posttraumatic stress disorder severity was associated with a bias for avoiding positive affective stimuli. These results provide further evidence that PTSD may relate to aberrant processing of positively valenced, or rewarding stimuli. Implicit responses to rewarding stimuli could be an important factor in PTSD pathology and treatment. Specifically, these findings have implications for recent endeavors in using computer-based interventions to influence automatic approach-avoidance tendencies. Anhedono reward . It is so reward . For exao reward  have beeo reward . NeurocoNeurocognitive research related to PTSD has focused primarily on responses to negative or trauma-relevant affective stimuli. Results from research investigating implicit attentional processes suggest that individuals with PTSD may experience greater attentional interference by negative or trauma-relevant affective stimuli . In othebehavioral tendencies to approach or avoid affective stimuli (The approach-avoidance task (AAT) was developed to identify automatic  stimuli . Subject stimuli  based on stimuli , and spi stimuli . Results stimuli  and posi stimuli .There have been two studies utilizing the AAT to investigate behavioral tendencies for individuals with PTSD associated with trauma-related, negative stimuli . One stuInvestigations using trauma-unrelated affective stimuli  are important for understanding whether observed behavioral tendencies may generalize to other situations and contexts. The investigation of behavioral tendencies to positively valenced stimuli or situations would also be important, as this could relate not only to underlying symptoms of anhedonia or emotional numbing, but perhaps also to motivation for treatment. There have been recent endeavors to use computer-based interventions  to modifIn the current study, we examined approach and avoidance tendencies in response to trauma-unrelated affective stimuli . This choice of stimuli was to identify whether PTSD relates to differences in approach and avoidance tendencies that are pervasive in generalizing to trauma-unrelated stimuli and situations. In addition, we aimed to understand how PTSD may relate to approach and avoidance tendencies to both positive and negative affective stimuli, which would be difficult to do if focusing exclusively on trauma-related stimuli. The use of emotional faces could also have implications for understanding interpersonal deficits that are commonly associated with PTSD . We hypoSD = 7.69; mean education = 15.55, SD = 1.85) with varying PTSD symptomology  – IV; a-approacha for angry conditions. More negative values reflect greater avoidance bias whereas larger positive scores reflect greater approach tendencies.The AAT was conducted similarly to previously described tasks , and inc1. Age and education were explored as possible covariates using Spearman’s rho. Three linear mixed effects analyses (LMEs) were used to investigate main and interaction effects for each psychological measure  and emotion type  on avoid-approach bias scores. While effect sizes for LME’s are controversial in the present literature given the impact and difficulty estimating random effects, conditional effects sizes for LME models were estimated. Results were considered significant at p < 0.05, Bonferroni corrected for three LMEs .Statistical analyses were conducted using R Statistical PackagePost hoc Spearman’s rho correlations were used to clarify LME results and identify potential relationships between the avoid-approach bias scores for each specific emotion type and psychological symptom severity including PTSD, depressive, and anger symptoms. To further delineate identified effects of implicit approach and avoidance tendencies, a second bias score was calculated (a–pushn).lculated . The secSD = 19.01), depression  and anger symptoms . Neither age nor education correlated with avoid-approach bias scores .Subjects endorsed mild levels of PTSD  = 4.80, p = 0.005, conditional effect Cohen’s d = 0.518], but no main effect for either PTSD symptoms  or emotion type . The interaction between depressive symptoms and emotion type was trending with a small to medium effect , but there was no main effect for depressive symptoms or emotion type . There were no main or interaction effects with anger symptoms, yielding small effects . Figure 1 depicts relationships between PTSD and depressive symptoms and approach/avoidance tendencies by emotion type.Linear mixed effects analyses analyses revealed a significant interaction with a medium effect between PTSD symptoms and emotion type [Post hoc correlations indicated that individuals with worse PTSD symptoms exhibited a greater avoidh-approachh bias (greater avoidance) for happy faces, and demonstrated a large effect . Despite a trending interaction between depressive symptoms and emotion type, there were no significant relationships between depressive symptoms and avoid-approach bias scores .h-approachh bias score, we examined the individual bias scores for happy faces for approach and avoidance response directions separately compared to neutral faces . Large effects were found for both approach and avoidance conditions, suggesting that those with worse PTSD symptoms were slower to approach , and quicker to avoid  happy faces.To further characterize the relationship between PTSD symptoms and the avoidavoidance tendencies to positive affective stimuli . These findings are consistent with reports of decreased behavioral and neural  responsivity to reward in PTSD . Our res stimuli , the prer = 0.35; In previous research, PTSD diagnosis has been reported to relate to greater attentional biases for negative affective stimuli . The fewpositive affective stimuli may be more robust than to negative affective social stimuli in PTSD, and warrant further investigation. In particular, further research is needed to understand whether the avoidance tendencies to positive affective stimuli are specific to the social domain  or if it generalizes to other domains. Notably, one previous study reported depressive symptom severity reported by individuals with PTSD to relate to increased avoidance of more general positive affective stimuli . While our findings related specifically to PTSD symptoms and not depressive symptom severity, both of these findings suggest that implicit behavioral tendencies to positive affective stimuli play a role in trauma-related psychological symptoms.In the present study, individuals with greater PTSD symptom severity exhibited behavioral biases associated with positive affective stimuli (happy faces). Our results suggest that avoidance tendencies to Evidence-based treatments for PTSD (i.e., Prolonged Exposure therapy; The current study was limited by a small sample and the lack of a non-trauma exposed group. Furthermore, all subjects were male combat veterans. Therefore, generalizability to other populations is unknown. In addition, subjects in the present study endorsed mild to moderate levels of PTSD symptomology. This limits generalizability to more severe PTSD populations and may provide a reasonable explanation for the lack of expected results related to negatively valenced stimuli. However, this also suggests that automatic approach-avoidance imbalances may be evident even for those with only moderate symptom levels. Nevertheless, utilizing a sample with more severe PTSD symptom severity or the use of trauma-relevant stimuli might yield different results and provide additional insights into the role of approach and avoidance behavior in PTSD populations. It is important for future, larger studies to investigate the impact of a broader range of PTSD symptomology and to examine the contribution of individual PTSD symptom clusters on approach and avoidance behavioral tendencies. Despite limitations, the current results provides initial evidence that PTSD symptoms are associated with differences in implicit behavioral tendencies in response to positively valenced stimuli, characterized by both reduced approach and increased avoidance tendencies. These results highlight the potential value of targeting responses to positive affective stimuli in PTSD treatment.AC: Substantial contributions to the conception and design of the work; data acquisition, data analysis and interpretation of results. Contributions to drafting, and revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. WY: Substantial contributions to data analysis. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. J-FS: Substantial contribution to data acquisition. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SB: Substantial contributions to the conception and design of the work. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CT: Substantial contributions to data analysis and interpretation of results. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. RA: Substantial contribution to the concept or design of the work, and interpretation of results. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.