Here, we study large areaMoS2-graphene-heterostructures formed by direct transfer ofchemical-vapor deposited MoS2 layer onto epitaxial graphene/SiC. We showthat via a direct transfer, which minimizes interface contamination, we can obtainhigh quality and homogeneous van der Waals heterostructures. Angle-resolvedphotoemission spectroscopy (ARPES) measurements combined with Density FunctionalTheory (DFT) calculations show that the transition from indirect to direct bandgapin monolayer MoS2 is maintained in these heterostructures due to the weakvan der Waals interaction with epitaxial graphene. A downshift of the Raman 2D bandof the graphene, an up shift of the A1g peak of MoS2 and asignificant photoluminescence quenching are observed for both monolayer and bilayerMoS2 as a result of charge transfer from MoS2 to epitaxialgraphene under illumination. Our work provides a possible route to modify the thinfilm TDMCs photoluminescence properties via substrate engineering for future devicedesign.Two-dimensional layered transition metal dichalcogenides (TMDCs) show great potentialfor optoelectronic devices due to their electronic and optical properties. Ametal-semiconductor interface, as epitaxial graphene - molybdenum disulfide(MoS Recently Han et al.developed a novel seeded CVD patterned growth method to obtain highly crystallineMoS2 flakes on oxidized silicon substrate2 grown by this approach has high crystallinity, with large flakes(between 20–100 μm) and electrical and opticalproperties comparable to exfoliated material. These CVD-grown flakes are suitable fortransfer onto epitaxial graphene/SiC substrate in order to obtain large area ofMoS2/graphene heterostructures. Using a transfer process  measurementswere used to study the electronic structure of the MoS2/epitaxial grapheneheterostructure. Due to a weak interlayer coupling the electronic structure of grapheneand MoS2 are well retained in their respective layers. However the bandalignment in the MoS2/graphene heterostructure allows under illumination acharge transfer process from MoS2 to graphene. This is revealed by adownshift of the graphene 2D Raman bands, an upshift of the A1g Raman band ofMoS2, and a strong quenching of photoluminescence (PL) ofMoS2/graphene heterostructure. Complementary we performed photocurrentmeasurements to elucidate how the presence of the semiconductor affects thephotoconductive properties of graphene. In that respect, this work may open a new way ingraphene optoelectronics by modulating the graphene photoelectric response through 2Dmaterials interfacing.One well-established method to produce high quality wafer-scale monolayer graphene is theepitaxial approach based on the graphitization of the Si face of SiC132 flakes grown bychemical vapor deposition on oxidized silicon substrates that were then transferredonto epitaxial graphene grown on SiC(0001) , we were ableto obtain several flakes with various stacking order and orientation in a singletransfer step .For the following experiments, the monolayer, bilayer and multilayer coverage wasestimated from optical analysis to be around 83%, 15% and 2% respectively.TMDCs/graphene heterostructures were made from MoSiC(0001) . ThegraiC(0001) . The CVDsolution . Afterwaubstrate . We finafer step . The MoS2/graphene/SiC heterostructure, we measured its band structure byangle-resolved photoemission spectroscopy (ARPES) at the Cassiopéebeamline of Synchrotron Soleil. The small x-ray spot size(50 × 50 μm2)allowed the measurement of the band structure of a single flake (monolayer orbilayer) forming the MoS2/graphene heterostructure. The photoelectronintensity is presented in 2 valence band. A close inspection of the K point of grapheneBrillouin zone is shown in 2 flake andgraphene underlayer is critical. In order to obtain a perfect alignment of theBrillouin zone of the two materials we need a mismatch angle of zero degree. As wecan see in 232 are not visibleindicating a non-zero mismatch angle for this flake. However we clearly see thegraphene band structure, in particular the π bands of graphene preservetheir linearity characteristic of a massless Dirac fermions signature, indicating ahigh structural quality of the MoS2/graphene heterostructure. Moreover,similar to pristine monolayer graphene, the Dirac point (ED) is locatedat 0.3 eV below the Fermi level (FL). From a linear fit, using therelation E = ħvFk, we obtain the value of the FermivelocityvF ~ 1.1 × 106 m/s,which matches the expected value for monolayer graphene on SiC. As the lineardispersion and Fermi velocity of the pristine graphene is preserved in theMoS2/graphene heterostructure, we can infer that the MoS2transfer does not affect the electronic structure of monolayer graphene in theheterostructure formation. In addition, this feature is expected theoretically sincein van der Waals heterostructures, the superposition of each layer electronicstructure constitutes a good approximation of the electronic structure of themultilayer. Differently from previous work of Diaz et al. no signature ofinterlayer hybridization2flake and the graphene underlayer. However identifying the dependence of this effecton the mismatch angle would clearly require a further work and go much beyond themain objective of this paper. 2 on epitaxial graphene,along the K-Γ-K direction in the hexagonal Brillouin zone, therespective second derivative are shown in 2 presents only one band atthe Γ point (maximum at Binding Energy (BE)~ −1.68 eV ± 0.05 eV),and this structure evolves into two branches in the case of bilayer MoS2(maximum at BE~ −1.25 eV ± 0.05 eV).This evolution is representative of the splitting of the bands due to the weak vander Waals interaction between the two MoS2 layers. The relative positionof the top of the valence bands at the Γ point in the bilayer film iscloser to the Fermi level than the one obtained from the monolayer  and Γ points. However, when considering the monolayerband structure, the top of the valence band becomes very flat near the Γpoint, leading to a direct gap at the K (K′) point. The evolution of thevalence band at the Γ point provides a straightforward method toidentify the thickness of ultrathin MoS2 films, and also proves the highquality of MoS2 transferred on epitaxial graphene. The epitaxial grapheneunderlayer does not affect the MoS2 band structure, as expected for a vander Waals heterostructure. However, we do not exclude the presence of the universalbuckled form of 2D crystal in our MoS2 layerIn order to better understand the electronic properties of theMoSonolayer . This in2/graphene heterostructure, micro photoluminescence (micro-PL) andmicro-Raman measurements are performed at room temperature (see method).To further investigate the electronic properties of mono and bilayerMoS2 todirect gap semiconductor in monolayer MoS2. The PL signal is stronglyenhanced when a direct band gap is present. The same behavior was also observed forthe as grown MoS2 on SiO2 , Γ−1 is between8–14, and presents a huge decrease as the thickness of graphene layer isreduced (~2 in the case of 5 nm FLG). In our case, we haveonly one graphene layer, meaning that as for SiO2 we can supposeΓ−1 ~ 1.Therefore, in both cases we can neglect the effect of optical interferences, anddirectly compare the PL raw data of MoS2/SiO2 andMoS2/graphene heterostructures as shown in 2/SiO2 heterostructure strain canexist due to the different thermal expansion coefficients of MoS2 and theSiO2 substrate during the MoS2 flakes growing2/graphene heterostructure38392 substrate). This phenomenonwas explored also as a function of the laser power. In 2 PL intensity evolveslinearly with increasing laser excitation412 and graphene. But in the case of graphene underlayer the PLsignal is quenched for each laser power.In the case of vertical vdW heterostructures, possible PL intensity variations couldarise from the interference effects due to different optical constants and thicknessof the different layers forming the heterostructure2 to thegraphene which hinders the recombination of electron−hole pairs createdby the photoexcitation322 and graphene , but rather to astandard hopping from an electron in MoS2 conduction band to anunoccupied state at the same energy in graphene43This phenomenon is the signature of electron transfer from MoS2/graphene heterostructure and pristine graphene layer−1. Besides thetypical second-order Raman bands that originate from the SiC substrate, the threemain structures typical of graphene are present on the pristine graphene andMoS2/graphene spectra: i) the D band (defect induced mode), ii) the Gband (in-plane vibration mode) and iii) the 2D band (two-phonon mode)2/graphene heterostructure within the wavenumberrange between 350–450 cm−1 twonew peaks are present. These two characteristic features correspond to the in-planevibration (1g) of Moand S atoms in the MoS2 film341g and 2 layers. The averageRaman spectra obtained from each layer are shown in −1,~21 cm−1 and24 cm−1 corresponding to monolayer,bilayer and multilayer (~three layers) MoS2,respectively342 on graphene and onSiO2 substrates result of a Lorentzian fitting, are narrower than those on SiO2. As in the case of the PL spectra, the upshift of the in plane Raman mode2, and confirms that the photoelectrons generated bythe Raman laser are transferred from the MoS2 to graphene. At the sametime the G peak presents an upshift. If we focus our attention on monolayerMoS2/graphene we have an upshift of~3 cm−1 ± 1cm−1.From this shift we can obtain a quantitative analysis of the level of electrondoping under illumination5357582/graphene as −1implies a change in the graphene Fermi level of ~70 meV.From these values we can calculate the variation in the n-doping of graphene usingthe relation vF = 1.1 × 106 m/s),we obtained an electron density for pristine graphene|N| ~ 7 × 1012 cm−2which increases to|N| ~ 1013 cm−2for 1ML MoS2/graphene under illumination.A detailed analysis of the Raman spectra of the monolayer and bilayerMoS2 decorated graphene) at roomtemperature while illuminating the sample with light energy(λ = 405 nm(hν ≈ 3 eV)) above theMoS2 band gap because of its thickness and limited coverage. The photocurrent generation in TMDCheterostructure generally suffers from two main limitations from an applied point ofview, which are their slow response time610.8. Such dependence isweaker than for large gain system like graphene–PbS quantum dot hybridsystem2 decorated graphenesystem over a larger range of light flux. While modulating the incident laserintensity thanks to a signal generator we can extract the frequency dependence ofthe graphene-MoS2 systemSince the spot diameter of the laser is 1 mm2 on epitaxial graphene layer. From the PL, ARPES and micro-Ramandata presented in 2/graphene heterostructurepresents a good long-range order at large scale. Our ARPES measurements on theheterostructure showed that graphene and MoS2 largely retained theiroriginal electronic structure indicating weak van der Waals interactions between thetwo crystals. The PL quenching in the MoS2-graphene heterostructure andthe upshift of the A1g Raman mode of MoS2 and the downshift ofthe 2D Raman mode of graphene confirm the charge transfer between theMoS2 and graphene layer. Our work suggests that the opticalproperties of MoS2 are strongly affected by the underlayer graphene. Thefact that an interaction is visible between MoS2 and graphene is a clearsignature of the quality of the wet transfer process and of the absence ofinterfacial contaminations. Moreover, this charge transfer can be influenced by theunderlayer graphene doping, varying the band alignment in the heterostructure, whichshould be considered in device design and fabrication. Furthermore, efficientphotoresponse was observed on large 2D MoS2/epitaxial graphene devices,opening a new way in graphene optoelectronics.In summary, we have studied the electronic properties of the wet transfer of largearea MoS2) at1550 °C to produce well-ordered atomic terraces of SiC.Subsequently, the SiC sample is heated to 1000 °C at apressure of about 10−5 mbar and thenfurther heated to 1550 °C in an Ar atmosphere. Thisgraphitization process results in the growth of an electrically active graphenelayer on top of the buffer layer, covalently bound to the substrateex-situ to perform different measurements.Monolayer graphene studied in this paper is produced via a two-step processbeginning with a starting substrate of 4H-SiC(0001)2/SiO2 samples were grown via CVD in a 1″quartz tube furnace. Microliter droplets of saturated ammonia heptamolybdatesolution were dried onto the corners of a Si/SiO2 growth substratethat had previously been coated with a layer of sodium cholate (1% solution spincoated 4000 rpm for 60 sec). Sodium cholate is a known growthpromoter, acting to increase diffusion of the molybdenum source by increasingthe surface adhesive energy relative to the adatom cohesive energy2) was used to bring sulfur vapor into the furnace fora 30 min growth period. The sample was then rapidly cooled bycracking open the furnace and sliding it downstream with respect to the quartztube.MoSThe PL measurements were carried out using a confocal commercial Renishawmicro-Raman microscope with a 100× objective and a Si detector(detection range up to ~2.2 eV). The Raman spectrameasurements were performed on the same microscope using a 532 nmlaser in an ambient environment at room temperature. To ensure thereproducibility of the data, we followed a careful alignment and optimizationprotocol. In addition, the excitation laser was focused onto the samples withspot diameter of ~1 μm and incident power of~5 mW. The integration time was optimized to obtain asatisfactory signal-to-noise ratio. We obtained Raman spatial maps by rasterscanning with 0.3 μm step size using a precision 2Dmapping stage.° and 16 meV. The meandiameter of the incident photon beam was smaller than50 μm. All ARPES experiments were done atroom temperature.The ARPES measurements were conducted at the CASSIOPEE beamline of SynchrotronSOLEIL . We used linearly polarized photons of50 eV and 90 eV and a hemispherical electron analyzerwith vertical slits to allow band mapping. The total angle and energyresolutions were 0.25For electrical measurements the samples are electrically characterized in air atroom temperature. Temporal and power dependence of the current are obtainedwhile biasing the sample and measuring the current with a Keithley 2634B assourcemeter. Illumination is ensured by a 405 nm blue laser diodewith tunable light intensity. The frequency dependence of the photocurrent ismeasured while the sample is biased using a Keithley 2634B. The output signal isamplified in a Keithley 427 current amplifier and the signal acquired on HPoscilloscope.3d5for S and Mo were used with cutoff radii (in atomic units)s = 4.3, p = 4.7,d = 5.5 (S) and s = 5.0,p = 5.6, d = 4.8 (Mo). In thisstudy we have considered standard unit cells of 3, 6 and 9 atoms to describerespectively a mono-bi- and trilayer of MoS2. Each configuration hasbeen relaxed using a sample of 32 k-points in the Brillouin zone. In case ofmultilayer MoS2, we have considered the most stable AB stacking, andthe equilibrium distance has been determined using theLCAO-S2 + vdW formalism712 are providedin First-principles calculations have been performed using a very efficient DFTlocalized orbital molecular dynamic technique (FIREBALL)676869How to cite this article: Pierucci, D. et al. Large area molybdenumdisulphide - epitaxial graphene vertical Van der Waals heterostructures. Sci.Rep.6, 26656; doi: 10.1038/srep26656 (2016).


Juvenile localised scleroderma (JLS) is a rare condition. Researchers and clinicians seeking to improve care for children and young people (CYP) with JLS face various challenges. JLS is often difficult to assess, both at disease onset and as the condition progresses. A variety of monitoring tools (mts) have been described for JLS.- To describe how mts are used and perceived by clinicians in the United Kingdom (UK).- To describe treatments used for JLS in CYP.We surveyed paediatric rheumatologists (prs) and dermatologists (dms) in the UK who manage CYP with JLS, via an an e-survey distributed to the membership of two national organisations representing these clinician groups. We asked respondents for their views and experience using 15 distinct JLS mts; about transition services and treatments used.48 clinicians  from across the UK responded. At time of survey response, dms reported managing a mean 4.2 CYP with JLS; prs 17.9. 26% dms managed all CYP in collaboration  whilst 31% prs managed all CYP in collaboration (with a DM or paediatrician). 43% of dms and 91% prs reported formal transition arrangements. 9% dms and 69% prs reported involving an adult rheumatologist in transition; 8% prs involved a DM in transition.Medical photography (100%), thermography (67%) and drawing in notes/use of pre-printed body map (both 56%) were the most regularly used mts by prs; photography (81%) drawing in notes (56%) and ultrasound (22%) were most regularly used by dms. Of 4 skin scores, the modified Rodnan skin score was the one clinicians used most frequently. However it was only used regularly by 33% prs and 3% dms. Familiarity, use  and perceived benefit of all other skin scores was low (0-40%). Laser doppler imaging, laser doppler flowmetry and 3.0T MRI were perceived useful by larger proportions (15-70%) of clinicians than reported using them  or regularly (0-11%)).Table A wide range of mts are used in the UK by prs and dms managing CYP with JLS. How these tools are accessed, used and perceived varies between and within these clinician groups. There are also differences in prescribing of treatments between prs and dms. These differences will impact on the feasibility of conducting clinical trials in JLS. Further work is needed to determine accessible and validated mts for CYP with JLS. Collaboration between dms and prs is an important factor in facilitating future high quality research and standardising care in JLS.None declared.


In this paper, we report how few layers graphene that can be produced in large quantity with low defect ratio from exfoliation of graphite by using a high intensity probe sonication in water containing liquid hand soap and PVP. It was founded that the graphene powder obtained by this simple exfoliation method after the heat treatment had an excellent exfoliation into a single or layered graphene sheets. The UV-visible spectroscopy, FESEM, TEM, X-ray powder diffraction and Raman spectroscopy was used to analyse the graphene product. The thermal diffusivity of the samples was analysed using a highly accurate thermal-wave cavity photothermal technique. The data obtained showed excellent enhancement in the thermal diffusivity of the graphene dispersion. This well-dispersed graphene was then used to fabricate an electrically conductive polymer-graphene film composite. The results demonstrated that this low cost and environmental friendly technique allowed to the production of high quality layered graphene sheets, improved the thermal and electrical properties. This may find use in the wide range of applications based on graphene. Recently, graphene as a single atomic layer of carbon has attracted much attention due to its unique nanostructure and novel thermal, electric and mechanical properties as well as its potential applications in physics, materials science, chemistry and biology . Graphen−4 m2 s −1, was about one order of magnitude lower than single layer graphene. Gupta et al [We suggest that the molecules in hand soap fluid may have similar interactions with graphite flakes to produce single or few layers graphene sheets. For testing this, hand soap—PVP (SP), as the solvent and dispersant and stabilizer, was selected to prepare a stable dispersion of the individual graphene sheets in water by using a high power sonicator. Benefits of hand soap include its low cost, established safety, non-pollutive properties and hence, a low environmental risks. In this Article, the probe ultrasonication technique produces the stable dispersions of a few layered graphene in SP solution with low defects, hence resulting in a high electrical conductivity. After wash, dry and thermal treatment, we obtained pure graphene powder, with high quality and with a low defect ratio. Kuang et al and other research groups –31 have ta et al  found thta et al –36.To outa et al –39. The The pure graphite powders  and Polyvinyl pyrrolidone  were purchased from Sigma- Aldrich company.Solvent: Liquid hand soap  was dissolved in deionized (DI) water to create a solution of hand soap-DI water, 0.5 wt%. For exfoliation and dispersion of graphite in the solution, a probe ultrasonic homogenizer (UH)  was used with high intensity. The sonication probe tip was provided in continuous mode for 60% amplitude modulation.The mixture, SP, containing 1g PVP with 400 ml of hand soap-DI water (0.5 wt%) was stirred for 1 h, then, 1 g of pure graphite (GT) powder was added to the SP solution to form graphite solution. The resulting solution was tip sonicated for 3 hrs, and then was left to stand for 24 hrs to separate the large graphite aggregates to the bottom. The homogenous dark dispersion remained suspended for at least five months after preparation without any significant precipitation  was dropped on the substrate during the spin coating process at 1000 rpm for 30 s, and finally, the film was subjected to heat treatment at 100°C under vacuum for 48 hrs to remove the solvent remnants and produce the multi-layered graphene. The quartz substrates were washed by distilled water and acetone and then placed in an ultrasonic cleaner for 30 min for the cleaning process.The optical absorption of the diluted solution before and after ultrasonic treatment was analysed using an UV−vis spectrophotometer (Perkin Elmer model Lambda 900). The powder morphology of the pure GT, the resulting graphitic powder and films were determined by field-emission scanning electron microscopy  and transmission electron microscopy  and X-ray diffraction (XRD)  equipped with a Cu Kα  radiation source. Raman spectroscopy was performed  which was used to analyze edge defects and estimate layer thickness. X-ray photoelectron spectroscopy (XPS) measurement was carried out using a Multifunctional XPS (Shimadzu Axis Ultra DLD). The electrical resistance of graphene composite film was characterized by four point probe measurement using a Keithley 2401 source meter.V was determined by the thickness and thermal diffusivity of sample, V(f) = V0eσL− where V (f) is the complex PE signal, σ is the complex TW diffusion coefficient and L is the liquid sample thickness. From a relation σ = (1 + i)/μ, the thermal diffusion length of sample is μ = (α/π f)1/2, where f is the modulation frequency, and α is the thermal diffusivity of sample. The amplitude and phase of the signal are respectively, [The experimental TWC technique for measuring the liquid thermal diffusivity was utilised as reported previously . In thisctively, |V(f)|=VS = L(π/α)1/2.From the graph of the ln(amplitude) and phase as a function of the square root of the frequency, the thermal diffusivity of liquid sample can be calculated from the slopes of The extent of exfoliation for graphite could be increased with increased ultrasonication time, due to the high energy activation of ultrasonication . This acFor investigating the graphene exfoliated by sonication treatment, the optical absorption of the SP-G solution before and after sonication of 3 hrs was analysed, TEM was employed to show the quality and morphology of the SP-G solution prepared with the ultrasonic method. The XRD patterns of pure GT, and the prepared samples without (SP-G) and with (G) heat treatment are displayed in -1, which corresponded to the D band and G band, respectively. The D band at 1350 cm-1 corresponds the breathing mode of sp2 carbon atoms, and it is absent without the presence of defects and edges in the graphitic structure [ID/IG = 0.15), while as expected a small ratio (GID/IG = 0.35) is found for the exfoliated graphene sheets. We propose that the ratio of the defects GID/IG to the defect GTID/IG (i.e. M = GID/IG /GTID/IG) can be used to quantitatively estimate the graphene sheets. We suggest using the ratio of the intensity of the D: G band ratio of graphene sheets, divided by the intensity of the D: G band ratio of the graphite source. This relationship  allows the measurement of the mean defects of the graphene sheets from the graphite used to produce the graphene. The edge defects factor of the exfoliated graphene in -1) is an effective means to determine the number of layers in the graphene sheets. As seen in Raman Spectroscopy is a powerful tool to determine the number of layers present along with the quality of the graphene present (relative defects in graphene) with a faster throughput than other techniques such as AFM . Fig 7A tructure . The pur2 (C-C) and consisted of peaks corresponding to carbon bound (C–C) at ~284.5 eV and (C-O) at around 286 eV, respectively [To further evaluate the quality and also the oxygen containing groups in the sheet, XPS analysis was performed for the pure GT and the G powder obtained by sonication after heat treatment. The high resolution of the sharp and large carbon 1s (C1s) peak of GT and G represented the graphitic carbon spectively ,51  Where αn, ρn and Cn are the thermal diffusivity, density and specific heat capacity of the nanofluids. It can be seen that in low concentration of grapheme obvious enhancement in thermal diffusivity can be expected with similar enhancements in thermal conductivity. To the best of our knowledge, there are no reports on thermal diffusivity of the exfoliated graphene nanofluids. Therefore, this method to measure thermal diffusivity of the graphene nanofluids may provide a new Insight to measure different properties of graphene nanofluids. This enhancement is in agreement with other reports in the literature for thermal conductivity of the graphene composites . While, These results confirm that the solution containing graphene had a large scale exfoliation into single or few layer of graphene that can be good candidates for high thermal conduction in graphene or graphene composite devices. The physical and chemical properties of graphene depend on its crystalline structure along with the number of layers. The combination of TEM observations along with, UV-vis absorption, conductivity measurements, and thermal properties clearly indicate a dispersed graphene solution with high stability, using this environmental solvent. The lack of any individual single layer graphene sheets suggested that the graphite cannot be fully exfoliated. Separation of graphene layers depends on the mechanical disruption of the graphene stacking by ultrasonication ,18. In oIn conclusion, a simple liquid exfoliation method of producing layered graphene with a low defect ratio from pure graphite powder was obtained by a sonication treatment in a mixture of SP surfactant in DI water. The presence of SP solution in this technique allowed an excellent dispersion of graphene sheets with a high stability, even after 6 months. In the thermal diffusivity of the SP solution with exfoliated graphene demonstrated a precise enhancement (by a factor of 3). This enhancement compared favourably to other graphene-loaded thermal conductive graphene composites. The fabricated film from SP-G solution containing graphene was utilized to make an electrical conductive polymer-graphene film composite, the sheet resistance was found to be 3 order magnitude lower than that of SP solution. Graphene with a precise dispersion was obtained from this technique, without any oxidization or significant defect formations. The grapheme produced is very promising for the development of the graphene based biomedical and electrical applications.S1 FigMore information about TWC technique(DOCX)Click here for additional data file.S2 FigThe frequency behavior of the Ln amplitude and phase of PE signal obtained from DI water (a) ln (amplitude), and (b) phase of PE signal versus the square root of the frequency for one of DI water.(DOCX)Click here for additional data file.


Due to the interactions from the substrate, a much large exchange field (about 280 meV) and an enhanced Rashba spin-orbit coupling are induced in graphene, leading to a topologically nontrivial QAH gap opened in the system. The avenues of enhancing the nontrivial gap are also proposed, from which nearly a gap one order large is achieved. Our work demonstrates that this graphene-based heterostructure is an appropriate candidate to be employed to experimentally observe the QAH effect and explore the promising applications.Quantum anomalous Hall (QAH) effect, with potential applications in low-power-consumption electronics, is predicted in the heterostructure of graphene on the (001) surface of a real antiferromagnetic insulator RbMnCl Through the proximity effect, the topmost FM Mn plane in RbMnCl3 may induce a strong FM exchange field in graphene, a key factor to achieve the QAH effect. This material can be grown easily by molecular beam epitaxy methods in experiments, whose (001) surface also has a hexagonal structure293 could be a suitable substrate for graphene to gain the QAH state. Based on density-functional theory (DFT) and Wannier function methods, we demonstrate the QAH effect in graphene and (001) RbMnCl3 heterostructure. A much larger exchange field (about 280 meV) is obtained in graphene. Berry curvature, Chern number, and anomalous Hall conductivity are calculated to confirm the topologically nontrivial band gap opened by the SOC. Our results demonstrate that this heterostructure is an appropriate candidate system for exploring the QAH effect and corresponding electronic applications.In this work, we tend to achieve the QAH state in the heterostructure of graphene on the (001) surface of a real antiferromagnetic (AFM) insulator RbMnCl3 compound has a hexagonal structure with the experimental lattice constant of a = 7.16 Å3 with Mn terminal, as illustrated in 3 lattices is about 2.5%. In our calculations, the graphene lattice is shrunk to fit the substrate lattice. The C atoms in graphene and the topmost Mn, Rb, and Cl atoms in the substrate were all relaxed to obtain the stable structure of the heterostructure. The magnetization of the Mn atoms close to the graphene in the heterostructure is found to be along the c-axis. Its energy is lower than that of the magnetization in the ab plane by 0.25 meV per unit cell. Three possible adsorption configurations were considered: the hollow, bridge, and top sites, which stand for the topmost Mn atoms of the substrate right below the hexagon centers (hollow sites), the hexagon edges (bridge sites), and the hexagon corners (top sites) of the graphene, respectively. The relative stability of the three configurations is judged by comparing their adsorption energies, defined as Eb = EG + ER − EG+R, where EG, ER, and EG+R represent the total energies of the bare graphene supercell, the bare RbMnCl3 film, and the graphene/RbMnCl3 heterostructure, respectively. The hollow site is found to be the most stable configuration, displayed in 0 (d0 = 2.03 Å) between the graphene plane and the topmost layer of Mn atoms in the substrate after the geometry optimization.The RbMnCl3 substrate, it is magnetized by the proximity coupled effect with the topmost ferromagnetic Mn layer. 3 film. We find that the Dirac bands of graphene are just located inside the bulk band gap of the RbMnCl3 film. Obvious spin splitting appears in the bands around the Dirac point. Since the K and K’ Dirac points of the pristine graphene are both folded into the Γ point of the 3 × 3 supercell of graphene, each spin-up and spin-down Dirac bands around the Γ point are twofold degeneracy. Magnifying the spin-polarized π bands around the Dirac point at the Γ point (12−3  meV)d states in the substrated orbit to the Dirac bands increases, leading to a larger exchange field and Rashba SOC. Thus, the opened bulk band gap could increase slightly to about 2.0 meV in this case. Γ point , we can The mechanism of the gap opening can be understood by a tight-binding (TB) model of graphene with a TM adsorbed at the hollow site in a 3 × 3 supercell. We consider the TM-induced on-site potential, magnetic exchange field, and Rashba SOC on the six carbon atoms nearest to the TM adatoms. The TB Hamiltonian can be written as719i, z axis, m(n) refers to the sites nearest to the TM atom, andm to site n. The first term represents the nearest-neighboring hopping with amplitude t = 2.6 eV, the second term is the intrinsic spin-orbit coupling. The last three terms correspond to the TM-induced extrinsic Rashba SOC, on-site potential and magnetic exchange field, respectively. For the TM-induced intrinsic SOC is usually much smaller than the induced Rashba SOC, we neglect the intrinsic SOC term by setting where ulations .k) is calculated by3132333435The Berry curvature and Chern number are calculated to identity whether the SOC-induced gap is topologically nontrivial. The Berry curvature Ω. For two-dimensional systems, the calculated Berry curvature is the z-component value. The Chern number C and Hall conductivity σxy can be obtained by integrating the Ω(k) over the first BZ, as 3 heterostructures. The peaks of the Berry curvature are primarily located at the k points with the SOC-induced band gaps. The corresponding distribution of Ω(k) in the two-dimensional (2D) momentum space is given in k) distributes very close to the Γ point in the first BZ. Integrating the obtained Ω(k) over the first BZ, the Chern number of C = 2 is obtained. This integer Chern number (C = 2) characterizes a quantized Hall conductivity and confirms the topological non-triviality of the gap opened by the SOC interaction. Two chiral edge channels will arise on each side of the sample. Therefore, the QAH effect can appear in the graphene/RbMnCl3 heterostructure. Since the nontrivial gap is opened below the Fermi energy (EF), hole doping is needed to observe this exotic effect in experiments. Our first-principles calculations indicate that doping one hole per unit cell could move the EF to the crossing points of spin-up and spin-down bands, as shown in 14 cm−2, which can be experimentally achieved through currently advanced gating technologies. For instance, employing an electrolytic gate, the doping concentration in graphene can be adjusted up to 4 × 1014 cm−2 in experimentsF.where the summation is over all the 3 substrate is strengthened, the exchange field and Rashba SOC in the graphene can be enhanced. To increase the interaction, we reduce the distance between the graphene sheet and the substrate, by applying an externally vertical stress to the heterostructure. As shown in g can be almost doubled (2.1 meV). In experiments, the exerted stress, however, cannot be too large. The structure of the systems may be destroyed under a very large strain.If the interaction between the graphene and RbMnCl3 heterostructure with Hf atoms adsorbed at the hollow site of the graphene. A relatively large band gap of about 9.0 meV appears in the system. Thus, compared to the original gap, nearly one order large gap is achieved by adding Hf atoms into the system. The origin of this large band gap comes from the enhanced Rashba SOC due to the adsorption of Hf atoms, as reported in Refs. k) and its corresponding 2D distribution in momentum space are illustrated in F is adjusted in the gap. The Ω(k) peaks are also localized around the SOC-induced band gap near the Γ point. The intrinsic anomalous Hall conductivity σxy as a function of the EF is plotted in F passes the SOC-induced bulk band gap, confirming the existence of the QAH effect in this proposed system. The Hall conductivity in F is larger than −1.24 eV, ascribing to the spin-direction reversal of the main bands around this energy point  surface of an AFM insulator RbMnCl3 film, opposite to the graphene interface, was passivated by hydrogen atoms. A vacuum space of larger than 15 Å was used to avoid the interaction between two adjacent heterostructure slabs. For the structural relaxation, the C atoms in graphene and the topmost Mn, Rb, and Cl atoms in the substrate were allowed to relax until the Hellmann-Feynman force on each atom was smaller than 0.01 eV/Å. All calculations were carried out with a plane-wave cutoff energy of 550 eV and 12 × 12 × 1 Monkhorst-Pack grides were adopted for the first Brillouin zone (BZ) integral. To take into account the correlation effects of Mn 3d electrons, the GGA + U methodThe geometry optimization and electronic structure calculations were performed by using the first-principles method based on density-functional theory (DFT) with the projector-augmented-wave (PAW) formalismHow to cite this article: Zhang, J. et al. Quantum Anomalous Hall Effect in Graphene-based Heterostructure. Sci. Rep.5, 10629; doi: 10.1038/srep10629 (2015).


Bone-morphogenetic protein-7 (BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. In osteoblasts, Forkhead box protein C2 (FoxC2) is a downstream target of BMPs and promotes cell proliferation and differentiation. However, the role FoxC2 may play in degenerative human intervertebral disc tissue and the relationship between FoxC2 and BMP-7 in nucleus pulposus (NP) cells remain to be elucidated. This study aims to investigate the presence and signaling mechanisms of FoxC2 in degenerative human intervertebral disc tissue and NP cells. Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses were used to measure FoxC2 expression in the NP tissue and cells. Transfections were carried out to measure the effect of FoxC2 on BMP-7-mediated extracellular matrix upregulation. Adenoviral knock-down of Smad1 was performed to investigate the mechanism of BMP-7-induced FoxC2 expression. In degenerative NP tissue, FoxC2 was markedly upregulated and positively correlated with increased disc degeneration. Induction of NP cell proliferation was confirmed by using cell counting kit-8 assay, immunocytochemistry and real-time qRT-PCR for Ki67. FoxC2 led to decreased noggin expression and increased Smad1/5/8 phosphorylation. During combined treatment with BMP-7, FoxC2 greatly potentiated anabolism through synergistic mechanisms on ECM formation. Combination therapy using BMP-7 and FoxC2 may be beneficial to the treatment of intervertebral disc degeneration. AlthougNP cells . In addiNP cells , 12. In NP cells . To amelNP cells . FurtherNP cells .et al. found BMP-7 has a similar anabolic effect on ECM metabolism in bovine intervertebral disc (IVD) cells [et al. report that after ECM depletion in response to IL-1, BMP-7 can effectively increase the expression of IVD ECM genes by rabbit NP cells[Bone-morphogenetic proteins (BMPs), which constitute the largest subgroup of the transforming growth factor-β (TGF-β) superfamily, are often used as therapeutic factors in treating disc degeneration , 17. BMPD) cells . FurtherD) cells , 22. Tak NP cells. These rFoxC2 is a member of the forkhead box (FOX) family of transcription factors. When the abbreviation of FOXC2 contains all uppercase letters, this refers to human form. When Foxc2 has only the first letter capitalized, this refers to mouse, and when FoxC2 has the first and subclass letters capitalized, this refers to all chordates . Foxc2 het al. have shown that following treatment of skeletal precursors with BMPs, Foxc2 may play a role in the early stage of chondrogenic differentiation, because increased expression of Foxc2 is observed in relation to SOX9, noggin and aggrecan [et al. have found that Foxc2 is a downstream target of BMP-2 and promotes the survival, proliferation and differentiation of osteoblasts [Recently, Nifuji aggrecan . Furthereoblasts . Recent eoblasts . A simileoblasts . Overallth, 2014 to March 8th,2015, following approval from the Institutional Review Board of the First Hospital of Jilin University. Patients consented to the collection and use of tissue samples for research and provided written informed consent in all cases. All the human tissue samples used for this study were obtained from patients as a standard part of the patients' spinal surgeries without additional biopsies. All animal experiments were carried out in a humane manner after receiving approval from the Institutional Animal Experiment Committee of Jilin University and in accordance with the Regulation for Animal Experiments and Fundamental Guideline for Proper Conduct of Animal Experiments of our university.The study protocol was approved by the Institutional Ethics Committee of the First Hospital of Jilin University. Human lumbar and cervical IVD samples were obtained from patients undergoing spinal surgeries during March 5BMP-7 inhibitor noggin was purchased from Protect . All other chemicals and reagents were purchased from Sigma  unless otherwise specified.et al[Patient samples were collected and isolated as previously described by Yu et al. We collet al, 7 of th3 blocks, which were treated separately with 0.1% collagenase II for 4‒6 hours [2 at 37°C. Cells were then passaged and further cultured in the presence of BMP-7 (10‒100 ng/ml). On reaching approximately 80% confluence, cells were subcultured in 60-mm culture dishes (4×105 cells/well). All experiments in this study used first- or second-generation cells.Disc tissue was harvested from male Wistar rats (200–250 g). All the rats were purchased from Animal Experimental Center of Jilin University and were killed with a lethal dose of 10% chloral hydrate. Lumbar and tail IVDs were separated from the spinal column under aseptic conditions. After separation of the NP, the tissue was cut into 1-mm‒6 hours . NP cellAccording to the manufacturer’s instructions, Lenti-FoxC2, Lenti-GFP or scramble was transfected into the cells by DharmaFECT1 Reagent  at a final oligonucleotide concentration of 10 nmol/L. For each cell transfection, two replication experiments were performed.Cell and tissue lysates were prepared using modified radioimmunoprecipitation assay (RIPA) buffer as previously described . Total pPCR amplification was carried out in accordance with the previously described method. Total RNA was isolated using TRIzol reagent  following the manufacturer’s instructions. cDNA was then reverse transcribed according to the manufacturer’s instructions. Quantitative PCR was carried out using a SYBR green Jumpstart Taq ReadyMix (TaKaRa) on a Roche LightCycler 480. A threshold cycle for each PCR amplification was subjected to 35 cycles of 95°C for 15 s and 54°C for 2 min. The primer sequences and their conditions for use were summarized in 3/ well) and treated with BMP-7 for 1‒24 h. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% triton-X 100 in PBS for 10 min, blocked with PBS containing 5% FBS, and incubated with antibodies against FoxC2 (1:200), aggrecan (1:100), SOX9 (1:200), and MMP-2 (1:200) at 4°C overnight. As a negative control, cells were reacted with isotype IgG under similar conditions. After washing, the cells were incubated with DyLightTM488-conjugated goat anti-rabbit antibody and fluorescein- or DyLightTM594-conjugated goat anti-mouse antibody (Jackson Laboratory), at a dilution of 1:200 for 45 min at room temperature. Fluorescence signals were captured using an Olympus Fluoview FV1000 confocal microscope and analyzed by the FV10-ASW 1.6 Viewer program .Cells were plated in flat bottom 96-well plates  values were read at 450 nm.All measurements were performed in triplicate and data were presented as means ± standard deviation (SD). Differences between groups were analyzed by Student’s t test; *p<0.05, ** p<0.01.Of the 36 DDD patients, 19 females and 17 males, the average age of these patients was 47.36±6.651 (range 33–57 years). The clinical findings for these patients were summarized in After approximately 7‒10 days, the Primary NP cells reached complete confluence. The cells were round or polygonal with a granular cytoplasm. Collagen II and aggrecan expression in these cells were observed by immunofluorescence . As earlTo determine whether up-regulation of FoxC2 in NP cells induce survival, proliferation and differentiation of NP cells, we transfected Lenti-FoxC2 in NP cells and transfected Lenti-GFP as the control. Transfecting Lenti-FoxC2 in NP cells significantly increased the expression of Ki-67, a famous proliferative marker, in immunofluorescence staining . CompareAs mentioned above, BMPs likely played an important role during the process of FoxC2 expression . We inveTo determine whether increased FoxC2 by BMP-7 is related to the BMP signaling pathway, we quantified FoxC2 mRNA expression in the presence and absence of BMP-7, SiRNA-Smad1, Scramble and the BMP inhibitor noggin . ConcordWe next examined the effects of FoxC2 on the expression of anabolic genes. As expected, the real-time qPCR results demonstrated BMP-7 stimulated the expression of ECM genes . SpecifiDisc degeneration is a complex process caused when the delicate balance between anabolic and catabolic processes is broken. From previous researches at the molecular level, we know that genes involved in NP cell degeneration are tightly regulated and coordinated by several signaling pathways , 36–38. We demonstrated that FoxC2 was frequently upregulated in human degenerative NP tissue and significantly associated with disc degeneration grade. We cultured primary NP cells from rat discs and characterized them by assessing cell morphology and expression of Collagen I, Collagen II and aggrecan. The characteristics of NP cells in our study were consistent with those of previous studies , 44. TheBMP signals are mediated by type I and II BMP receptors and their downstream molecules Smad1, 5 and 8. Thereafter, when type I receptor kinase is activated, Smad1, 5 and 8 proteins are phosphorylated and form a complex with Smad4 to translocate into the nucleus. Within the nucleus, they then interact with other transcription factors and regulate expression of target genes. Together with other transcription factors and transcription coupling factors, the Smad complexes bind to Smad binding elements (SBEs) in the promoters of target genes whereby they regulate expression of these genes. The anaTo further understand the relationship between FoxC2 and BMP pathways, we demonstrated that overexpression of FoxC2 activated a positive regulation mechanism. Noggin was known to inhibit the BMP pathways in a negative feedback system. Overexpression of FoxC2 was found to efficiently downregulate noggin expression, thereby indirectly reducing the inhibition effect of noggin within the BMP pathways. Furthermore, overexpression of FoxC2 resulted in phosphorylation of BMP7-responsive SMAD proteins (Smad1/5/8), that modulated BMP7-SMAD1/5/8-dependent anabolic target genes in NP cells. We therefore speculated that FoxC2 played a crucial role in sustaining activation of the BMP pathways.Results from previous studies demonstrated that BMP-7 had the ability to stimulate aggrecan and collagen synthesis in human disc cells. In this study, further evidence demonstrated that both FoxC2 and BMP-7 had potent anabolic effects on rat NP cells. We found that FoxC2 enhanced the upregulation effect of BMP-7 for ECM proteins like Collagen II and aggrecan. These results not only supported our previous conclusion about the FoxC2 positive regulation effect in BMP signaling, but also provided evidence for the benefits of a novel combination treatment in disc degeneration.et al. demonstrated that Foxc2 was a common transcriptional activator of TGF-signaling [et al. have shown that forced expression of FOXC2 promoted upregulation of Wnt4 expression and activation of increased levels of BMP [Several studies have previously shown that FoxC2 can induce the BMP pathway through crosstalk with other signaling pathways. Fujita ignaling . Althougignaling . Gozo ets of BMP . It is ws of BMP , 47. ThiIn conclusion, we identified that the transcription factor FoxC2 was over expressed in human degenerative discs. The mRNA expression level of FoxC2 was positively correlated with disc degeneration grade. Furthermore, we demonstrated that overexpression of FoxC2 increased the proliferation of NP cells. Importantly, BMP-7 induced FoxC2 expression, which was a positive feedback for maintaining signal activation . Our fin


Giardia intestinalis is a parasitic unicellular eukaryote with a highly reduced genome, in which only six cis-spliced and four trans-spliced introns have been discovered. However, we anticipate that more cis- and trans-spliced introns likely remain unidentified in genes encoding hypothetical proteins that occupy ca. 2/3 of all of the open reading frames (ORFs) in the Giardia genome. Consequently, comprehensive surveys of introns in ORFs for hypothetical proteins are critical for better understanding of the intron evolution in this organism.cis-spliced introns in the draft genome data of G. intestinalis strain WB, by surveying the conserved sequence motifs shared amongst the previously known introns. G. intestinalis strains can be divided into phylogenetically distinct assemblages A–H, and all the introns identified in past studies are shared among the published genome data from strains WB, DH, GS, and P15 representing assemblages A1, A2, B, and E, respectively. Nevertheless one of the two novel introns identified in this study was found to be absent in strain P15.In this study, we identified two novel G. intestinalis assemblages A1, A2, B, and E, one of the two introns identified in this study has highly likely been lost after the divergence of the assemblages. On the basis of a sequence comparison between the intron-bearing loci in WB, DH, and GS genomes and the homologous but intron-free locus in P15 genome, we propose that the loss of this particular intron was mediated by integration of the DNA fragment reverse-transcribed from mature mRNAs.By considering the organismal relationship among  Giardia intestinalis, a unicellular eukaryotic parasite belonging to the Diplomonadida (Excavata) is known to possess a highly reduced genome of only 12 Mbp in length G. intestrains DH  as well,Giardia introns/splintrons known to date bear conserved sequence motifs at the 5′ and 3′ termini, 5′-STATG-3′ and 5′-HCTRACMCVCAG-3′ , respectively. Furthermore, the two motifs may be flanked with each other within 300 bp, since all of the known introns in the Giardia genome range from 29 to 220 bp in length. We searched for genome segments that satisfied the above criteria in the draft genome data of G. intestinalis strain WB .G. intestinalis strain WB (ATCC50803) was cultivated as described previously , although reverse transcription activity of these encoded proteins has yet to be experimentally confirmed in G. intestinalis cells [There are three major models to explain how eukaryotic genomes lost spliceosomal introns: (i) ‘de-intronization’ by mutations, (ii) non-homologous end joining (NHEJ) repair of double strand break (DSB) in an intron sequence, and (iii) homologous recombination of the cDNA e.g., -23; see is cells .cis-spliced introns and their punctate distribution in the genomes of G. intestinalis assemblies. Together with the recently found trans-spliced introns, the data presented here suggest that the intron evolution in this organism is more complex than we previously thought.In this study, we found two novel The authors declare that they have no competing interests.RK determined the sequences and analyzed the data. YI and TH provided research materials. RK, YI, and TH prepared the manuscript. All the authors read and approved the final manuscript.Giardia intestinalis strain WB and the corresponding ORFs in strains GS and P15. A. orfA. Amino acid residues and nucleotides identical among the three sequences are shaded in black background. Asterisks indicate stop codons. The inserted position and nucleotide sequences of orfA intron are presented in the balloon. ORF nos. of orfA homologues in strains WB, DH, GS, and P15 are AACB02000068-1-10039-10248, AHGT01000085-3-18630-18839, ACGJ01001903-2-4190-4396, and ACVC01000007-3-18771-18962, respectively. B. orfB. The details of this figure are the same as described in A. ORF nos. of orfB homologues in strains WB, DH, GS, and P15 are AACB02000001-6-305427-304747, AHGT01000002-2-45140-45820, ACGJ01002919-1-19018-19737, and ACVC01000101-5-40937-40224, respectively. The intron sequences, which are a part of Figure Amino acid sequences of ORFs AACB02000068-1-10039-10248 (orfA) and AACB02000001-6-305427-304747 (orfB) in Click here for fileA. Deintronization by substitutions. An intron sequence (red) is changed to an exon sequence (light blue) by nucleotide substitutions (askerisks), resulting in extension of exon sequence. B. Non-homologous end joining repair of double strand break in intron sequence. In this model, ‘microhomology’ pairing between 5′ and 3′ splice sites anchors the upstream and downstream exons, which are split by double strand break. Subsequently, the broken strands are repaired, resulting in elimination of the entire intron sequence. C. Homologous recombination between an intron-containing gDNA and the corresponding intron-free cDNA (dark blue). This model assumes that the cDNA fragment, which is reverse-transcribed from a mature mRNA bearing no intron, is recombined into the corresponding intron-containing locus in the genome, resulting in elimination of the entire intron sequence.Proposed models for intron loss. Click here for file


Nanoscale carbon-based fillers are known to significantly alter the mechanical and electrical properties of polymers even at relatively low loadings. We report results from extensive molecular-dynamics simulations of mechanical testing of model polymer (high-density polyethylene) nanocomposites reinforced by nanocarbon fillers consisting of graphene flakes and fullerenes. By systematically varying filler concentration, morphology, and size, we identify clear trends in composite stiffness with reinforcement. To within statistical error, spherical fullerenes provide a nearly size-independent level of reinforcement. In contrast, two-dimensional graphene flakes induce a strongly size-dependent response: we find that flakes with radii in the 2–4 nm range provide appreciable enhancement in stiffness, which scales linearly with flake radius. Thus, with flakes approaching typical experimental sizes (~0.1–1 μm), we expect graphene fillers to provide substantial reinforcement, which also is much greater than what could be achieved with fullerene fillers. We identify the atomic-scale features responsible for this size- and morphology-dependent response, notably, ordering and densification of polymer chains at the filler–matrix interface, thereby providing insights into avenues for further control and enhancement of the mechanical properties of polymer nanocomposites. Using MD simulations, we study the mechanical response of a model polymer , reinforced by graphene flakes. The choice of HDPE as the polymer matrix is simply one of convenience that allows us to focus on the essential physics of matrix–filler interactions without additional complications arising from the matrix itself. To understand the importance of polymer chain packing at the filler surface in controlling the mechanical response, we also study the mechanical response of polymer-matrix composites with chemically comparable but morphologically distinct fullerene fillers. By systematically varying filler concentration, morphology, and size, as well as inter-chain and chain-filler interactions, we identify certain clear trends in composite stiffness with reinforcement. We have undertaken an extensive statistical sampling over atomic configurations and trajectories relative to the parameters noted above, which establishes clear confidence levels for the observed material response. In particular, we find that, to within statistical error, spherical fullerenes provide a nearly size-independent level of reinforcement of the matrix. In contrast, composites with graphene fillers exhibit reinforcement that depends strongly on the graphene filler size: although sub-nanometer graphene flakes are found to be ineffective fillers, flake sizes in the 2–4 nm range lead to appreciable reinforcement, which scales linearly with flake size, with graphene flakes outperforming with increasing size all the fullerenes examined. We elucidate the fundamental mechanisms responsible for this size- and morphology-dependent response, thereby providing insights into processing strategies for further control and enhancement of the mechanical properties of nanocarbon-reinforced polymers.2 and CH3 groups) as a single entity. Such united-atom models are commonly described using the Dreiding potentialEbond, Eangle, and Edihedral, arising from changes in the bond length (r), bond angle (θ), and dihedral angle (ϕ), respectively, which are given by the expressionsThe interatomic potentials for both the HDPE matrix and carbon fillers are based on a united-atom model, which treats individual hydrocarbon sub-units 212291011121314et al.et al.2-CH2 non-bonded interaction for the potential by Capaldi et al. is about 20% stronger than that for the potential by Buell et al.; since CH2 groups are the dominant component of the polymer matrix, this difference in non-bonded interaction strength results in a stiffer response of the matrix according to the former potential relative to that according to the latter as shown subsequently.where Nanocarbon fillers also are described using a Dreiding potentialTable S2 in the Supplementary Information14Table S3 in the Supplementary Informationet al. as HDPE1 and to that by Capaldi et al. as HDPE2; the corresponding parameterizations for the interactions in the composite systems will be referred to as HDPE1/C and HDPE2/C.Henceforth, for convenience, we will refer to the parameterization by Buell 3-(CH2)n-CH3  chains. The carbon fillers used in our study are “zero-dimensional”, cage-like fullerenes and two-dimensional (2D), planar graphene flakes. We considered fullerenes of three different sizes, C60, C180, and C540, and graphene flakes of five different sizes, C61, C181, C541, C1087, and C2161; the three smallest graphene flakes were chosen to closely mimic the carbon content of the three fullerenes examined, thereby facilitating a direct comparison of the role of filler morphology in the composite’s elastic response. The fullerene structures were prepared using the routines by Schwerdtfeger et al.Computational models of material samples were prepared with the aim of gathering extensive statistics and identifying clear trends in the elastic response as a function of filler size, concentration, and geometry. The polyethylene matrix was modeled using unbranched CHr0) with changes in orientation at each step being equal to the equilibrium bond angle (θ0); no two non-bonded atoms were allowed to approach within a distance of one bond length of each other in the SAW. For preparing composite system supercells, we first dispersed fillers in the supercell and thereafter inserted the polymer matrix using a SAW scheme under additional constraints that prohibited collisions between the polymer chains and fillers. Table S4 in the Supplementary InformationWe used a self-avoiding-random-walk (SAW) algorithm to generate supercell models of random polymer matrices. The SAW step size was chosen as the equilibrium C-C bond length of the polymer backbone (3) and, hence, required proper thermal equilibration and relaxation. To this end, we first carried out a conjugate-gradient energy minimization of the initial configuration with a force tolerance of 0.01 eV/Å implemented in the MD simulation software LAMMPSNPT) dynamics, implemented in LAMMPS, at 500 K and equilibrated over 100 picoseconds (with a time step of 1 femtosecond) to a pressure of 1 atm using a Nosé-Hoover thermostat and barostatNPT) dynamics, the samples were quenched by 25 K every 100 ps. Tg) as indicated by the example constructions in Tg to lie over the range 240–265 K, which contains the typical experimentally reported value of 250 KTg to the range of 260–295 K. The root mean square (RMS) displacement of the polymer united atoms computed over the (NPT) annealing schedule exceeds the radius of gyration of the polymer chains in our computational samples, which confirms proper initial equilibration of our samples prior to mechanical testingTo ensure reasonably fast convergence of the SAW algorithm, samples were produced at a low initial density (0.5 g/cmNPT) dynamics. Fixed strain increments were applied to a chosen simulation supercell vector at intervals of 10 ps to result in an overall strain rate of 109/s, while the remaining cell vectors were relaxed to attain a pressure of 1 atm. The virial stress (σij), defined asThe mechanical response of polymers is extremely sensitive to temperature. Here, we chose to carry out all of our tests at a temperature of 150 K such that all of our samples were well within the glassy regime see . The choV is the volume of the simulation cell; mp and i-th component of the velocity of particle p; i-th component of the force and the j-th component of the separation, respectively, between particles p and q; with the summations being over all particles in the simulation cell.was calculated by averaging over the last 5 ps of every applied strain increment. In Eq. (3), 2-CH2 interactions for HDPE2 are about 20% stronger than those for HDPE1, which accounts for the observed stiffer response in the former case. Specifically, at the chosen temperature of 150 K (below Tg), the calculated Young’s modulus for the neat polymer is 1.62 ± 0.02 GPa with HDPE1 parameters and 2.02 ± 0.03 GPa with HDPE2 parameters. These values are typical of Young’s moduli of glassy amorphous polymersFirst, we consider the mechanical response of neat HDPE to establish the baseline properties of the matrix prior to insertion of fillers. 61 and C181), the role of the filler is, to within statistical error, inconsequential; it is not until the graphene flake size increases to C541 that we begin to see an appreciable improvement in the composite modulus with filler concentration. On the contrary, fullerenes act as effective fillers even at small sizes  at enhancing the composite’s modulus. However, the filler-size effect for fullerenes appears to be much less significant than that for graphene flakes, with results for C60, C180, and C540 spanning a rather narrow band of moduli (with appreciable overlap of error bars) as indicated by the grey shaded regions in 541 flakes begin to result in comparable modulus enhancement as that of the fullerene fillers, while C1087 and C2161 flakes outperform the largest fullerenes considered here.Next, we analyze systematically the influence of filler size, morphology, and concentration on the response of nanocarbon-reinforced HDPE. r or, equivalently, as depicted in the insets in Ngf, in the flake. This E/E0 ~ r or E/E0 ~ Ngf1/2 at given filler concentration is an important scaling relation for the elastic response of the HDPE-graphene composites. We note that this scaling relation is as yet based only on a limited set of flake sizes in the 1–4 nm range; larger-scale MD simulations with flakes in the 10–100 nm range will be reported in a future publication to verify the validity of this scaling relation over a couple of orders of magnitude of graphene filler size. In any case, the results here are very promising as they establish a clear trend for the enhancement of polymer–graphene composite stiffness, as experimentally relevant graphene flakes have sizes that are typically in the sub-micron range. On the contrary, the limited scope for modulus enhancement with increasing fullerene filler size that is evident from our results—in conjunction with the fundamental challenges associated with the synthesis of such large fullerenes—is indicative of their reduced utility as fillers compared to graphene flakes.The size-dependent stiffening of graphene-reinforced HDPE is elucidated further in The main computational findings thus far, namely, the linear scaling of composite stiffness with graphene concentration as well as the linear dependence of composite stiffness on the lateral dimensions of the filler are generally consistent with classical composite theory61 and C181) dispersed in HDPE: 1087 and C2161 flakes are dispersed in the HDPE matrix [1087 flake [540 fullerenes, whose surfaces tend to undergo faceting and deviation from strictly spherical geometry, we also observe some layering and densification of the matrix in the vicinity of the filler’s facets [60 and C180 fullerenes. Nevertheless, it is clear that a graphene flake provides a much larger planar area relative to a faceted fullerene of comparable carbon content for adsorption and subsequent densification of polymer chains. However, due to their low surface area, small graphene flakes cannot induce such a local ordering and densification of the polymer chains.While continuum theory provides a broad understanding of the composite response, there are additional microscopic aspects of the matrix–filler interface, which may become important for load transfer at the nanoscale. For example, consider the case of small graphene flakes , we see that the effect of randomness versus ordering is insignificant, to within statistical error; with larger fillers, the responses are more intelligible although there is still some overlap of error bars. For ordered graphene flakes, we see that the response for loading along the flakes , comparable to those of typical fullerenes, there is little to no improvement in the stiffness of the composite with increasing graphene filler concentration. With increasing flake size, however, we have found an appreciable improvement in the composite modulus, with the effect of using C541 flakes being comparable to those of the fullerenes and with larger flakes  outperforming the fullerenes as fillers. This composite stiffness enhancement likely arises from more efficient load-transfer via interfacial shear (shear-lag) as well as ordering and densification of the polymer chains at the larger planar surfaces provided by larger graphene flakes. Within the limited statistics of graphene flake sizes considered in this study, the increase in composite modulus scales linearly with the flake size (circular flake radius). Given that typical solution-processed graphene flakes are much largerIn summary, we have conducted an extensive molecular-dynamics parametric study of the elastic properties of fullerene- and graphene-reinforced HDPE composites. By systematically varying the concentration, morphology, and size of fillers, as well as the non-bonded interaction strength expressed through different interatomic potentials, we have identified the contrasting roles of spherical fullerene and layered graphene fillers. CStudies of HDPE composites with larger graphene fillers are underway with the aim of establishing scaling laws for modulus enhancement over a couple of orders of magnitude of flake size as well as elucidating the chain dynamics and organization at the graphene–PE interface. Studies of nonlinear deformation and failure of fullerene- and graphene-reinforced HDPE composites also are underway and will be reported in future publications. Finally, in the present study, we have focused on the simplest model for matrix–filler interactions governed by weak dispersive forces. To the extent that strong covalent bonding between the filler and matrix is not significant, we expect that the qualitative behavior of polymer densification at the graphene–polymer interface as well as the quantitative power-law scaling relationship for composite stiffness should hold for other modes of filler–matrix interactions including, for example, hydrogen bonding between graphene oxide flakes and the polymer matrix. The use of functionalized graphene fillers could of course result in other modes of chemical bonding with the polymer matrix, and the effects of such stronger bonding of the composite’s constituents on its mechanical properties will be explored elsewhere.How to cite this article: Lu, C.-T. et al. A Comparison of the Elastic Properties of Graphene- and Fullerene-Reinforced Polymer Composites: The Role of Filler Morphology and Size. Sci. Rep.6, 31735; doi: 10.1038/srep31735 (2016).


CD274/PD-L1 gene copy number status was assessed by fluorescence in situ hybridization analysis. PD-L1 expression was detected in 36/80 (45%) cases and concordance of PD-L1 expression in primary tumor and corresponding nodal metastasis was present in only 20/28 (72%) cases. PD-1 expression was found in tumor-infiltrating lymphocytes (TILs) but not in tumor cells. CD274/PD-L1 gene amplification was detected in 19% of cases, with high level PD-L1 amplification present in 12/80 (15%), and low level amplification in 3/80 (4%). Interestingly, CD274/PD-L1 gene amplification was associated with positive PD-L1 immunostaining in only 73% of cases. PD-L1 copy number status was concordant in primary tumor and associated metastases. Clinically, PD-L1 tumor immunopositivity was associated with a higher risk for nodal metastasis at diagnosis, overall tumor related death und recurrence. Based on our findings we propose to include PD-L1 copy number status in addition to protein status in screening programs for future clinical trials with immunotherapeutic strategies targeting the PD-1/PD-L1 axis.Immunomodulatory therapies, targeting the immune checkpoint receptor-ligand complex PD-1/PD-L1 have shown promising results in early phase clinical trials in solid malignancies, including carcinomas of the head and neck. In this context, PD-L1 protein expression has been proposed as a potentially valuable predictive marker. In the present study, expression of PD-L1 and PD-1 was evaluated by immunohistochemistry in 80 patients with predominantly HPV-negative oral squamous cell carcinomas and associated nodal metastasis. In addition,  Squamous cell carcinomas comprise over 95% of malignant tumors in the head and neck region, including the oral cavity. The disease is associated with a poor prognosis, with an overall 5-year survival rate of 60%. Two-thirds of the tumors present with locally advanced or metastatic disease (stages III and IV). Historically, oral cavity squamous cell carcinoma (OCSCC) was viewed as a tobacco- and alcohol-related cancer, but infection with human papillomaviruses (HPVs) has emerged as an important risk factor for OCSCCs (reviewed in ) TreatmeCD274/PD-L1 gene, located on chromosome 9p24.1, in close proximity to PDCD1LG2 (programmed cell death ligand 2). PD-L1 can be upregulated in tumor cells and in a number of different tissue types in response to INF-γ and other inflammatory mediators  [24 [2431]  28] wit wit28] wUp to date, there is limited data concerning changes of PD-L1 expression in tumors during metastatic progression and none of the studies up to date have evaluated SCCs of the head and neck. Results of our own study show that in only 72% of cases, the PD-L1-status was retained in nodal metastatic lesions. In two studies comparing matched tumor samples with corresponding metastatic lymph nodes involving patients with RCC and triple-negative breast cancer, respectively, the concordance rates were considerably higher , 35. TisTolerance of PD-L1 positive tumors by the immune system is mediated by interaction of PD-L1 on tumor cells with its receptor PD-1, preferentially expressed on Tregs and other immune cells. Upon activation of PD-1/PD-L1, the anti-tumor response of the immune system is attenuated as T-cell functions are suppressed by the mechanisms named above. In HNSCC, co-receptor signals on T-cells mediated by PD-1 can inhibit antitumor response in a pre-clinical model , 36, 37.In our OCSCC patient cohort, PD-L1 positive carcinomas had a significantly higher risk for nodal metastasis at the time of diagnosis . Moreover, the risk of disease-and overall tumor-related death was significantly higher in PD-L1 positive OCSCC. In comparison, results of previous studies have been contradicting in this regard, probably reflecting differences in methodologies used to assess PD-L1 status and differences in patient characteristics, including percentage of HPV-related SCC tumor subgroups  28]. It. It28]. CD274/PD-L1, PD-L2 and JAK2 genes [Expression of PD-L1 in tumors cells is mediated by different mechanisms. It can be induced by autocrine or paracrine factors within the tumor microenvironment, especially INF-γ or HIF1α. In this case, PD-L1 expression is dynamic, with variable, time-dependent expression. On the other hand, expression can be driven by gene amplification events involving the 9p24.1 locus. The 9p24.1 chromosomal locus contains the K2 genes , 23. SelK2 genes  and has K2 genes , 23. OthK2 genes , 38-40.CD274/PD-L1 gene locus, in 19% of OCSCC cases. The amplification driven PD-L1 expression in a subgroup of OCSCC may identify a new subgroup of HNSCC with a disease-specific genetic alteration. Further studies are needed to evaluate the impact of PD-L1 amplification on pathogenesis and disease progression and on prognosis of this newly identified subgroup of HNSCC.Using FISH analysis, we found amplification of 9p24.1, including the PD-L1 amplification [CD274/PD-L1 gene copy number gain as a potential mechanism for PD-L1 overexpression in the present study may provide a rationale for treatment of HNSCC patients, especially in a subgroup of OCSCC with PD-L1 amplification. Future studies have to investigate whether PD-L1 copy number status, which is stable in the primary tumor and corresponding lymph node metastasis, might be a better predictive marker for tumor response to blockage of PD-1/PD-L1 pathway [CD274/PD-L1 gene amplification and the PD-L1 immunohistochemical status is surprisingly poor, with 4 amplified cases being PD-L1 immunonegative. The reason for this discrepancy is currently not clear, but may involve technical problems with the antibody clone used or posttranscriptional or posttranslational modifications.Blocking the PD-1/PD-L1 axis is a promising treatment option in multiple tumor types. Up to date, several clinical studies have been conducted, including especially patients with melanoma and non-small cell lung cancer (NSCLC), but also HNSCC (reviewed in ). These fication . Responsfication , 42-45. fication , 46, 47. pathway  than PD-CD274/PD-L1 amplification is a novel finding in OCSCC and is a frequent event, often  associated with high PD-L1 expression levels and stable during metastatic progression. Based on these data, we propose to include CD274/PD-L1 amplification analyses into studies recruiting patients for immune checkpoint protocols.In summary, we show that PD-L1 is expressed in 45% of OCSCC, when a 5% cut-off of positive cells is applied. PD-L1 positive cases have a significantly higher risk for nodal metastasis at diagnosis. PD-L1 expression is associated with higher risk for overall tumor-related death and recurrence. Although PD-L1 expression status in corresponding nodal metastasis is equivalent to expression in primary tumors in the majority of cases, a considerable number of cases (32%) were discordant. th edition). Complete clinicopathologic data including age, gender, localization of primary tumor, local, nodal and distant recurrences and overall and disease-free survival were available for all patients . Grading and staging was undertaken according to the current World Health Organization Classification of Tumors of Head and Neck (2004) and the AJCC tumor, node, metastasis (TNM) classification  using a Tissue Microarrayer  with a core size of 0,6 mm. A minimum of 2 and (where feasible) up to 4 tumor cores from tumor invasion front and tumor center were taken from the primary tumors in areas previously marked by two pathologists . Corresponding regional nodal metastasis from 28 patients were also included in the TMA, selecting 1 or (where feasible) 2 different areas from the metastatic deposits, depending on size of metastasis.IHC was performed on 2 μm sections from each TMA using a PD-L1 primary antibody  at a dilution of 1:100 and a PD-1 primary antibody  at a dilution of 1:50. Stainings were run on an automated immunostainer with an iVIEW DAB detection kit . Immunohistochemical expression of PD-L1 in tumor cells was assessed by counting the percentage of positive tumor cells and quantifying staining intensity in a 4-tiered grading system including “no staining” (0), “weak staining” (1+), “intermediate staining” (2+), “strong staining” (3+). PD-L1 positivity was arbitrarily defined as at least 5% tumor cells displaying membranous PD-L1 staining of any intensity. The cut-off value of 5% was chosen, because many clinical trials involving immunomodulatory therapeutics have chosen this value. Immunohistochemical expression of PD-1 in TILs was assessed by counting the percentage of PD-1 positive TILs. PD-1 positivity was counted as at least 1% positive TILs.in situ hybridization, the SPEC CD274, PDCD1LG2/CEN9 Dual Color Probe containing a mixture of fluorochrome direct labelled SPEC CD274, PDCD1LG2 probe specific for the CDC274/PD-L1 and PDCD1LG2/PD-L2 genes located at 9p24.1 and an orange fluorochrome labeled CEN 9 probe specific for the classical satellite III region of chromosome 9 (D9Z3) at 9q12  was used according to the manufacturer´s instructions. At least 20 nuclei per sample were counted. PD-L1 amplification was defined as PD-L1/CEP9 ratio ≥ 2.0, with high level amplification defined as ≥ 4.0 and low level amplification defined as ≥ 2.0 and ≤ 4.0. Polysomy 9 was defined as average PD-L1 copy number > 3 signals/cell.Dual-color FISH analysis was performed on 2μm sections from formalin-fixed paraffin-embedded tissue. Sections were deparaffinized, dehydrated in 100% ethanol and air-dried. For DNA was isolated from formalin-fixed, paraffin-embedded tissue of primary tumor samples using the QIAamp DNA FFPE Tissue Kit  in combination with the QiaCube  according to the supplier's instructions. HPV analysis and determination of HPV subtypes was performed with the HPV LCD 3.5 Array - Kit  according to the manufacturer's recommendations.2-test or Fisher's exact test, depending on the cell counts of corresponding contingency tables. Likewise, quantitative data was compared between groups using the non-parametric Mann-Whitney U test. Cumulative incidence functions were estimated for censored outcomes to account for competing risks such as death. All statistical tests were performed on exploratory two-sided 5% significance levels.Descriptive and exploratory statistical analyses were performed using SPSS 21  and R 3.2.0 . The distribution of qualitative data was compared between groups using χ


Immunotherapy has become a key strategy for cancer treatment, and two immune checkpoints, namely, programmed cell death 1 (PD-1) and its ligand (PD-L1), have recently emerged as important targets. The interaction blockade of PD-1 and PD-L1 demonstrated promising activity and antitumor efficacy in early phase clinical trials for advanced solid tumors such as non-small cell lung cancer (NSCLC). Many cell types in multiple tissues express PD-L1 as well as several tumor types, thereby suggesting that the ligand may play important roles in inhibiting immune responses throughout the body. Therefore, PD-L1 is a critical immunomodulating component within the lung microenvironment, but the correlation between PD-L1 expression and prognosis is controversial. More evidence is required to support the use of PD-L1 as a potential predictive biomarker. Clinical trials have measured PD-L1 in tumor tissues by immunohistochemistry (IHC) with different antibodies, but the assessment of PD-L1 is not yet standardized. Some commercial antibodies lack specificity and their reproducibility has not been fully evaluated. Further studies are required to clarify the optimal IHC assay as well as to predict and monitor the immune responses of the PD-1/PD-L1 pathway. Among the many molecularly defined checkpoint proteins,PD-1 is a type 1 transmembrane protein of the immunoglobulin superfamily,,PD-1 binds two B7 family ligands, namely, PD-L1 and PD-L2 (B7-DC or CD273),Cancer cells can activate PD-L1 expression through various oncogenic signaling pathways, such as phosphoinositide 3-kinase/protein kinase B (PI3K/PKB)Antibodies that target either PD-1 or PD-L1 are being developed to block ligand-receptor interaction and to improve antitumor immune response by allowing T cells to attack the tumor. To date, these antibodies have demonstrated exciting clinical responses against many cancer types.,-PD-L1 is expressed in several tumor types, such as melanoma, glioblastoma, and cancers in lung, kidney, head and neck, stomach, colon, pancreas, breast, cervix, cervical, and ovarian cancer. This protein has also been observed in hematologic malignancies, such as multiple myeloma, lymphoma, and various leukemia types,,PD-L1-positive cancers may indicate immune active tumors that could be sensitive to anti-PD-1 and/or PD-L1 therapies because of their correlation with poor prognosis in many of these malignancies, including lung adenocarcinomaPD-L1 has dynamic expression, and its evaluation by IHC is not well standardized. Previous studies have used a range of various antibodies, treatments, tumor types, and criteria to determine the positivity of samples. Therefore, a coherent definition of PD-L1 positivity must be established to facilitate further study of PD-L1 as a potential biomarker for the PD-1/PD-L1 pathway blockade. Given the intrinsic heterogeneity of PD-L1 expression in many tumors, the present results must be interpreted with caution. However, a biomarker of the response to a specific immunotherapy treatment is yet to be found.,,In contrast, although testing the biopsied tumor tissue remains a recommended method for mutation analysis, challenges associated with serial tumor biopsy, particularly in NSCLC, have spurred the search for non-invasive blood-based assays that allow the frequent assessment of biomarkers as a part of routine clinical carevs. investigator’s choice of chemotherapy  as first-line therapy for stage IV or recurrent PD-L1-positive NSCLC, and a phase I study of nivolumab in combination with gemcitabine/cisplatin, pemetrexed/cisplatin, carboplatin/paclitaxel, bevacizumab maintenance, erlotinib, ipilimumab, or as monotherapy in patients with stage IIIb/IV NSCLC.Immunotherapy has shown promising results in early NSCLC clinical trials involving PD-1 or PD-L1 antibodieset al.vs. docetaxel for patients previously treated with PD-L1 positive NSCLC (NCT01905657), and combination studies with ipilimumab or chemotherapy for NSCLC patients (NCT02039674).As another anti-PD-1 mAb, pembrolizumab received FDA approval in October 2014 and can be used for treating epidermal growth factor receptor (EGFR) mutation-negative and ALK rearrangement-negative NSCLC that has progressed on or after platinum-based chemotherapy. Approval was granted based on the results of a phase I trial by Garon BMS-936559 and MPDL3280A are anti-PD-L1 mAbs. BMS-936559 showed modest activity (ORR of 6%-17%) among patients with advanced cancers, including NSCLC, in a phase I multicenter trial (NCT00729664). Objective response  was observed in 5 of 49 evaluable NSCLC patients,,PD-L1 is up-regulated in cancer and is expressed in tumor cells in 40%-50% of NSCLCs independent of tumor histology,Several new immune-based treatments for small cell lung cancer (SCLC) are currently in clinical development. These treatments include the mAb-targeting Delta-like ligand 4 (DLL4) demcizumab (NCT01859741) and nivolumab with or without ipilimumab (a mAb antibody against CTLA-4) (NCT01928394),Several companies have developed different primary antibodies for analyzing both PD-1 and PD-L1 proteins by IHC. Some studies suggest that tumor PD-L1 expression that is detected by IHC may predict clinical responses to anti-PD-1/PD-L1 therapyet al.The similarities among the PD-L1 antibodies that are used in trials, as well as the staining localization, threshold for signal detection, and test conditions, need to be investigated to obtain a robust protocol. Gadiot et al.The phase I study of Topalian A recently published patent describes antibodies with specific sequences that bind to human PD-L1 as well as reveals the benefit of detecting PD-L1 expression in FFPE human tissue samples by IHC (WO/2014/100079)Roche/Genentech and Bristol–Myers Squibb have developed different companion assays for PD-L1 expression, each with its own experimental PD-L1 antibody. These antibodies include Spring Biosciences clone SP142 , Spring Biosciences clone SP263 , and Dako clone 28-8 . To date, the properties and concordance between these IHC antibodies have not been reported, and only clone SP142 is commercially available. The characteristics of all antibodies that are discussed in this section are listed in ,,et al.For an antibody to be considered as a favorable diagnostic tool, it must show sensitivity, specificity, reproducibility, and robustness in detecting the target by IHC. Some of the numerous available assays for detecting PD-L1 expression by IHC can only stain cancer cells, whereas the other assays can stain tumor-infiltrating immune cells. Therefore, patients with tumor cell staining and/or immune cell staining were examined in some studies, whereas the other studies only included patients with PD-L1 expression in tumor cellset al.Two patterns of cellular distribution of PD-L1, namely, membranous (cell surface) and cytoplasmic, have been described in tumor cells to indicate PD-L1 positivity. Membranous PD-L1 expression is present in tumors and inflammatory cells. PD-L1 staining pattern also differs between the assays. While some assays only evaluated membranous staining, others considered both membranous and cytoplasmic staining. PD-L1 is a type 1 transmembrane protein, and its cytoplasmic localization can represent intracellular stores of ligand that may relocate to the cell surface depending on cell stimulation. Interestingly, Brahmer ,,,The literature provides four definitions of PD-L1 sample positivity that are independent of the sample location and the staining of cells, i.e., whether ≥1, ≥5, ≥10, or ≥50 of cells per area are stained positive for PD-L1Immunotherapy for lung cancer is a new and exciting therapeutic modality. Multiple mAb candidates that target the PD-1/PD-L1 immune checkpoint have demonstrated reliable responses in tumors, including lung cancer, whereas some mAb candidates have shown remarkable antitumor effects in different clinical trials. Unfortunately, the lack of a reliable biomarker of response obscures such a scenario. Data on the correlation between PD-L1 positivity and patient responses to the different PD-1/PD-L1 blocking agents are also conflicting. PD-L1 is up-regulated in many cells and cancer types and contributes to the malignancy of these cancers by interacting with PD-1 and inhibiting T cell activation, thereby limiting the detection and destruction of tumor cells by the immune system. This ligand may play important roles in the inhibition of immune responses in both lymphoid and non-lymphoid organs. If PD-L1 is up-regulated in a tumor without an appropriate immune infiltrate, the blockade may have no effect because the tumor lacks the effector cells that fight the cancer.Several immunohistochemical antibodies have been developed for detecting PD-L1 expression in FFPE tissue. Characterizing tumors and immune cells via PD-L1 protein expression by IHC may help identify those patients who can benefit from the mAb candidate anti-PD-1 and anti-PD-L1 agents. As a result, the ability of PD-L1 protein expression to become a favorable predictive marker of response has been measured in various ways in several clinical trials. The conflicting results of PD-L1 staining may be attributed to the small sample sizes that are tested in different assays and/or to the variability of the used antibodies. No precise cut-off has also been established for determining PD-L1 positivity by IHC. The limitations in the specificity and reproducibility of some antibodies may explain the contradictory relationships between assays.Confirming PD-L1 as a predictive biomarker presents a promising new therapeutic opportunity to administer those agents that prevent PD-1/PD-L1 pathway interaction in advanced or metastatic lung cancers and other tumors. Further studies must be conducted to clarify the optimal IHC assay, validate and standardize the definition of PD-L1 positivity, and explore the relationship among various expression levels of the PD-L1 protein, as well as the effect of such levels on the prognosis of lung cancer patients with PD-1/PD-L1-directed therapies.