A simulated four-stage graphene distributed amplifier achieved up to 4 dB gain and 3.5 GHz bandwidth, which could be realized with future IC processes. A PCB level graphene distributed amplifier was fabricated as a proof of circuit concept.Ever since its discovery, graphene bears great expectations in high frequency electronics due to its irreplaceably high carrier mobility. However, it has long been blamed for the weakness in generating gains, which seriously limits its pace of development. Distributed amplification, on the other hand, has successfully been used in conventional semiconductors to increase the amplifiers’ gain-bandwidth product. In this paper, distributed amplification is first applied to graphene. Transmission lines phase-synchronize paralleled graphene field-effect transistors (GFETs), combining the gain of each stage in an additive manner. Simulations were based on fabricated GFETs whose  Their work achieved S21 of 10 at 1 GHzet al. fabricated graphene voltage amplifiers in complementary push-pull configuration, which generated a voltage gain of 11.4 dB and a −3 dB bandwidth of only 70 kHzHowever, the overall progress in electronics is greatly hindered by graphene’s gapless band structure1et al. and the authors’ group have proposed the multi-layer-routing inverted process for graphene integration, which serves as the foundation for future novel graphene circuit architectures1219On the other hand, graphene’s IC process has witnessed substantial improvements1219gm of each transistor in a phase-synchronized manner. Therefore, the bandwidth-gain-product could be increased. While the gain of a conventional cascade amplifier is the product of the gain of each stage, distributed amplifiers’ gain is directly proprotional to the number of stages. This trait makes distributed amplification scheme especially suitable for graphene, as each GFET stage generates very modest or less-than-unity gains. The otherwise product of the gain of each stage would be mediocre.In this paper, the graphene distributed amplifer is proposed to solve its long-standing weakness in amplification. Engineers have successfully applied distributed amplication to conventional semiconductor technologies to increase the amplifiers’ gain-bandwidth-product232425fT ranging from 8.2 GHz to 10.6 GHz and fmax 12.4 GHz to 16.6 GHz without de-embedding . The fmax exceeded fT in these works due the low resistance of the buried gatesFour GFETs with gate lengths of 300 nm through 500 nm have been fabricated, which generated fmax point of view. The GFETs employed a two-finger structure with each finger 6 μm wide. The gate dielectric employed HfO2 with equivalent oxide thickness (EOT) of 2 nm formed directly on the buried gate. Graphene was synthensized by CVD method and transferred by “bubbling” method as previously reported (details in Method)27GFETs in buried-gate structure have been fabricated. Cross-section and top views of a 400 nm-gate-length GFET structure are shown in 3N42O3fT/fmax of 8.2 GHz/16 GHz, 8.4 GHz/12.5 GHz, 9.1 GHz/12.4 GHz, 10.6 GHz/16.6 GHz, respectively, as shown in The probing pads were 80 μm × 80 μm with 100 μm pitch in ground-signal-ground (GSG) layout, as shown in d, Cg, Ld and Lg, constitute the gate and drain artificial lines. R1 and R2 form the two terminating resistances.1, R2, and the lumped passive components, Cg, Cd, Lg, Ld (details in Method). Source and load impedances were set as R1 and R2, respectively. Simulation #1 achieved S21 of roughly 4 dB and 3.5 GHz bandwidth, as shown in 1, R2, Cg, Cd, Lg and Ld resulted in 1.28 kΩ, 78.5 Ω, 0.01 pF, 0.7 pF, 105 nH and 7.6 nH, respectively. Simultion #1 represents situations when GFETs are exactly identical (T1~4: GFET #4). After the demonstration of high-performance individual GFETs in many lab works, efforts in graphene electronics society should now emphasize reproducibility and reliability to support the potential mass production.Circuit simulation based on measured S-parameters of the GFETs was performed in a standard circuit simulator, Agilent Advanced Design System (ADS). In Simulation #1, T1 through T4 all employed GFET #4, respresenting ideal situations when GFETs are exactly identical. The optimization was performed with three goals: 1) S21 > 5 dB; 2) S11 < −10 dB; 3) S22 < −10 dB. Variables were the terminating resistances, R1, R2, Cg, Cd, Lg and Ld resulted in 1.28 kΩ, 58 Ω, 0.01 pF, 1.2 pF, 86 nH and 7.2 nH, respectively, as shown in Another simulation, Simulation #2, was launched closer to real circumstances. GFET #1 through #4 were assigned to T1 through T4, respectively. The setup was the same with Simulation #1, which generated S21 of 3 dB and 3.5 GHz bandwidth, as shown in Simulation #1 and #2 for the first time demonstrated wide-band graphene amplifiers in literature. Both simulations were based on measured S-paramenters of the GFETs, illustrating the feasibilty of wide-band graphene amplifiers with present averge-performance GFETs. The passive component were subjected to the following equation that guarantees the phase-synchronization requirement:g, Cd, Lg and Ld were 0.01 pF, 0.7 pF, 105 nH and 7.6 nH, respectively. Miller effects obscured Cdrain and the gate oxide capacitance was roughly 0.08 pF, caculated according to the dimension and gate dielectric of the 400 nm GFET. Cgate was further decreased due to quantum capacitance effect321 and R2, which roughly equaled characteristic impedences of the gate and drain transmission lines, respectively, caculated as where C and L represents the capacitance and inductance at each node. For Simulation #1, Cg in Simulation #1 and #2.On the other hand, efforts have been made in this work to realize a PCB-level graphene distributed amplifier, which meets problems such as precise GFET models, the choice of discrete lumped passive components, and bonding wires. Any variations are likely to destroy the phase-synchronization between the gate and drain transmission lines, especially when any of the passive components values are too small, e.g., Cg, Cd, Lg, Ld are avoided in the simulations, Simulation #3 and #4. In particular, Cg was fixed at 1 pF . The terminating impedances were both set at 50 Ω. Simulation #3 used Cg = Cd = 1 pF and R1 = R2 = 50 Ω. The simulation result is shown in g, the gain decreased to −10~−5 dB and the bandwidth remained at 3 GHz. Simulation #4 further avoided matching sections at the input and output terminals by employing 50 Ω source and load impedence. Cg and Cd were 1 pF, and Lg and Ld 10 nH, which are common discrete devices. The simulation result is demonstrated in Firstly, small values for any of CA PCB-level graphene distributed amplifier was fabricated according to Simulation #4, which featured approximately 5 cm × 3 cm dimension, as shown in g. It increases with each GFET’s transconductance. The charateristic impedences of the artificial transmission lines are approximated as Test results are shown in Even though the fabricated work somewhat reassembles a real amplifier, future graphene distributed amplifers should be realized in IC technologies, where precise models of GFETs and passive components should be addressed. Besides, on-chip waveguides could be considered to replace artifical transmission lines. They might further reduces losses.Distributed amplifiers over the past decades have successfully been applied in compound semiconductor and CMOS technologies2324254/H2 flow rates were set at 4.5/500 sccm. After growth, Pt foils were quickly pulled out of the high temperature area27Graphene in this work was synthesized by CVD method on Pt foils as previously reported. Large scale monolayer graphene films were grown on 180 μm thick Pt foils  by ambient-pressure chemical vapor deposition (APCVD) method. The growth temperature was 1000 °C and CH2 with equivalent oxide thickness (EOT) of 2 nm was deposited as the gate oxide by atomic layer deposition (ALD) method. After graphene transfer, the channel of the GFETs was defined by contact photolithography and the residual graphene was removed by oxygen plasma etching. The source/drain contact was defined by electron-beam-lithography (EBL). Due to misalignment, certain source-gate and drain-gate overlaps existed in this patch of fabrication. The contacts were formed by lift-off process. The source/drain regions underwent 5 min UVO treatment before the sputtering of 40 nm Pt. The high work function of W induced more doping to graphene and UVO treatment enhanced the metal-graphene’s binding to each other. TML pattern characterization showed the lowest contact resistance of 500 Ωμm.The fabrication was based on 200 mm CMOS platform. Conventional BEOL processes fabricated buried gate/source/drain structures made of W. HfOThe Advanced Design System software was version 2012.08. In optimization toolset, the simulated anealing algorithm was used. Three goals were typically used: 1) S21 > 5 dB; 2) S11 < −10 dB; 3) S22 < −10 dB, in the frequency span from 500 MHz to 3 GHz. The weight of the each goals could be independently swiched.How to cite this article: Lyu, H. et al. Graphene Distributed Amplifiers: Generating Desirable Gain for Graphene Field-Effect Transistors. Sci. Rep.5, 17649; doi: 10.1038/srep17649 (2015).


In the race of gaining higher energy density, carbon’s capacity to retain power density is generally lost due to defect incorporation and resistance increment in carbon electrode. Herein, a relationship between charge carrier density/charge movement and supercapacitance performance is established. For this purpose we have incorporated the most defect-free pristine graphene into defective/sacrificial graphene oxide. A unique co-solvent-based technique is applied to get a homogeneous suspension of single to bi-layer graphene and graphene oxide. This suspension is then transformed into a 3D composite structure of pristine graphene sheets (GSs) and defective N-doped reduced graphene oxide (N-RGO), which is the first stable and homogenous 3D composite between GS and RGO to the best of our knowledge. It is found that incorporation of pristine graphene can drastically decrease defect density and thus decrease relaxation time due to improved associations between electrons in GS and ions in electrolyte. Furthermore, N doping is implemented selectively only on RGO and such doping is shown to improve the charge carrier density of the composite, which eventually improves the energy density. After all, the novel 3D composite structure of N-RGO and GS greatly improves energy and power density even at high current density (20 A/g). In supercapacitors, also known as double layer capacitors, the movement of charge determines both the energy and power densities. Unlike batteries, supercapacitors store their energy in electrode-electrolyte interphase generated due to the application of voltage bias12356782 g−1, is much higher than that of activated graphene oxide (GO) with surface area of 3100 m2 g−1 13et al.z orbital, and the fifth is an additional electron that actually occupies a π*-donor state, providing n-type doping to graphene. In contrast, pyridinic and pyrrolic nitrogen atoms are found near a carbon vacancy and so they have only two carbon atoms as neighbors. As a consequence, they contribute 2 electrons with only two σ bonds along with one electron in the pz orbital forming a π-bond with its neighbor, and thus leave two electrons as a lone pair. Imparting N-doping in carbon, can therefore have huge implication on its surface morphology as well as its electrochemical performance. Hence, it can be concluded that in carbon materials like graphene, one can achieve superior capacitance/energy density by simply increasing the charge carrier density as well as sheet exposure to electrolyte. Therefore, graphene can be an excellent candidate for obtaining higher energy density without significantly disrupting the power density.Charge storage and utilization in supercapacitor devices, are highly dependent on the defect density of the carbon. It has been reported that the volumetric capacitance of the supercapacitors gets saturated, if surface area of carbon increases beyond a certain limit, especially when pores have thin walls similar to the thickness of grapheneet al. showed a tremendous specific capacitance improvement after PANI incorporation in carbon aerogel. However the capacitance retention of the material was quite low and it retained only 66% of the initial capacity at 10 A/gHerein the major challenge is to incorporate N heteroatom into the graphene matrix as pristine graphene has a perfect structure, and it is difficult to introduce heteroatom without disrupting its lattice121719202122242526Herein, we take the advantage of π electronic interaction of graphitic carbon to obtain a stable uniform 3D composite of N- RGO and pristine GS via freeze drying for generation of high surface area and macropores. Giving a 3D structure to the composite is essential to exploit the complete surface of GSs. To obtain this 3D structure by freeze drying, it is of greatest importance to identify the solvent, which can disperse and keep both graphene and GO sheets stable and be compatible for freeze drying process as well. Therefore, the use of normal organic solvents, which are generally being used for dispersing graphite, is out of question as they can introduce other functional groups onto graphene sheets during processinga (HSP distance) curve shows the parabolic behavior with minimum at 40% ethanol concentration in water, which clearly illustrates that the adaptability of co-solvent system for graphite is maximum at this ethanol concentration. On the basis of cohesive energy, calculated by considering atomic and molecular bonding, such as dispersive δD, polar δP, and hydrogen bonding δH, . Therefore, the smaller the value of Ra, the better the adaptation of solvent for solute. For the present system on the basis of Ra value, we have tried to determine the exact concentration of ethanol and water in the co-solvent system, required for the efficient dispersion of graphite. For our present system, 40% of ethanol in water was found to give the best dispersion of graphite. The concentration of graphite in 40% ethanol-60% water system is calculated to be 0.32 mg/ml as shown in where RQuality of the dispersed graphene was further evaluated. Figure S2 of SI shows that dispersed sample mainly comprises of 1–2 layers of graphene. The electron diffraction pattern shows that the mixed solvent system does not disrupt the chemical integrity of the obtained graphene. From atomic resolution HR-TEM images, the lattice constant for graphene is found to be 0.25 nm, which is consistent with that of pristine graphene. On the other hand, Figure S3 of SI shows the dispersed GO morphology. It is seen that GO sheets are efficiently dispersed in water-ethanol azeotropic system like in pure water system.Topographical features of the freeze-dried N-RGO-GS composite and N-RGO are shown in 3234The effectiveness of present reduction process on the prepared composite material is analyzed by XPS analysis. 3g−1 and from 190 to 171 m2g−1, is observed after graphene sheet incorporation. However, micropore volume is found to increase in N-RGO-GS . Interestingly, very pronounced G band and 2D band are observed in N-RGO-GS in comparison to N-RGO, which clearly indicates the efficient incorporation of GSs into GO matrix. BET surface area of the prepared samples was also evaluated as shown in Figure S7 of SI. An obvious decrement in mesopores volume and surface area, from 0.79 to 0.51 cmN-RGO-GS . This vaThe electrochemical testing was performed in a three-electrode setup by keeping Pt as a counter and SCE as a reference electrode. The effect of GS addition on the electrochemical behavior can be easily seen from the CV performance in max of To understand this excellent stability in capacitance in N-RGO-GS as compared with N-RGO at higher current density, we have carried out the impedance analysis on the samples. As a result, from the lower relaxation time and higher frequency response of N-RGO-GS as compared with N-RGO, it can be understood that at lower frequency alternating current (AC) field, almost all the polarized ions in N-RGO are fully aligned, and hence N-RGO possesses better capacitance than N-RGO-GS. It is necessary to mention here that the defect density in N-RGO is much higher  as compared to N-RGO-GS, which enables much higher charge storage in this material at lower frequency/current density. However, as the AC frequency increases, most of the polarized ions in N-RGO cannot follow the higher frequency field at each direction reversal, due to poor conductivity of this sample as compared with its counterpart. As the high-frequency AC field changes direction faster, these ions “relax” to nonaligned positions, where N-RGO cannot store energy as much as N-RGO-GS can. Hence, a decrement in the N-RGO performance is observed at higher frequencyTo get a better picture of the origin of this phenomenon, Mott-Schottky plots are obtained for N-RGO and N-RGO-GS . Before 43444546In conclusions, herein, with the motive of finding a common ground of improving energy density without significantly losing the power density and proving the importance of defect-free conductive surface in supercapacitors, we have developed for the first time a novel uniform 3D high surface area structure of N-doped reduced graphene oxide–pristine graphene sheet (N-RGO-GS) composite, via simple freeze drying process, using a common ethanol-water azeotrope solvent. In the resultant novel 3D composite, the graphene sheets are present between two graphene oxide layers, and this kind of packing can keep the graphene sheets separate, thus offering efficient electrical transmission path in the composite scaffold with high surface area. The well-separated sheet structure provides much higher surface accessibility to the electrolyte ions, improving the electrolyte-electrode interaction. Furthermore, it was found that N-doping induces n-type charge carriers and helps in increasing the capacitance (energy density) of the N-RGO-GS. On the other hand, due to the incorporation of highly conductive pristine graphene, the defect density, which affects the frequency response of the capacitance, has been reduced, causing a drastic reduction in relaxation time. Therefore, much improved overall supercapacitance performance is observed even at higher current density (20 A/g), illustrating superior energy density retention. Through this study, we have also shown that in addition to higher surface area, a defect-free conductive surface is very essential for obtaining an excellent candidate for supercapacitor. This facile method of incorporating pristine graphene into disordered graphene oxide is expected to have huge implication towards the development of high efficient supercapacitor and battery materials in the energy storage field and can definitely open new dimensions in both synthetic and electro-chemistry of new carbon composite materials.4 in H2SO4:H3PO4 (9:1) solution. The obtained mixture was then stirred for 12 h at 50 °C. After getting brownish colour, 400 ml ice was added to the solution followed by dropwise addition of 3 ml H2O2. Obtained yellowish solution was then stirred for 2 h more. The final product was then obtained by centrifuging and washing with HCl and water. The obtained graphene oxide (GO) product was then dried at 70 °C overnight.The graphene oxide was synthesized using improved Hummers method. In brief 3.0 gm of graphite , was mixed with 18.0 gm of KMNOFirstly, 10 mg graphite powder was dispersed in 50 ml ethanol/water mixture with 40 vol% ethanol volume, using a bath sonicator operated at low power for 8 h continuously. The obtained dispersion was then centrifuged at 3000 rpm for 30 mins, and the supernatant was collected. On the other hand, the GO product was dispersed in the same azeotropic solvent (50 ml ethanol/water mixture with 40 vol% ethanol volume), by 2 h of sonication for 25 mg GO. The GO dispersant was then centrifuged at 4000 rpm for 45 mins to get stable GO solution. To obtain the final composite, both graphene and graphene oxide solutions are then mixed together under sonication for 30 mins. The obtained composite solution was then freeze-dried in a freezer (operated at −40 °C) and dryer (operated at 5 m torr) . The obtained graphene oxide-graphene sheet product is termed at GO-GS composite. For comparison, only GO dispersion was also freeze-dried by keeping all the conditions similar to those of GO-GS. In addition, we have tried to synthesize only 3D graphene by keeping all the conditions same as above, but in absence of graphene oxide.The reduction of GO was carried by vapor hydrazine reduction. In brief, the GO or GO-GS sample, kept in a separate beaker, was treated with hydrazine vapor generated by the blowing Ar gas into 10 ml 50% hydrazine hydrate (Sigma Aldrich) for 8 h. During reduction process the samples were heated at 60 °C to stop the condensation of hydrazine on carbon samples. The completion of reduction was judged by the change of brown color of GO to black color of RGO. The reduced hydrazine-treated samples were then termed as N-RGO and N-RGO-GS for the corresponding GO and GO-GS, respectively. At the same time, N-doping was realized on the reduced GO during hydrazine process.The morphology and microstructure of the obtained samples were investigated by scanning electron microscopy (SEM) Hitachi  microscope, Transmission electron microscopy (TEM) EM912 Omega and High resolution-TEM (HR-TEM) JEOL FE-2010 microscope. X-ray photoelectron spectroscopy (XPS) analyses were carried out with an ESCALAB 250 XPS System using a monochromated Al Kα (150 W) source. Raman spectra were recorded with a Renishaw spectrometer using an Ar ion laser (λ = 514.5 nm). The absorption spectra of the samples were recorded by using an Ultraviolet-Visible-Near Infrared Spectrophotometer (CARY 5000) manufactured by Agilent Technology. The specific surface areas of all the samples were measured with ASAP 2020 Physisorption Analyzer  at −196 °C, based on the Brunauer– Emmett–Teller (BET) method from nitrogen adsorption data in the relative pressure range from 0.05 to 0.2. Total pore volumes were determined from the amount of gas adsorbed at the relative pressure of 0.99. The pore size distribution was derived from adsorption branches by the Horvath-kawazoe (HK) report.2) and dried at room temperature overnight, followed by further drying at 80 °C in a vacuum oven for 12 h. The sample loading in all samples are kept constant at 10 mg. The electrochemical performances of the electrodes were tested using a three-electrode configuration in aqueous 6.0 M KOH electrolyte, where prepared carbon sample acts as a working electrode with SCE as a reference and Pt wire as a counter electrode. Cyclic voltammetry (CV), galvanostatic charge–discharge (CD) and electrochemical impedance spectroscopy (EIS) methods were employed for measurement of the electrochemical performance. The CV and CD curves were recorded between −1.0 to 0.0 V at potential scan rates of 10, 50 and 100 mVs−1 and at current densities of 0.1 to 1.0 Ag−1. The EIS measurements were carried out in the frequency range from 10 KHz to 100 mHz with a sinusoidal amplitude of 10 mV. The alternating current (AC) complex impedance was measured based on the following equation Z* = Z′−iZ″. The complex AC capacitance C* = C′−iC″ was calculated from the impedance data, as C′ = Z″/ω|Z|2 and C″ = Z′/ω|Z|2, where ω = 2πf, and f is frequency. The relaxation times τ = 1/fm were calculated from the relaxation frequencies fm, corresponding to the C″ maxima.All the electrochemical measurements were carried out with a Biologic electrochemical workstation (Biologic VSP). N-RGO and N-RGO-GS carbon electrodes were fabricated as follows. The electrode slurry was prepared by mixing 90 wt% active materials and 10 wt% polyvinylidene fluoride (PVdF) binder in a mortar and pestle using a few drops of N-methylpyrrolidinone (NMP) as a solvent. The resulting uniform slurry was then coated onto a nickel foam current collector .


Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva. Two connexons from neighboring cells can also dock to form GJs, which provide conduits for direct exchange of small (<1 kDa) signaling molecules between communicating cells [Connexins (C×s) are a family of transmembrane proteins that assemble to form connexons (hemichannels) or gap junctions (GJs). Each connexin protein is composed of four transmembrane-spanning domains, two extracellular loops, and the cytoplasmic domains including N-terminus, C-terminal domain and the cytoplasmic loop. Assembly of six connexin subunits generates one connexon or hemichannel, which functions in autocrine and paracrine signaling by providing a pathway for transfer of signaling molecules, including ATP, NADf small < kDa signng cells . In adding cells –6.Connexins are expressed virtually by all cells in the body, and play crucial role during development and normal tissue function, and contribute to development of various pathologies ,6. In adIn general, fibroblasts in normal skin express connexins, and appear connected to each other by GJs –19. The in vitro [Based on the above studies, C×43 appears a key connexin expressed by skin cells, and it is strongly down regulated in the wound epithelium at the early stages of wound healing. To study its role in more detail, different strategies to further suppress its function or expression in experimental murine skin wounds have been used. For instance, transient blocking of C×43 function at the early stage of wound healing by ACT1, a peptide that binds to the cytoplasmic carboxy-tail of C×43 , or tranin vitro ,25,32,33Interestingly, wound healing in human and pig oral mucosal attached gingiva is faster and results in significantly reduced scar formation as compared to similar skin wounds –37. TherTissue sections from experimental wounds created in palatal attached gingival mucosa in three healthy males (mean age 38 years) that have been previously extensively characterized were used ,42–46. B2. Cells were routinely seeded for experiments when they reached about 95% confluence. Experiments were performed at passages 5 to 10.Four gingival fibroblast lines (GFBLs) were isolated from clinically healthy attached gingiva from healthy human donors, as previously described  S1 Tabl. Cells wGingival tissue donors provided a written informed consent, and procedures were reviewed and approved by the Office of Research Ethics of the University of British Columbia, and complies with the ethical rules for human experimentation that are stated in the 1975 Declaration of Helsinki.2) in their normal growth medium. At day 2 when cultures became confluent, culture medium was replaced with serum-free medium. At day 3, cells were treated with 150 μM of Gap27  [2O) for 24 h before RNA isolation.To block the C×43 function, GFBLs were seeded on 6-well plates  ,49, equa Canada) –50, or c Canada)  or Gap26 Canada) , or corrTo block the expression of C×43, GFBLs were seeded in 6-well plates as described above. After 24 h, siRNA transfection was carried out using Lipofectamine RNAiMax reagent . To this end, 22 μl of Lipofectamine RNAiMax and 150 picomoles of C×43 siRNAs  or control siRNAs  were mixed with 2.4 ml of Opti-MEM medium (Invitrogen), and incubated at room temperature for 5 min. The mixtures of transfection reagent and individual siRNAs were combined and incubated at room temperature for 20 min for complex formation. Then the reagent was diluted with Opti-MEM to yield final siRNA concentration of 30 nM, and added to the cells. After 5 h in cell culture incubator, the transfection reagent was removed, and cells fed with their normal growth medium overnight. Medium was then replaced with serum-free growth medium, and RNA isolation and sample collection for Western blotting was performed after 48 h.http://www.leonxie.com/referencegene.php). Non-transcribed RNA samples were used as a negative control. The PCR reactions were performed in triplicate for each sample. The data was analyzed and is presented based on the comparative Ct method .Real-time PCR analysis was performed according to MIQE guidelines  as we ha2O. Lysates were collected using a rubber policeman, and filtered through a NucleoSpin Filter (Macherey-Nagel) by centrifugation at 5,000g for 10 min.Confluent GFBL cultures were treated as described above. The conditioned medium was then collected and immediately treated with Complete Protease Inhibitor Cocktail . The samples were concentrated (30–40 times) by centrifugation  using Centrifugal Filter Units  for 3 h, and stored at −80°C until use. To collect cell lysates, cells were washed with ice-cold phosphate-buffered saline (PBS), and lysed with a buffer containing 25 mM Tris-HCL (pH 7.6), 100 mM Octyl β̃D-glucopyranoside, 5 mM NaF, 1 mM Na3VO4, , and the Complete Protease Inhibitor Cocktail (Roche Diagnostics), dissolved in HImmunoblotting analysis was conducted as described in detail previously . BrieflyThe activation of signaling pathways by Gap27 treatment was studied in cell lysates obtained as described above. For the experiments, GFBLs were seeded on 6-well plates, treated with Gap27 (150 μM) or equal amount of control peptide for 1, 2, 6, and 24 h, and cell lysates collected, as above. Western blotting was performed with antibodies against total or phosphorylated forms of β-Catenin and GSK3α/β (β-Catenin pathway), SMAD3 (TGF-β pathway), ERK1/2 and p38 (MAPK pathway) . β-TubulTo identify latent and active MMPs, a set of cell/conditioned medium samples was treated with or without p-aminophenylmercury acetate  at 37°C for 4 h to activate latent enzymes  prior toTo determine the role of key signaling pathways in Gap27-induced gene expression, we blocked TGF-β pathway with SB431542 , MEK1/2 with PD184352 , p38 with SB203580 , GSK3α/β with SB415286 , AP1 with curcumin , and SP1 with WP631  in Gap27-treated cells, respectively. To this end, confluent GFBL cultures were pre-incubated with inhibitors at 37°C for 1 h, and then treated with Gap27 (150 μM) with or without the inhibitors in serum-free growth medium for 24 h. All inhibitors were dissolved in DMSO, and control samples were treated with respective amounts of DMSO only. Total RNA was collected for real-time PCR as described above.2O; 1 h) in DMEM at 37°C, media was removed, and a scrape wound was created through the cell layer with a 10 μL pipette tip, and cells incubated as above with 0.5% Lucifer Yellow  in PBS+ (containing 1 mM Ca2+ and Mg2+) for 5 min at 37°C. Cells were rinsed once with PBS+ and then fixed with 4% formaldehyde at room temperature for 20 min. Nuclei were then stained with 300 nM DAPI (Molecular Probes Inc.) in PBS for 5 min. In a set of experiments, cells were transfected with C×43 siRNA-1, siRNA-2 or control siRNA (30 nM) as above before seeding on the coverslips for 24 h, serum-starved for 24 h, and then subjected to the dye transfer experiment as above. Samples were mounted with Immu-Mount solution , examined using the ECLIPSE 80i Microscope , and images captured using NIS-Elements BR software .To assess the GJ function of C×43, dye transfer assays were performed. To this end, GFBL cultures were generated on gelatin-coated glass coverslips in 24-well plates as described previously . Brieflyhttps://www.adobe.com).To assess role of C×43 in cell migration, GFBLs were grown on gelatin-coated glass coverslips, serum-starved, and pretreated with Gap27 or control peptides (150 μM) for 24 h as above. A wound was then created through the cell layer using a 100 μL pipette tip. Cells were then cultured in DMEM with the peptides in a cell culture incubator, and wound closure recorded over time by standardized digital images taken from the samples using a phase contrast microscope  and a digital camera (Nikon Coolpix 995). In a set of experiments, cells were transfected with C×43 siRNA-1 or -2 and control siRNA-1 or -2 (30 nM) separately as above, seeded on gelatin-coated glass coverslips for 24 h, and serum-starved for 6 h before wounding. Experiments were performed in triplicate per treatment group, and images were captured from three to four different areas of the wound on each coverslip. Wound closure rate was determined measuring the area of the open wound at each time point relative to the area of the wound at the time of wounding using the Adobe Photoshop for Mac software  and then serum starved (24 h) as described above, fixed with 4% formaldehyde at room temperature for 20 min, and then permeabilized using 0.5% Triton X-100 in PBS for 4 min. All samples were then blocked with PBS+ containing BSA (10 mg/ml) and glycine (1 mg/ml) at room temperature for 30 min, followed by an incubation with the primary antibody  diluted For immunostaining of human gingival tissue samples, sections were fixed with cold acetone (−20°C) at room temperature for 5 min. Samples were then washed, blocked, and incubated separately with each primary antibody  overnighAll data is presented as mean +/− standard error of the mean (SEM) from repeated experiments. Statistical analysis was performed by using Student’s t-test, p<0.05 was considered statistically significant. Values obtained from the real-time PCR by the comparative Ct-method were Log2 transformed for statistical testing .Previous studies have shown that C×43 is present in human skin, periodontal ligament and gingival fibroblasts, but that skin and periodontal ligament fibroblasts also express C×32, C×40 and C×45 ,17,57. TIn order to assess expression of C×43 during gingival wound healing, we compared localization of C×43 in unwounded human gingiva, and in experimental excisional gingival wounds 3, 7, 14, 28 and 60 days after wounding by immunostaining . Fibroblin vivo [Having established that C×43 is the major connexin expressed by GFBLs, and that abundance of C×43 plaques is strongly reduced during gingival wound healing, we wanted to find out the functional significance of this down regulation. Down regulation of the number of C×43-positive plaques in wound fibroblasts can result in reduced hemichannel and GJ function. Treatment of cells with C×43 mimetic peptides can be used to specifically block these channel functions , which pin vivo . Therefoin vivo ,61. Treain vivo , also inin vivo . Thus, rin vivo . Therefoin vivo ,63, molein vivo  , and studied the expression of the above wound healing-associated genes. Treatment of GFBLs with both C×43 siRNAs consistently resulted in about 80% down regulation of C×43 at both mRNA and protein levels, while expression of the two other connexins expressed by these cells, C×32 and C×45, were not affected (data not shown). C×43 siRNA treatment with both C×43 siRNA-1 and -2 effectively suppressed GJ-mediated dye transfer , but didAs gene expression analysis showed that blocking of C×43 function by Gap27 strongly regulated expression of several genes Tables –4, we fuHaving established that during gingival wound healing, C×43 abundance and/or GJ and hemichannel functions maybe strongly reduced, and that suppressing C×43 function in cultured fibroblasts distinctly regulates expression of a set of wound healing-associated genes, we wanted to find out which intracellular signaling pathways are involved. To this end, we treated GFBLs with Gap27 and assessed phosphorylation changes in TGF-β (SMAD3), MAPK (ERK1/2 and p38), GSK3α/β and β-Catenin pathways that have been previously associated with C×43-mediated signaling ,67–73, aIn order to further assess the role of the above pathways in Gap27-induced gene expression changes, we blocked TGF-β signaling by SB431542, MEK1/2 by PD184352, p38 by SB203580, and GSK3α/β by SB415286 in Gap27-treated cells, and assessed gene expression changes relative to untreated cells by real-time PCR. In addition, we assessed involvement of AP1 and SP1, transcription factors previously linked to C×43 signaling ,79,80, b2) hemichannel plaques [in vivo likely represent C×43 hemichannel and/or GJ plaques on the cell membrane.Gingival fibroblasts that participate in scarless oral wound healing expressed C×43 as the major connexin, which is similar to skin fibroblasts ,21. In o plaques . Connexi plaques . Thus, tin vivo are not clear, but are likely cell type-specific and could include effects of wound healing-related cytokines, growth factors and mechanosignaling [Interestingly, the C×43-positive plaques found in fibroblasts in unwounded tissue were missing from fibroblasts at the wound edge and those migrating into the wound at days 3 and 7 post-wounding. This could be explained by reduced recruitment of C×43 to hemichannel and GJ plaques, or their redistribution, resulting to smaller plaques undetectable by immunostaining, or from down regulation of C×43 expression and/or its increased turnover. The factors that modulate these processes during wound healing ignaling ,84. In aignaling , blockinignaling ,86. Repaignaling . Howeverignaling . Our preignaling ,44,45. Hignaling , suggestignaling , and in ignaling . In addiignaling . Thus, rignaling , 43–46, i.e. it is needed to suppress expression of these genes. Therefore, during gingival wound healing reduced expression/function of C×43 in fibroblasts may allow increased expression of these molecules. This group of molecules includes several MMPs, TIMP-1 and -3, Tenascin-C, TGF-β1 and VEGF-A, which are important modulators of inflammation, cell migration, angiogenesis and ECM deposition [in vivo. The hallmark of scar formation and fibrosis is increased accumulation of ECM and increased cell contractility [in vivo [To study the significance of reduced C×43 function for these later stages of wound healing, we blocked its function in human gingival fibroblasts by C×43 mimetic peptide Gap27 [position , and mayposition ,37. Of nposition ,42, suggactility . Therefoactility , while u[in vivo , were si[in vivo . There i[in vivo ,37,53,93The mechanisms of the mimetic peptide-induced gene expression change in gingival fibroblasts may include blocking of transfer of signaling molecules via hemichannels and/or GJs, peptide-induced changes in C×43 levels, conformation and/or phosphorylation of its cytoplasmic tail that interacts with the signaling molecules ,72,94. TIn order to study the role of the above signaling pathways in regulation of Gap27-induced fibroblast gene expression in more detail, we used pathway-specific pharmacological inhibitors. In addition, we blocked two transcriptional regulators (AP1 and SP1) that associate with C×43 signaling ,79,80. BAs mentioned above, C×43 can also promote TGF-β-induced signaling . InteresTreatment of gingival fibroblasts with Gap27 also induced fast phosphorylation of GSK3α/β, but this did not associate with marked changes in the phosphorylation or levels of its downstream target β-Catenin. Phosphorylation of GSK3α/β renders it inactive removing its inhibitory effect on its targets, which include more than 40 proteins and transcription factors . TherefoInterestingly, Gap27 treatment also significantly increased expression of C×43 at both mRNA and total protein levels, although it did not affect relative proportions of the differently phosphorylated forms of the protein in the Western blots. Increased C×43 mRNA expression was also noted after MFA treatment. These findings are different from previous observations where treatment of human skin fibroblasts with Gap27 in a scratch wound model did not upregulate C×43 levels, but promoted its phosphorylation at S368 . It is pIn order to assess the mechanisms of Gap27-induced C×43 upregulation in gingival fibroblasts we used pharmacological inhibitors to key signaling pathways that associate with C×43. Previous findings have indicated that transcriptional modulator AP1 regulates C×43 expression by various signals . Accordiin vivo warrant further investigation.To summarize, our findings demonstrate that C×43 shows similar spatiotemporal regulation in gingival wound epithelium over time as previously describe for skin. In addition, we showed for the first time that the abundance of C×43-positive plaques is strongly reduced in fibroblasts at the early stages of human gingival wound healing, returning to the level of normal tissue by day 60 post-wounding. Thus, wounding-induced suppression of C×43 in wound fibroblasts leads to disruption of the connexin-mediated intercellular communication network in the connective tissue, resulting to a gene expression change that maybe important for the fast and scarless wound healing outcome in gingiva. Interestingly, blocking of C×43 function by mimetic peptides strongly regulated expression of a number of wound healing and scar formation-associated genes in human gingival fibroblasts. These changes involved p38, MEK1/2-ERK1/2, TGF-β-SMAD3 and GSK3α/β mediated signaling pathways, and AP1 and SP1 transcription factors. Among these pathways, ERK1/2-MEK1/2 appeared to be a key regulator of C×43 mimetic peptide-modulated gene expression. Thus, C×43 mimetic peptides may provide an efficient tool to modulate fibroblast gene expression during wound healing. The exact mechanisms by which the C×43 mimetic peptides cause these effects, and the mechanisms and importance of C×43 down regulation for fast and scarless wound healing outcome in human gingiva S1 FigRepresentative immunostainings of C×43 (red) and vimentin  in unwounded human oral mucosal tissue (attached gingiva) (A–C), and in gingival wounds 3- (D-F), 7- (G-I), 14- (J-L), 28- (M-O) and 60-days (P-Q) post-wounding. (A–C) In unwounded gingiva, abundant C×43 staining was localized in suprabasal epithelial cells. Most intensely stained cells were located in the stratum spinosum, but weak staining was also noted in basal epithelial cells. Inserts in (B) and (C) show higher magnification images of C×43 localization in basal cells at the connective tissue papilla and rete peg areas, respectively. (D–F) At day 3 post-wounding, C×43 was down regulated in migrating epithelial cells (D and E) and fibroblasts at wound edge . (G–I) At day 7 post-wounding, when the wound was completely covered with a new epithelium, 2–3 most basal epithelial cell layers showed C×43 staining in the wound epithelium, while there was only a very weak immunoreactivity for C×43 in the spinous layer (G and H). Very little C×43 immunoreactivity was noted in fibroblasts at the wound edge (I). (J–L) At day 14 post-wounding, C×43 was confined to the 2–3 most basal layers of wound epithelium (J and K). At this stage, immunoreactivity for C×43 was slightly increased at the wound edge connective tissue (L) as compared to 7-day wounds (I). (M–O) At day 28 after wounding, C×43 immunoreactivity was normalized in the epithelium at the wound site, being present mainly in suprabasal cells of the stratum spinosum (M and N). Abundance of C×43-positive plaques in connective tissue cells at the wound edge (O) was increased as compared to earlier time points (I and L). (P–Q) At day 60 after wounding, structure of the epithelium and connective tissue formed at the wound area was closely similar to unwounded tissue. C×43 immunoreactivity was also similar to unwounded tissue in the epithelium at the wound site (P and Q). (R) Negative control staining of C×43 in unwounded epithelium. Data shown represents minimum of three sections stained in parallel samples from two to three individual donors at each time point. Arrowheads  indicate wound edge. E: epithelium; CT: connective tissue; FC: fibrin clot; W Edge: wound edge; WE: wound epithelium; GT: granulation tissue; WCT: wound connective tissue. Nuclear staining (blue) was performed using DAPI. Magnification bars in the inserts in B, C and Q: 50 μm.(TIF)Click here for additional data file.S2 Fig(A and B) Representative images of wound samples double immunostained with anti-C×43 (red) and anti-Clever-1  antibodies. (A) At day 14 post-wounding, very few C×43-positive structures (arrowheads) were noted in some of the macrophages located in the newly made wound connective tissue. (B) At day 28 post-wounding, number of M2 macrophages was strongly reduced compared to day 14, with very little macrophage-associated C×43 immunoreactivity. Magnification bar: 10 μm.(TIF)Click here for additional data file.S3 Fig(A and B) Representative images of the same wound location in parallel day 14 wound sections stained with an antibody against α-SMA (A) and C×43 (B). At day 14 post-wounding, the wound contained numerous α-SMA-positive myofibroblasts. However, very few C×43-positive structures (arrowheads) were noted in cells in the myofibroblast-rich area. Magnification bar: 10 μm.(TIF)Click here for additional data file.S4 FigConfluent fibroblast cultures (GFBL-DC) were cultured in their normal growth medium (DMEM) (A), or treated with control peptide (B) or Gap27 (C) (150 μM), and images acquired 24 h after treatment. Magnification bar: 50 μm.(TIF)Click here for additional data file.S5 Fig(A) Representative images of human gingival fibroblast (GFBL-DC) migration in the presence of Gap27 or control peptide (150 μM) across a scrape wound over time. Lines indicate original wound margins. Magnification bar: 20 μm. (B) Quantification of Gap-27-induced fibroblast migration over time. (C) Representative images of human gingival fibroblast (GFBL-DC) migration in the presence of control siRNA-1 or -2 or C×43 siRNA-1 or -2 (30 nM) across a scrape wound over time. Wounds were completely closed in all groups at 24 h. Lines indicate original wound margins. Magnification bar: 40 μm. (D) Quantification of cell migration in C×43 and control siRNA treated samples over time. Results show pooled data for C×43 siRNA-1 and -2, and control siRNA-1 and -2 treated samples, respectively. Wounds were completely closed in all groups at 24 h. For the experiments, siRNA transfection was performed 30 h before wounding. Wound closure rate was determined measuring the area of the open wound at each time point relative to the area of the same wound at the time of wounding. Results show mean +/− SEM from minimum of triplicate samples. Statistical testing was performed comparing test and control samples at the given time point . Non-treated samples (incubated in DMEM only) did not show difference to control peptide or control siRNA-treated samples, and are not shown.(TIF)Click here for additional data file.S6 FigConfluent fibroblast cultures (GFBL-DC) were treated with Gap26 or control peptide (300 μM), and Gap27 or control peptide (150 μM) for 24 h, and expression of a set of genes was analyzed by real-time PCR. Results show mean mRNA expression relative to control-peptide treated samples from triplicate samples from one experiment. Horizontal line indicates relative mRNA expression for the control-peptide treated samples. EDA-FN: Extra Domain A-Fibronectin; EDB-FN: Extra Domain B-Fibronectin; TN-C: Tenascin-C; α-SMA: α-Smooth Muscle Actin; VEGF-A: Vascular Endothelial Growth Factor-A.(TIF)Click here for additional data file.S7 FigReal-time PCR results from GFBL-DC cultures treated with increasing concentrations of MFA for 24 h relative to vehicle-treated samples are shown. Results represent mean of triplicate samples in one experiment. MFA induced a concentration-dependent increase in expression of MMP-1, MMP-10, Vascular Endothelial Growth Factor-A (VEGF-A), Tenascin-C (TN-C) and C×43, and down regulation of CXCL12 (SDF-1α).(TIF)Click here for additional data file.S8 FigConfluent GFBL-DC cultures transfected with control siRNA-1 (A and B), C×43 siRNA-1 (C) or C×43 siRNA-2 (D) were scrape-loaded with Lucifer Yellow (green), and dye transfer was followed for 5 min. Treatment of cells with C×43 siRNA-1 and -2 reduced markedly dye transfer as compared to control siRNA-1 . Results show representative images from triplicate samples. For the experiments, siRNA transfections were performed 48 h before the experiment. Magnification bars: 50 μm.(TIF)Click here for additional data file.S9 FigConfluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 (150 μM) with or without curcumin (AP1 inhibitor), WP631 (SP1 inhibitor), SB431542 (TGF-β inhibitor), PD184352 (p38 inhibitor), SB203580 (MEK1/2 inhibitor) or SB415286 (GSK3α/β inhibitor) for 24 h, and expression of MMPs and TIMPs (A), ECM proteins and contractility-associated genes (B), TGF-β1 and growth factors (C), and cell-cell junction proteins (D) was analyzed by real-time PCR. Results represent mean mRNA expression relative to non-treated cells from triplicate samples in one experiment. DMSO: Cells treated with the vehicle (DMSO) only; TN-C: Tenascin-C; DCN: Decorin; FMOD: Fibromodulin; α-SMA: α-Smooth Muscle Actin; NMMIIB: Non-Muscle Myosin IIB; VEGF-A: Vascular Endothelial Growth Factor-A.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). In addition to its nervous system functions, TrkB is also expressed in the aortic endothelium. However, the effects of endothelial TrkB signaling on atherosclerosis remained unknown. Immunofluorescence analysis revealed that TrkB expression is downregulated in the endothelium of atherosclerotic lesions from ApoE−/− mice compared with the atheroma-free aorta of WT mice. Endothelial TrkB knockdown led to increased lesion size, lipid deposition and inflammatory responses in the atherosclerotic lesions of the ApoE−/− mice compared with the control mice. Mechanistic studies showed that TrkB activation prevented VE-cadherin shedding by enhancing the interaction between vascular endothelial protein tyrosine phosphatase and VE-cadherin, maintaining VE-cadherin in a dephosphorylated state. Our data demonstrate that TrkB is an endothelial injury-response molecule in atherogenesis. Endothelial BDNF/TrkB signaling reduces VE-cadherin shedding and protects against atherosclerotic lesion development in ApoE−/− mice. Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). The BDNF/TrkB pathway plays critical roles in the survival, growth, and maintenance of central and peripheral neurons . In addiIn this study, we compared TrkB expression in aortas with and without atherosclerotic lesions in mice and investigated atherosclerotic lesion size, lipid and macrophage components, proinflammatory markers expression in ApoE−/− mice after knocking down endothelial TrkB, seeking to reveal the role and mechanism of endothelial TrkB signaling in the development of atherosclerotic lesions.We performed immunofluorescence staining to compare the expression of TrkB in C57BL/6 mice (controls) and ApoE−/− mice fed with an atherogenic diet for 8 weeks. In agreement with previous results , TrkB exOur previous study revealed that TrkB maintained endothelial barrier integrity . Then thThen the lipid and macrophage deposition in the atherosclerotic lesions of ApoE−/− mice was evaluated. Lipid deposition in the lesion, as demonstrated by the Oil Red O-positive region, was significantly increased in ApoE−/− mice infected with AAV9-shTrkB Figure . The infOur previous study revealed that TrkB promoted VE-cadherin synthesis in HAECs . As expeIt has been reported that an extracellular fragment of VE-cadherin can be cleaved and detected in medium . Then weTyrosine phosphorylation processes of VE-cadherin are required for the VE-cadherin cleavage . Then, wAs a transmembrane binding partner of VE-cadherin, vascular endothelial protein tyrosine phosphatase (VE-PTP) binds directly to VE-cadherin. The binding maintains VE-cadherin in a dephosphorylated state . Then, wIn this study, we investigated the role of endothelial TrkB signals in the development of atherosclerotic lesion in ApoE−/− mice. Our data showed that TrkB expression in the endothelium was downregulated in atherosclerotic lesions. Endothelial TrkB knockdown led to increased lesion size, lipid deposition and inflammatory responses in the atherosclerotic lesions of the ApoE−/− mice compared with the control mice. Finally, TrkB activation prevented the VE-cadherin shedding by maintaining the interaction between VE-PTP and VE-cadherin and reducing tyrosine phosphorylation of VE-cadherin. Our data supported that endothelial TrkB signaling may be a novel therapeutic target for atherosclerosis.In vitro experiments we further demonstrated that the TrkB levels in HAECs were significantly decreased by proinflammatory factors. Our data suggest that TrkB is an endothelial injury-response molecule in atherogenesis and TrkB may participate in the pathologic process of atherosclerosis. Consistently, the decreased BDNF/TrkB levels were also found in the tissues of aged or patients with CAD-related complications. Decreased expression of BDNF in the endothelium was associated with hypertension [TrkB is a high-affinity receptor for BDNF with well-established functions in the nervous system. However, TrkB is also expressed in the aortic endothelium. In this study, we found that TrkB expression in the endothelium to be downregulated in atherosclerotic lesions of ApoE−/− mice compared with the atheroma-free aorta of WT mice. rtension . In humartension . In the rtension . The reaIn this study, we revealed a novel role of endothelial TrkB in cardiovascular system as an atheroprotector. Our data demonstrated that endothelial BDNF-TrkB pathway deficiency led to accelerated development of atherosclerotic lesions in the aortic trees and aortic root of ApoE−/− mice. Similarly, as a growth factor signal, angiopoietin-1 has been reported to protect adult vasculature from leakage . The admIn this study we found that BDNF/TrkB signaling prevented the TNF-α induced-VE-cadherin shedding. The elevated shedding fragment of VE-cadherin has been found to be associated with diabetic retinopathy and coronary atherosclerosis , 24. ThuOur data demonstrate that TrkB expression in the endothelium is downregulated in the atherosclerotic lesions of ApoE−/− mice. In addition to promoting the synthesis of VE-cadherin, endothelial BDNF/TrkB signaling also regulates the shedding of VE-cadherin and protects against atherosclerotic lesion development in ApoE−/− mice. Endothelial TrkB may be used as a novel therapeutic target in atherosclerosis.11 vg/g body weight particles of adeno-associated virus serotype-9 (AAV9) with or without shRNA specifically targeting TrkB via the tail vein [All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health  and were approved by the Ethics Committee of Shandong University. Eight-week-old male ApoE−/− mice received a single systemic administration of 2.1×10Oil Red O staining was used to assess the size of the atherosclerotic lesion as previously described . BrieflyImmunofluorescence staining was used to show the expression of TrkB and VE-cadherin, and macrophage content of the lesions. Briefly, the cryosections of the aortic sinus were fixed with 4% paraformaldehyde. After washing with PBS and blocking with serum, the sections were incubated with anti-TrkB (sc-12), anti-CD31 (Abcam), anti-VE-cadherin , or anti-mouse macrophage  antibodies. Then, the FITC or TRITC-conjugated secondary antibodies were used. Finally, the sections were observed using fluorescence microscopy.Antibodies for anti-TrkB (sc-12), anti-VE-cadherin (C-19), and anti-vascular endothelial protein tyrosine phosphatase (sc-65228) were purchased from Santa Cruz. The preparation of the cell lysates, measurement of the protein concentrations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, sequential incubation with primary and second antibodies and film development were performed as previously described .ΔΔCt calculation.Total RNA was isolated from the aortas of the mice. First cDNA was synthesized using AMV reverse transcriptase. PCR was performed using the following primers: forward, 5′-AGC AGG GAA ACA TCT ATA ACG-3′ and reverse, 5′-CTT GAA CTT TGG GTT TAC TGG-3′ for mouse VE-cadherin; 5′-AGG CTT CTG GGC CTT ATG TG-3′ and 5′-GTA TTC CTG GCG AGA GAA GCA-3′ for NF-κB; 5′-GTG AAG GGA ATG GGT GTT-3′ and 5′-GGT CAC TGT CCC AGC ATC-3′ for TNF-α; 5′-TTC CAG AAA CCG CTA TGA-3′ and 5′-GGT TGT CAC CAG CAT CAG-3′ for IL-6; 5′-GGC ACC CAG CAG AAG TTG TT-3′ and 5′-CTT GGT AGA GGT GAC TGA GG-3′ for ICAM-1; 5′-TCT CTC AGG AAA TGC CAC CC-3′ and 5′-GTG TGC TGC TAT TGG CTG TG-3′ for VCAM-1; 5′-TCC TCT GGA GAG TGG AGT GC-3′ and 5′-ATG TGA AGC TTT GAC CCA CC-3′ for E-selectin as well as 5′ -TGT CTC CTG CGA CTT CAA CA-3′ and 5′-GGT GGT CCA GGG TTT CTT ACT-3′ for mouse GAPDH. The relative expression of the genes was obtained through the 2Human aortic endothelial cells (HAEC) were purchased from ATCC . The HAECs were cultured in endothelial cell medium. All cells were cultured at 37°C with 5% CO2. All assays were performed in triplicate.P < 0.05 set as the significance level, and they were performed using the SPSS 15.0 software .The animal study data are presented as the mean ± SEM. For comparisons between two groups, Student's t test was employed. All statistical tests were 2-tailed with


The properties of graphene can vary as a function of the number of layers (NOL). Controlling the NOL in large area graphene is still challenging. In this work, we demonstrate a picosecond (ps) laser thinning removal of graphene layers from multi-layered graphene to obtain desired NOL when appropriate pulse threshold energy is adopted. The thinning process is conducted in atmosphere without any coating and it is applicable for graphene films on arbitrary substrates. This method provides many advantages such as one-step process, non-contact operation, substrate and environment-friendly, and patternable, which will enable its potential applications in the manufacturing of graphene-based electronic devices. Graphene was peeled layer-by-layer using Joule heating to induce the atom sublimation and electron beam irradiation to facilitate the atom displacement. Yang, et al.2. Nitrogen plasma attacked the top-layer plane, introducing defects into the lattice; oxygen molecules etched out the carbon atoms at the edge of defect sites, finally removing the damaged top layer. All other top-down methods also included inducing the defects on the top layers of graphene through oxidation or plasma treatments7et al.et al.There are several studies which reported the top-down methods that used some methods reducing graphene thickness to achieve the specific number of layers. Huang, et al.et al.et al.Laser has been employed in graphene treatments, including direct laser writing, laser patterning and laser induced synthesis. For examples, EI-Kady, Here, a novel yet simple method of picosecond (ps) laser thinning is proposed to accurately control the number of graphene layers. The graphene films can be thinned to the specific number of layers using the scanning laser radiation with tunable pulse threshold energy in atmospheric condition. In the process, arbitrary patterns are achievable if the unique scanning paths are set up. Because this process/method has many advantages, such as one-step process, non-contact operation, substrate and environmental-friendly, and patternable thinning, it will be potentially used in the fabrication of graphene-based electronic devices.The ps laser thinning process of graphene is demonstrated in e.g. poly(dimethylsiloxane)(PDMS). The pristine (5-layer) to monolayer graphene are displayed from left to right. A typical optical image of graphene with different layer numbers is shown in 2/Si) indicates the number of layers, which is further confirmed by Raman  reflects the number of graphene layers. The position, full width at half maximum (FWHM), and Lorentzian fitting of 2D peak supply further evidences for determining the layer number of grapheneIG/I2D from 1.3 to 0.3 (−1 (5-layer) to 2690 cm−1 (1-layer) and the FWHM (2D) decreases from 58 cm−1 (5-layer) to 28 cm−1 (1-layer), which is consistent with the result that the 2D peak position bluely shift following the layer decreaseRaman is a powerful tool to determine the number of graphene layers and evaluate their qualities3 to 0.3  reveals To accurately determine the number of graphene layers, the visible transmittances of graphene samples were measured. The optical image shows that the graphene films are uniform after the same laser thinning process, and the transmittance of graphene monotonically decreases following the layer increase of graphene. As shown in IG/I2D. The purple area represents the lowest IG/I2D radio, which indicates the presence of monolayer graphene. The yellow area represents the 5-layer graphene. The word of “TSINGHUA UNIVERSITY” with monolayer graphene is patterned on a bilayer graphene sample  is widely applied to characterize the crystallization quality of grapheneID/IG keeps fairly low during all the thinning process, which is different from the layer- by-layer methodsThe mechanism of the one-step laser thinning process is further proposed. i.e., graphene grain residues which were not being completely peeled off  was utilized to prove this layer thinning mechanism. 2, which is much higher than the threshold for obtaining a 4-layer graphene (0.5 J/cm2). Dhar, et al.The threshold of the laser energy density to thin graphene into specific layers is summarized in 2. Then, graphene was thinned to the accurate NOL by laser thinning. As the channel materials and the electrodes, graphene was patterned by lithography, which is displayed in the top-right inset of 2/Vs. Nagashio, et al.21Due to the unique thickness-dependent electronic properties, high flexibility, and transparency, graphene and its relatives are fantastic materials for both electron active channels and electrodes in various electronic devicesTo conclude, a one-step laser thinning process, with features of non-contact operation, substrate and environment-friendly and patternable, is demonstrated to obtain the accurate number of graphene layers. A “peeling off” mechanism of the laser process is proposed. The thinning process is conducted in atmosphere without any coating. Besides, the laser scanning is applicable for graphene films on arbitrary substrates. All of these advantages make this method have broad potential applications in graphene-based electronic devices.2 substrates2/min. The whole process was conducted in the air ambient.Pristine graphene samples were prepared by CVD on copper substrates. The numbers of layers of as-prepared graphene films were confirmed to be 5 by optical microscopy, Raman spectroscopy and optical absorption. Graphene films were then transferred onto Si/SiOThe optical images were taken using a Nikon microscope. SEM images were obtained using a LEO-1530 microscope (LEO Electron Microscopy. GERMANY). Raman spectra were collected with a HORIBA Lab RAM HR spectrometer (514 nm laser excitation) (HORIBA Jobin Yvon. FRANCE). Raman mapping images were recorded by a Raman 11 spectroscopy (Nanophonon. JAPAN). AFM images were acquired by a SPM-960 instrument (SHIMADZU. JAPAN). The transmission spectra of graphene were obtained with a UV-visible spectrophotometer.How to cite this article: Lin, Z. et al. Precise Control of the Number of Layers of Graphene by Picosecond Laser Thinning. Sci. Rep.5, 11662; doi: 10.1038/srep11662 (2015).


We study optical response of all-dielectric multilayer structures and demonstrate that the total scattering of such structures can be suppressed leading to optimal invisibility cloaking. We use experimental material data and a genetic algorithm to reduce the total scattering by adjusting the material and thickness of various layers for several types of dielectric cores at telecommunication wavelengths. Our approach demonstrates 80-fold suppression of the total scattering cross-section by employing just a few dielectric layers. The possibility to suppress the total scattering cross-section of an object has attracted significant attention in recent years. One of the approaches to achieve nearly ideal invisibility is based on the so-called Transformation Optics124Another technique is scattering cancellation with plasmonic or dielectric multilayer structures. One of the first examples was demonstrated by Kerker for core-shell spherical particles consisting of metallic core and dielectric shell111213141516181920The possibility to realize a structure which is invisible in the optical range raises several important questions. (i) Is it possible to use real conventional materials to control SCS practically? (ii) How many layers are required to achieve the best invisibility possible? (iii) Can we design all-dielectric structures to exhibit lossless invisibility in the optical range? In this work we answer these questions by employing a genetic algorithm to optimize SCS properties of a dielectric cylinder. We use experimental data for some commonly used dielectrics as well as for the materials used in the shells, and show that one can achieve up to 80 fold total scattering cross-section suppression. We find that the optimum invisibility is achievable with three or even smaller number of dielectric shells.24x axis with the magnetic field polarized along the cylinder axis , âz is a unit vector along z axis, and H0 is the amplitude of the incident magnetic field. We assume that our multi-layer structure contains L layers, and it is placed in free space. By decomposing the electromagnetic fields into a superposition of cylindrical harmonics, the field in layer l is presented asnJ and n-th order Bessel and Hankel functions of the first kind, respectively; n is the mode number, r is the radial coordinate within the l-th layer, n-th mode coefficients in the l-th layer which will be found by solving the boundary condition equations for the tangential components of the fields, ϕEn, so that our equation also describes the incident plane wave as a superposition of cylindrical waves. Coefficient N(n) = 1 for n = 0 and for all other modes N(n) = 2 due to the symmetric relation of the Bessel functions with positive and negative indices.The incident electromagnetic plane wave is propagating along the By taking into account R1 such that the nanowire exhibits the maximum of SCS at Invisibility implies that the SCS of the whole structure is minimized. We aim to achieve this by the proper design of multilayer dielectric shells. Here we use a genetic optimization algorithm293031The genetic algorithm uses a database of several dielectric materials, and it chooses the ones that are more suitable for cloaking. We noticed that the GA always chooses two types of materials with the lowest and highest dielectric constants to form the highest possible index contrast between shell layers, which in our database are fused-silica and silicon. Materials with dielectric constant values in between are not chosen by the GA and adding such materials to our set does not help improving the cloaking quality. Now we would like to understand the physical origin of the observed invisibility. One of the obvious reasons could be that the reduction of scattering is caused by a shift of the resonance of the core caused by adding a dielectric layer. To have more in-depth physical interpretation of the optimization process, we calculate the stored energy inside the core and the shell of the introduced GaAs-Si core-shell structure separately for different modes, as demonstrated in 3334The field profiles of bare nanowires and core-shell structures with cores made of AlAs and GaAs are calculated in both near and far field regions using the optimized parameters from In conclusion, we have designed and optimized the invisibility condition for all-dielectric multi-layer structures at a given frequency by employing a genetic algorithm. By using experimental data to describe materials, we have demonstrated it is possible to achieve nearly 80 fold suppression of total scattering cross section by using three or less dielectric layers. We have designed the optimized structures with minimal SCS for AlAs, TlBr and GaAs scatterers using alternating silicon and fused-silica coatings and proved that increasing the number of layers in the dielectric multi-layer shell, leads to saturation of the minimal scattering cross-section.Genetic algorithms (GAs)293031New generations are born through some known procedures. In this work we use ‘crossover’ . In the discussed results, the fitness function is defined based on the NSCS in TE polarization by having magnetic field parallel to the axis of the nanowires. However, similar results can be obtained for TM polarization, as we demonstrate in To employ our genetic algorithm to minimize the scattering cross section, we need to define a ‘fitness function’, asl’ of a multi-layer nanowire, we usen is the mode number.In the discussion section, to calculate the stored energy in layer ‘A.M. wrote the main manuscript and prepared the figures. A.E.M. and I.S. added more scientific detailed descriptions and completed the discussion section. A.E.M., I.S. and Y.K. reviewed and revised the manuscript. All authors discussed the results and contributed to the scientific interpretation.


Sarcoidosis is a rare condition that is often misdiagnosed as malignant tumors due to the similar clinical manifestations and imaging findings.We encountered a 56-year-old Chinese woman who had a chief complaint of a persistent cough. The chest computer tomography (CT) revealed mediastinal and bilateral hilar lymph node enlargement, and positron emission tomography-computer tomography (PET-CT) revealed abnormal fluorodeoxyglucose (FDG) uptake in the lymph nodes of the chest and abdomen. To further clarify the diagnosis, a lymph node sampling was performed by video-assisted thoracoscopic surgery (VATS) and the histopathologic diagnosis of sarcoidosis was confirmed.VATS could be an effective and minimally invasive diagnostic method to discriminate pulmonary sarcoidosis with other malignant tumors. Sarcoidosis is an uncommon disease of unknown etiology that is characterized by the presence of noncaseating granulomas . SarcoidA 56-year-old Chinese woman was admitted with cough and expectoration (white phlegm) for 1 month. These symptoms developed without apparent cause and in the absence of other problems, such as chest pain and/or distress, breathlessness, fever, hemoptysis, nausea, or vomiting. She denied the presence of any chronic disorders and smoking and had no family history of cancers. An enhanced chest computed tomography (CT) scan imaged a right hilar mass and a small amount of bilateral pleural effusions, accompanied with the enlargement of multiple lymph nodes in the mediastinal and bilateral hilar areas, as shown in Fig. To further clarify the diagnosis, a subcarinal lymph node resection was performed by right-sided video-assisted thoracoscopic (VATS). In brief, a 40-mm incision was made in the fifth intercostal space of the right anterior axillary line for the instrumental operation. A 30-degree 10 mm high definition thoracoscope was employed through the seventh intercostal space of the right mid-axillary line. It was observed from the thoracoscope that mediastinal lymph nodes were diffusely enlarged, and lymph node biopsy was applied by a harmonic scalpel. As shown in Fig. Sarcoidosis is a multisystemic disorder of unknown etiology that is characterized by the presence of noncaseating granulomas and the proliferation of epithelioid cells . Sarcoid18F-FDG PET has been proven to be a very useful imaging technique in diagnosis, disease activity assessment, monitoring treatment response, and risk assessment in patients with sarcoidosis [In recent years, PET-CT has become a superior means of diagnosis of sarcoidosis. Moreover, coidosis . In suspcoidosis . In atypcoidosis . Althougcoidosis . Pleuralcoidosis –16. A plcoidosis , 18.In conclusion, sarcoidosis is difficult to be differentiated with other malignant tumors by radiological and PET-CT examinations. VATS biopsy is a minimally invasive, safe, and effective procedure in the diagnosis of pulmonary sarcoidosis.The patient granted written informed consent for publication of this manuscript and the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.This material has not been published and is not under consideration elsewhere. There is no financial disclosure from each author.


In particular, we show that the process was successfully used to realize and characterize a simple, optically transparent graphene-based wire-grid polarizer at microwave frequencies (X band). The availability of graphene operating in a  Up to now, graphene properties in the microwave range have been studied by means of theoretical models68lossy-dielectric region0/2), which strongly affects the performance of any microwave device that requires metallic components.Chemical Vapour Deposition (CVD) graphene has been indicated as the ideal conducting material for future 2D technologies. However, the most current and lowest reported experimental values of its sheet resistance around 1000 Ω/sq) literally invalidate any such prospects000 Ω/sq 2 V−1 s−1) is approximately one order of magnitude lower than the carrier mobility of the mechanically exfoliated graphene, if measured inside a large single CVD graphene grainIn this regard, the transport properties of CVD graphene are determined by both intrinsic and extrinsic factors such as (i) its polycrystalline nature and (ii) the lack of reproducible methods that would allow its transfer to substrates of technological interest without generating holes, cracks and ripples, especially on large areas. These defects, as well as grain boundaries, act as barriers to charge transport and result in sheet resistance higher than that of mechanically exfoliated micrometer grapheneIn theory, the use of multiple layers of CVD graphene acting as parallel resistances should amount to a simple method to lower the sheet resistance. However, experiments suggest that adding parallel CVD graphene layers does not necessarily decrease sheet resistance, since the partial contact or the presence of additional impurities  contribute contact/series resistances between graphene layers12) has been successfully used for hole-doping of graphene2 consists of the exploitation of the intrinsic defects in CVD graphene as enhanced reactive centers for the attachment of dopant functionalities. This provides the stability of covalent chemical doping without detrimental effects on material mobility due to the creation of new C-sp3 acting as charge scattering centers in the C-sp2 lattice.Chemical doping is an effective way to enhance graphene sheet conductivity via the attachment of electron donors or acceptors. For example, the treatments with thionyl chloride . In contrast, chemically doped graphene can overcome the low threshold limit with only two layers. The minimum sheet resistance value measured for chemically doped graphene is 27 Ω/sq, and was achieved with five-layer graphene samples. If additional graphene layers are added, the effect of series resistances between adjacent graphene layers becomes predominant, resulting in higher Rs.The effect of the combined use of SOCl2 treatment.The optical transmittance of chemically doped graphene from one to five layers is reported in quasi-metallic graphene) allowed us to test the electromagnetic response of hole-doped graphene at microwave frequencies. In particular, we measured reflectance, transmittance, and absorbance of samples with different sheet-resistances. The measurements were carried out by means of a microwave setup consisting of a klystron connected to a WR90 rectangular waveguide (the waveguide supports only the TE10 mode). A slotted-line acquired the electromagnetic power (square law) with a spatial resolution of 1 mm in the rectangular waveguide at 9 GHz . Further details on the measurement setup are reported in the The possibility to operate with very-low sheet resistance samples . The comparison between the analytical model and the experimental results shows very good agreement.quasi-metallic region acts as a metal by efficiently reflecting the impinging electromagnetic field. For example, when Rs = 27 Ω/sq the reflectance is larger than 80%. At the same time, the doped graphene sheet conductivity is independent over a wide frequency range  in the stripes over an area of about 50 × 55 mm2 , while the electric field with vertical polarization (EV) is reflected /T90 of the polarizer Tpol with respect to transmission at 90° (T90). We adopted this normalization to emphasize the difference at 0° . In fact, the graphene-based wire-grid polarizer (red curve) shows a near-unity transmission at 90°.We compared the graphene-based wire-grid polarizer with a wire-grid polarizer made with copper stripes of identical dimensions (5 mm width) and identical foot-print with respect to the graphene-based polarizer. The comparison reveals that the two polarizers only show a constant 3 dB difference over the entire angular range while their angular behavior is identical. This result is quite remarkable, considering that the copper layer is 35 μm thick, while graphene thickness is about 1 nm.In conclusion, we have detailed a chemical protocol for the optimization of graphene in terms of (i) doping stability, (ii) scalability to large areas, and (iii) reduction of the series resistances between graphene layers in multilayer graphene, thus achieving multilayer graphene with sheet resistance values below 30 Ω/sq.lossy-dielectric to that of a quasi-metal.This protocol allowed us to experimentally verify the optimized graphene behavior in terms of reflectance, transmittance, and absorbance of hole-doped graphene when the sheet resistance ranges from that of a Finally, the optimized graphene was exploited for the realization of an optically transparent graphene-based polarizer. The comparison between the graphene-based polarizer and its copper-based counterpart revealed that the two polarizers show only a constant 3 dB difference over the entire angular range while their angular behavior remains identical.quasi-metallic region with very-low sheet resistance graphene may allow the realization of microwave devices (e.g. microstrip patch antennas) and the dynamical tuning of their S-parameters over a wide-range. In this regard, graphene-based antennas with low-sheet resistance graphene may help overcome the limitation of low gain23The possibility to operate in the 4/H2 as precursors. The graphene was transferred onto corning glass substrate (treated by O2 plasma to improve graphene adhesion) by the thermal tape method. The copper was etched in a solution of ammonium persulfate. Multilayer graphene samples were fabricated by transferring of additional graphene layers onto graphene/glass substrates.Graphene was grown by CVD on a 25 μm thick copper foils in a typical quartz tube CVD reactor at 1000 °C using CH2 treatments were performed in a dry chamber by placing graphene/glass substrate and 1 mL of liquid SOCl2 (avoiding direct contact) at 105 °C for 60 min. Doping of multilayer samples was performed by repeating SOCl2 treatment after transferring and stacking each graphene layer.SOCl2 in air and at room temperature  media (impinging from medium 1 to medium 2), that introduces a finite sheet conductivity ση1 and η2 are the wave impedances in the two media, respectively, and σD2 ≈ σDC = 1/Rs. Moreover, the sheet conductivity is independent over a wide frequency range, as found in references 6 at microwave frequencies (X-band). If we apply the boundary conditions for the electric and magnetic fields at the interface z = 0 we obtain:where where Γ and T are the reflection and transmission coefficients, respectively.Solving the Equation GR and transmittance TGR, GR may be evaluated as AGR = 1 – RGR – TGR. If we assume η1 = η2 = η0 (where η0 corresponds to the impedance of free space) it is possible to define two different regions for a single graphene sheet: the lossy-dielectric region quasi-metallic region where we set How to cite this article: Grande, M. et al. Optically Transparent Microwave Polarizer Based On Quasi-Metallic Graphene. Sci. Rep.5, 17083; doi: 10.1038/srep17083 (2015).