Graphene supports long spin lifetimes and long diffusion lengths at room temperature, making it highly promising for spintronics. However, making graphene magnetic remains a principal challenge despite the many proposed solutions. Among these, graphene with zig-zag edges and ripples are the most promising candidates, as zig-zag edges are predicted to host spin-polarized electronic states, and spin–orbit coupling can be induced by ripples. Here we investigate the magnetoresistance of graphene grown on technologically relevant SiC/Si(001) wafers, where inherent nanodomain boundaries sandwich zig-zag structures between adjacent ripples of large curvature. Localized states at the nanodomain boundaries result in an unprecedented positive in-plane magnetoresistance with a strong temperature dependence. Our work may offer a tantalizing way to add the spin degree of freedom to graphene. Owing to long spin lifetime and diffusion length, graphene holds promise for spintronics, yet it does not possess intrinsic magnetic properties. Here, the authors observe experimentally a large, positive magnetoresistance at graphene nanodomain boundaries. This means that inducing magnetic moments in graphene is non-trivial, and there have been many studies suggesting how this could be achieved, such as the introduction of vacancy defects131415171819212324252627pz orbitals with px and py orbitals from the σ-band30323132343637232425262728Spin-transport electronics, or spintronics, is a subject of active international interest and investigation. Utilizing electrons' spin degree of freedom would give rise to new and more efficient forms of logic and storage devices245678911In this paper, we investigate the magnetoresistance (MR) of graphene synthesized on cubic-SiC(001). STM characterization indicates that a zig-zag structure forms on one side of our NBs and, moreover, ripples with large curvature are formed on both sides adjacent to the NBs. An unprecedented positive in-plane MR with a strong temperature dependence was observed. This positive MR decreases as the temperature is reduced from 300 to 100 K, reaching zero at 100 K, and increases again in the temperature range from 100 to 10 K. From non-equilibrium Green's function calculations, we show that the MR arises from the contributions of the NBs, which can explain the one-dimensional (1D) transport behaviour we observe at low temperatures. Above 100 K, the Zeeman effect leads to the inequivalent confinement of electrons with different spins from the two-dimensional (2D) sheet to the 1D NBs, which produces the positive MR observed at higher temperatures. This behaviour shows that it is possible to engineer new tunable electronic and magnetic nanostructures purely from graphene.2 V−1 s−1 at 10 K and 60 cm2 V−1 s−1 at 300 K, and the corresponding current densities are 2 × 1012 cm−2 at 10 K and 2 × 1013 cm−2 at room temperature. This value for electron mobility is notably less than the ∼1,000 cm2 V−1 s−1 observed for trilayer graphene prepared by mechanical exfoliationB2) in B dependence at low field and a quadratic B2 dependence at high field. To rule out the possibility that this effect might be due to misalignment between the magnetic field and graphene plane, θ=0°) to an in-plane  configuration, where θ is the angle between the direction of the magnetic field and the direction normal to the graphene plane. The MR exhibits a strong dependence on θ and the MR ratio increases with decreasing θ. For θ<60°, the MR ratio is always negative because of a reduction in scattering at the NBs. Therefore, misalignment between magnetic field and graphene plane can be ruled out as the source of the effect. A similar phenomenon was observed at other temperatures  modelR0 and C are the fitting constants, indicating that the transport below 150 K is restricted to the NBs. In contrast to the low-temperature data, the R–T curve above 150 K has a different slope and is fitted better by the cube root expression z direction at high enough temperatures. To verify the nature of the transport mechanisms below and above 150 K, I–V curves and the corresponding dV/dI measured at different temperatures. Above 150 K, the current increases linearly with applied voltage, clearly highlighting the 2D transport mechanism in this temperature range. The linear I–V curves also indicate good Ohmic contact between the electrodes and graphene. Interestingly, below 100 K, nonlinear I–V curves were observed and the dV/dI shows a maximum at I=0 mA, indicating the presence of a charge transport gap below 100 K. At low temperatures, the NBs have 1D localized edge statesTo investigate the effect of the NBs on the magneto-transport properties, σ under an in-plane field for massless Dirac fermions in pristine graphene should give a positive Δσ(B) and thus a negative MRσ(B), which requires a sublinear behaviour σ(μ)∝μα with 0<α<1, where μ is the chemical potential. Details of the calculation can be found in the B at low fields and a quadratic B dependence at high field. Moreover, a rather strong temperature dependence is also observed. The most reasonable explanation for the positive MR comes from the intrinsic properties of the NBs themselves and that the carriers have different transport mechanisms below and above 150 K due to the presence of the NBs. Here we explain how the NBs can modify the Δσ(μ) and the MR of graphene by calculating the transport properties of graphene containing a single NB based on a NEGF calculation and the Landauer–Keldysh formalismB dependence at low field and B2 dependence at high field is predicted by the calculation in agreement with what was experimentally observed. We simulated different lengths of the pristine graphene while maintaining the dimensions of the NB and found that the MR dependence remains exactly the same (σ(μ) as a function of μ for B=4 T, confirming that for graphene with NBs, σ(μ) has a sublinear behaviour σ(μ)∝μα with α≈0.839. Thus, we can conclude that the positive MR under an in-plane magnetic field at low temperatures arises predominately from the NB itself. 58In general, according to the Kubo–Greenwood formulathe same . Figure I–V characterizationE0) and the first excited state (E1) for each quantum well will thus be on the order of 0.1 eV as estimated from the finite-depth quantum well confinement. Once energized by an applied bias or thermal agitation, electrons can jump to the first excited state, and the wave functions of the localized edge states associated with the NBs begin to smear out more readily . Under an in-plane field, we observed a positive MR with a strong temperature dependence. Using NEGF calculations combined with experimental data, we have shown that the NBs with ripples have the potential to work as a spin filter and can result in a positive MR at low temperature. Moreover, our work suggests that graphene with NBs has localized states and large spin–orbit interaction at the ripples. The confinement of electrons of a particular spin direction from 2D to 1D NBs by the Zeeman effect is responsible for the positive MR observed at high temperatures.−11 Torr. The pressure during the graphene synthesis at sample temperatures of 1,300–1,350 °C did not exceed 4 × 10−10 Torr. STM characterization of the graphene trilayer synthesized on SiC(001) was carried out in situ at room temperature using single crystalline W[111] and W[001] tips prepared using chemical etching and UHV sharpening. Low energy electron microscopy (LEEM) measurements were done using a SPELEEM microscope (Elmitec GmbH) installed on I311 at the MAX laboratory in Sweden. Before the LEEM characterization, the trilayer graphene/SiC(001) samples were annealed in the microscope's UHV chamber at temperatures ranging from 600 to 1,000 °C to remove possible contamination.The uniform trilayer graphene was fabricated on a cubic-SiC thin film with a thickness of ∼1 μm grown on on-plane Si(001) wafers using Si-atom sublimation followed by high-temperature surface graphitization in the ultra-high vacuum (UHV) chamber of a room-temperature GPI-300 scanning tunnelling microscope352 plasma etching for 60 s (8 W at P=20 mTorr). The nanocontact fabrication was carried out by electron beam lithography using single-layer-positive tone resist poly(methyl methacrylate) (PMMA) supplied by MicroChem Corp. After development, thick metal contacts consisting of Ti (5 nm)/Au (45 nm) were deposited through e-beam evaporation.The field effect transistor Hall bar structure was fabricated by electron beam lithography using a single-layer negative tone resist ma-N 2403 supplied by MicroChem Corp, followed by Ot equal to 2.7 eV, and the system Hamiltonian isA tight-binding model was used to describe the system with a nearest neighbour hopping energy Ci†(Ci) creates (annihilates) an electron at site i and Ui is the onsite energy at site i. The onsite energy in the graphene system is the energy of the Pz orbital due to position variance6263t and Ui values can be calculated by taking into account the position variance caused by NBs or their substrate and so ong is the Landé g-factor, B is the external magnetic field and σ is the Pauli matrix. The last term in the Hamiltonian is Rashba spin–orbital coupling, induced by the ripple near the NB, αR is the SOC strength. In the simulation we consider that the NB and its nearby graphene two-layer neighbours both have Rashba SOC. Note that no phonons were involved in our calculations. We use the NEGF formalism to calculate magnetoresistence53where GR (GA) is the retarded (advanced) Green's function of the systemΓp(q) is defined as Σp(q) is the self-energy of the lead. From the transmision T(E), we can get the conductance G(E)=G0T(E), where fp(q) is Fermi distrbution of each lead. In our MR calculations, the bias V is fixed at 0.4 V and the resistance is obtained by R(B)=V/I(B). The conductivity σ(μ) is proportional to the transmission and can be calculated from σ(μ)∼T(E=μ). The charge density is calculated by the local occupation number at each site. The occupation number operator at site i is G< is the lesser Green's function whose formula is G<(E)=GR(E)Σ<(E)GA(E), where i is σ= are the Pauli matrices, and Trs[…] denotes the trace in the 2 × 2 spin −½ space.where The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Wu, H.-C. et al. Large positive in-plane magnetoresistance induced by localized states at nanodomain boundaries in graphene. Nat. Commun.8, 14453 doi: 10.1038/ncomms14453 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References


Climate change is expected to influence spatial and temporal distributions of fish stocks. The aim of this paper is to compare climate change impact on a fishery with other factors impacting the performance of fishing fleets. The fishery in question is the Northeast Arctic cod fishery, a well-documented fishery where data on spatial and temporal distributions are available. A cellular automata model is developed for the purpose of mimicking possible distributional patterns and different management alternatives are studied under varying assumptions on the fleets’ fishing aptitude. Fisheries management and fishing aptitude, also including technological development and local knowledge, turn out to have the greatest impact on the spatial distribution of the fishing effort, when comparing the IPCC’s SRES A1B scenario with repeated sequences of the current environmental situation over a period of 45 years. In both cases, the highest profits in the simulation period of 45 years are obtained at low exploitation levels and moderate fishing aptitude.The online version of this article (doi:10.1007/s13280-017-0955-1) contains supplementary material, which is available to authorised users. It is difficult to predict future development of Arctic marine ecosystems and, even more so, how these are affected by human interactions. Immediate effects of such interactions are not only functions of the level and profile of the human activity but also of current state and dynamics of the natural system. Spatial and temporal distributions of prey and predator species vary, depending both on external drivers . Climate change could cause system perturbations, redistributing some, or large, parts of the systems sample space. A dramatic change in the sample space of the system may be referred to as an ecosystem shift  stock in the Barents Sea, the resilience of the Northern cod fishery is confirmed by archaeological fishbone analyses  cod fishery, the most important fishery in the Barents Sea. The NEA cod stock employs a variety of different coping strategies to adapt to a fluctuating physical and biological environment such as spawning and feeding migratory patterns, cannibalism, maturation dynamics and opportunistic feeding strategies . The climate scenario is based on the IPCC AR4 SRES A1B scenario Anon.  which atThe focus on spatial distributions and fleet diversity is motivated from the widespread expectation that northern fish species will shift to a more northern distribution caused by increased water temperatures  covering biological growth and spatial and temporal distribution of the cod stock. This model is run together with an agent-based model (ABe: Agent-Based economic model) defined within the same lattice, covering the economic exploitation of the stock. The flow chart of the combined CAb-ABe-model and the connected SinMod model is shown in Fig. SinMod model  with rules based on a Moore neighbourhood of range two. Each cell is defined in terms of geographical coordinates and the state variable of the cell is the cod biomass in the water column at the geographical position of the cell. Hence, the spatial distribution of the cod biomass at one point in time is given by the matrix of state variables in the lattice. According to the definition of Moore neighbourhoods  and ecosystem survey (during August/September). Age-structured data from these data sources have been aggregated for the purpose of parameterising the CAb-ABe model. Registered catches and survey data have been spatially interpolated by Radial Basis Function interpolation .Information on spatial distribution of NEA cod for the period 2004–2010 has been provided through the on Myers  followedThe data sample from the period 2004 to 2010 was considered to represent the current environmental situation, rather than reflecting ongoing changes in climate. There are several reasons for this. The period is rather short and the datasets, although displaying significant variations in the distributional patterns, do not show any significant trends or systematic changes. The seasonal variations are extreme but the seasonal biomass centres of gravity are almost identical each year during the period. In terms of weighted biomass distances for each quarter during the time period from a given geographical point , cluster analyses did not reveal any systematic changes and different years constituted the main cluster for each of the four quarters.SinMod and FishExchanges are made publicly available through UiT Open Research Data.Based on this, the data sample was considered as a representative distribution related to the current climatic conditions. The average monthly spatial distributions of NEA cod stock biomasses during the period 2004–2010 were found by merging the different sources of information relevant for each month as explained in Eide . ResultiSpatial and temporal distributions of the NEA cod environmental carrying capacity levels for each scenario have been estimated on the basis of constraining physical and biological factors in addition to the observed distributional patterns Fig. . The NEASinMod datasets of bathymetry, temperatures and zooplankton biomasses over the simulation period, representing the changes in environmental carrying capacities corresponding to the A1B scenario. As stochastic element is added to estimated environmental carrying capacities. As the mean deviation of the carrying capacity of each cell varies between 20 and 30 %  during the period of observations (2004–2010), a normally distributed stochastic element with a mean value of one and a standard deviation of 10 % is assumed. The stochastic element also serves to establish the zero scenario monthly carrying capacities, repeating the current climate with the minor perturbations caused by the stochastic process.Monthly estimated current carrying capacities are then modified according to Figure When having established the cellular automata lattice with cell-specific carrying capacities, which develop according to environmental variables and observed distributional pattern in the cod population, the next step is to establish cellular automata distributional rules. Essentially, the rules describe how individual cod moves in terms of directions and distances within the time frame of 1 month. According to Rose et al. , NEA codThe rules should in principle be able to move the cod biomasses over time according to previous observations. This boils down to a straight forward statistical problem minimising the sum of squared distances between the observed centres of gravity in the observed cod biomass and the centres of gravity in the by rule distributed cod biomass  and two fleet types  placed in each of these ports  per unit of harvest. The fleet revenue (re) obtained in cell i isvc) of the fishing operation isi.Similarly to Heen and Flaaten , Hannesscm) of all cells isThe fleet contribution margin of all cells are found by Eqs.  and 3)  3) when e and x give the fishing effort of the fleet and stock biomasses distributed on cells and months. Index m indicates month number and n is the total number of cells available for the given fleet. Number of available cells depends both on the physical range of the vessel  from the contribution margin described in Eq. :5\documet (a given month in a given year) is the sum of the fishing effort distributed on all available cells:Total fleet fishing effort at time V. The relation between absolute fleet size, V, and utilised fishing effort, E, isThe fleet capacity in terms of maximum fishing effort which may be produced during a single month is E/V) may, however, bring the resulting dynamics closer to the dynamics assumed by Smith, since also fleet utilisation varies in space and time (e.g. negative contribution margins keep vessels in harbour). After introducing the entry (fg) and exit (fd) rates, the fleet dynamics are given byThis study assumes a pure or quota-regulated, open access fishery. Entry to and exit from the fishery are driven by profits beyond the normal level or negative profits, respectively. While Vernon Smith in his seminal paper Smith  assumed Entry rates are often expected to be higher than exit rates as in Eide .s. The core expression for each vessel group in the model is given bys, reflecting the fleets aptitude of identifying the most profitable (in terms of the revenue/cost ratio) fishing grounds. The smartness parameter (s) is a lump-based parameter where a number of features are reduced down to the value of this single parameter. The two extremes (s = 0 and s = ∞) go from a uniform distribution of fishing activities in the area available for the fleet  to placing all fishing activities into one single cell . For the special situation s = 1, the distribution of fishing activities exactly follows the distribution of profit opportunities (expressed by revenue/cost ratios).A reasonable assumption is that the fishers attempt to maximise their economic performance by fishing at the most profitable areas (e.g. within the most profitable cells). The problem is however to identify where the most profitable cells are positioned. The fishers search to solve this problem through the use of their best knowledge, experience and skills, including the use of fish finding technology, the information that may be obtained within the fishing community and from other sources, attitude towards risk and the economic factors constraining their activity. How successful the fishers are in identifying the most profitable areas depends in this model on the value of a single parameter, the smartness parameter entclass1pt{minima of Eqs.  and the s = 1 is regarded to be the lowest smartness level of interest, while a possibly unrealistically high level of s = 10 is the highest smartness level included in the study. The range In the following, F), assuming perfect stock information.The study includes different governmental constraints represented by four different management regimes of which one is no management (open access). The other three management regimes are all in principle structured similarly to the current management system, assuming different exploitation rates and perfect management control. A total allowable catch (TAC) is set according to a given target levels of the fishing mortality rate  vessels and high sea (large) vessel in a fixed ratio (60/40), which is a slight simplification of the current quota allocation system. The high sea vessels are not allowed to fish inside four nautical miles from the baseline, which is implemented by limited access  to cells along the coast.This study investigates and compares distribution and variability in the two scenarios, in particular emphasising fleet diversity and spatial distribution of the fishing activity. While previous studies Eide , 2008 sus-values 1, 1.5, 2, 3, 5 and 10) and four management regimes . The fleet dynamics is in all simulations controlled as described above, inducing a total fleet capacity  which may be larger or equal to the active fleet E: V) and the fishing effort (E) follow different paths in different fishing ports and for the two types of fleets. The Shannon function H is used as a fleet diversity index  equal 0.2 and 0.4. This effect is however not apparent in the case of F = 0.1 in which available stock biomass already is stabilised on a quite high level . When comparing the profit surfaces of the two scenarios for all the years  with the last 25 years of the simulation , it becomes visible how the environmental effect contributes in lifting the whole profit surface of the A1B scenario.The years after the environmental shift in the mid-thirties provides however the fleets with considerably higher profits in the A1B scenario than what is obtained in the zero scenario . In general, the A1B scenario seems to give relatively larger benefits to higher smartness levels than the zero scenario does. In both cases, highest profits are found at the fishing mortality rate (F) 0.2 but while the smartness level in A1B scenario maximum is 5 it is 1.5 in the zero scenario.Open access fishery combined with high smartness levels results in higher profits in the zero scenario than in the A1B scenario throughout the simulation period. At lower smartness levels, however, the profit surface area within the open access area reaches surprisingly high levels as seen in the lower-right table in Fig. S1 also indicates, for both scenarios, that the largest profits are obtained at moderate levels of the smartness parameter s, in the range 1–3 in the zero scenario and 1–5 in the A1B scenario. The exception is for the last 25 years of the simulation period, when also higher exploitation levels contribute in large profits in the A1B scenario.Figure S2 of the Electronic Supplementary Material, differing between vessels belonging to the coastal and high sea fishing fleets. As higher values of the diversity index indicate higher diversity, clearly the coastal fleet exhibits the highest diversity at low exploitation levels and for low smartness values at all levels of exploitation. The two scenarios follow the same pattern in this respect and also regarding the trends while increasing smartness levels. As higher smartness levels seem to contribute in increased fleet diversity for the high sea vessels (for exploitation rates at F = 0.2 and above), the opposite is the case for the coastal fleet, though not so pronounced at F = 0.2 as for the highest exploitation levels. Although the fleet diversity for high smartness levels and open access seems to drop below the corresponding levels of the zero scenario and the opposite for F = 0.4, the general impression is that the fleet diversity in the A1B scenario corresponds very closely to the fleet diversities found in the zero scenario simulations.Eide  introducS2. Each graphical plot in Fig. S2 is divided by the diagonal into two sector where the upper sector is the area where the high sea fleet exhibits a higher diversity than the coastal fleet, while it is opposite in the sector below. In Fig. S2, for the zero scenario as well as for the A1B scenario, at high exploitation rates (open access and when F = 04) and for s-values higher than 1.5, all points lay above the diagonal and hence indicate that the high sea fleet is more diverse than the coastal fleet.The declining diversity for small-scale vessels at higher smartness values and exploitation rates is also clearly visible in Fig. The fact that the high sea fleet has a wider range than the coastal fleet may be the simple explanation of the higher diversity at high levels of exploitation. The advantage of a higher mobility combined with a minimum level of fishing aptitude becomes relatively a more important advantage as the exploitation level increases. As seen in Fig. At low s-values in open access, the fleet diversities of the two scenarios are virtually identical. Overall, the highest fleet diversities are found at low exploitation levels and high smartness levels. The fleet diversities of the two scenarios follow each other closely but from the zero to the A1B scenario, the tendency is increasing diversity in the coastal fleet at low smartness while it is the high sea vessel diversity which increases at high smartness levels.S3 of the Electronic Supplementary Material shows the vertical and horizontal distributional ranges of the gravity centres of stock biomass and fishing effort distributions over the 45 simulated years. The stock distribution in terms of centres of gravity turns out to be very stable, almost not affected of fishing intensity and levels of smartness. A slight North-eastern movement is indicated for the A1B scenario compared with the zero scenario but the main impression is that the stock biomass distribution does not change. In the case of open access, the two scenarios are practically equal in terms of stock biomass distribution.Figure Significantly larger changes are seen in the distribution of fishing effort, also reflecting the changing fleet compositions due to stock properties, exploitation levels and smartness. At increasing levels of smartness, there is a light tendency towards a more South-western distribution of fishing effort in both scenarios, even for the A1B scenario where the stock distribution slightly moves in the opposite direction. This indicates that the effect of reducing cost related to distance from port may be a more important factor than the possibly more North-eastern stock distribution.S3 come out in Fig. S4 of the Electronic Supplementary Material, showing how the stock biomass distribution clusters for the two scenarios and their different combinations of fishing intensity and smartness levels. The two scenarios come out as independent clusters for all smartness levels at the lowest exploitation rate (F = 0.1), while a more mixed picture is seen at higher exploitation levels. At the higher exploitation levels, the differences between the different scenarios and combinations are smaller but still a distinct clustering between scenarios are visible.Some details of the information embedded in Fig. S5 and S6 of the Electronic Supplementary Material), which for natural reasons are closely related. In these cases, the lowest exploitation level and low smartness levels cluster independent of climate scenario. It seems also to be a combined clustering tendency for both scenarios at higher exploitation levels and higher smartness levels, suggesting that the distribution of effort and hence catches is more depending on smartness levels and fishing intensities than marginal changes in the distribution of stock biomasses.This is however not the case for the distribution of catch and effort Below is the link to the electronic supplementary material.


The aim of our study was to investigate the role of TrkB pathway in tumor occurrence and development for in order to provide theoretical basis to laryngeal cancer therapy.in vivo experiments were conducted in syngeneic BALB/c mice.Biological characteristics of the cells were studied by migration tests and colony forming assay. Gene and protein expression analysis was performed by RT-PCR or western blot. Significant changes in protein and gene expression, including higher expression level of TrkB, were found in cells and laryngeal cancer specimens. we demonstrated that TrkB activates AKT via c-Src, leading to increased proliferation. Also, TrkB induced EMT via increased expression of EMT related transcription factors such as Twist-1 and Twist-2.Our data indicate TrkB are overexpressed in laryngeal cancer, and TrkB signaling is involved in tumorigenicity of laryngeal cancer. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer.What is already known about this subject?• Cancer of the larynx is one of the most common types of head and neck cancer.• The survival rate of advanced laryngeal cancer is only 30 to 40%.• The tropomyosin-related kinase B receptor (TrkB), together with TrkA and TrkC, are neurotrophin receptors regulating the proliferation and differentiation of neuronal cells.What are the new findings?• TrkB are overexpressed in laryngeal cancer.• TrkB signaling is involved in tumorigenicity of laryngeal cancer.• TrkB acts as a key regulator of the PI3K/AKT signal pathway-mediated tumor metastasis.How might these results change the focus of research or clinical practice?• These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer. Our study provides molecular insight into the tumor metastasis and has important implications in elucidating oncogenic processes. Cancer of the larynx is one of the most common types of head and neck cancer. Laryngeal cancer is also the second most common tumor of the respiratory tract and is the eleventh most common cancer worldwide with high mortality rate. Among them, the laryngeal squamous cell carcinoma accounts for approximately 95% of all malignant tumors of the larynx . LaryngeSeveral risk factors have been implicated in the pathogenesis of laryngeal cancer, including tobacco use, alcohol consumption, air pollution, genetics, nutrition, bacterial infection, life-style and occupational factors –9. Moreovia mitochondrion-driven apoptotic pathway, rendering cancer cells survival, chemoresistance and invasiveness [The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly mutated pathway in head and neck squamous cell carcinoma . Activatsiveness . Previousiveness . The ovesiveness . HoweverImmunohistochemical assay was performed to investigate the protein expression of TrkB in 69 carcinomas and 32 tumor-adjacent normal laryngeal tissues.69 samples of laryngeal cancer and 32 samples of tumor-adjacent normal laryngeal tissues were used to detect the expression level of TrkB by immunohistochemistry. It showed that the expression of TrkB was significantly increased in laryngeal cancer than that in tumor-adjacent normal tissue Figures  and B. TFirstly, the western blotting analysis showed that the expression level of TrkB in Hep-2 was higher than that in other laryngeal tumor cell lines, including TU686 cells and M4e cells. Thus, Hep-2 cell line was used in the following studies. Secondly, Transwell assays showed that the down-regulation of TrkB expression could inhibit the migration of Hep-2 cells Figure . SubsequFigure We have demonstrated that c-Src activation by TrkB induces activation of the PI3K/AKT and cyclin D1 expression. To investigate the relationship between Runx3 or Keap1 and TrkB-induced tumorigenesis, we first examined whether the expression of TrkB influences Runx3 and Keap1 expression. We found that Runx3 and Keap1 mRNA expression were significantly increased in Hep-2 TrkB-sh RNA cells Figure . We alsoWe examined whether expression of TrkB was able to promote an EMT in Hep-2 cells. The results showed that the expression of proteins E-cadherin (E-cad) was down-regulated after TrkB overexpression and the expression of proteins of N-cadherin and Vimentin were up-regulated in Hep-2-TrkB cells Figure  and 5B. To investigate the role of TrkB in tumor formation, Hep-2 or Hep-2-shTrkB cells were injected into of BALB/ c mice to establish tumor animal mode. After 30th days, the tumor volume and weight in Hep-2-shTrkB groups were significantly smaller than that in control group, while the inhibitory rate was markedly higher than control group Figure . We nextLaryngeal cancer is one of the most common malignant tumors of the head and neck, accounting for about 1% to 5% of the systemic tumors in our body, and the pathological type of squamous cell carcinoma, accounts for 93% to 99% of all laryngeal cancel higher incidence of laryngeal cancer is in northern China, and the age of onset is 50 to 60 years old and male with a high incidence than women . In receIn this study, we show that TrkB is frequently overexpressed in highly metastatic laryngeal cancer cell lines and clinical laryngeal cancer samples. Furthermore, TrkB expression was significantly up-regulated in laryngeal cancer samples in comparison with normal laryngeal tissue. Activation of the PI3K/AKT pathway through TrkB-mediated c-Src activation induced tumorigenic and metastatic potential of laryngeal cancer.Tumor metastasis is an important reason causing tumor morbidity and mortality. Several studies have shown that TrkB play an essential role in invasion and metastasis of various tumor cells , 21. In Studies have shown that PI3K/AKT signaling pathway involved in development of human malignant tumors, and the pathway is related with tumor proliferation, apoptosis, metastasis, invasion and other cellular processes , 23. PI3c-Src played essential roles in multiple aspects of tumor progression, including proliferation, disruption of cell/cell contacts, migration, invasiveness, resistance to apoptosis, and angiogenesis . MoreoveIn conclusion, TrkB are overexpressed in laryngeal cancer, and TrkB signaling is involved in tumorigenesis and metastasis of laryngeal cancer. It indicates that TrkB pathway may be a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer. The future study should focus on the targeting regulation of TrkB pathway via non-coding RNA which may provide a novel therapeutic plan for laryngeal cancer.2. The inhibitors of protein kinase (K252a and SU6656) and PI3K inhibitor (LY294002) was purchased from Calbiochem .Human laryngeal cancer cell lines  were obtained from the Cell Bank of the Chinese Academy of Sciences  and were maintained as previously described –35. BrieFresh-frozen human laryngeal cancer samples and tumor-adjacent normal laryngeal tissues were obtained from the Affiliated Hospital of Southwest Medical University. The subject was approved by the institutional review board and the ethics committee of the Affiliated Hospital of Southwest Medical University (K2013166) and all patients have signed informed consents.2O2 and 10% goat serum and they were subsequently incubated overnight with a primary antibody for TrkB  at 4°C. The samples were incubated with HISTOFINE simple stain MAX-PO (R)  and finally visualized by 3,3’diaminobenzidine (DAB) with hematoxylin-counterstain.The paraffin sections of laryngeal cancer were deparaffinized and rehydrated routinely. The sections were immersed with 3% HShort-hairpin RNA (shRNA) constructs targeting TrkB (shTrkB) and AKT1 (shAKT1) and pLKO were obtained from Sigma-Aldrich  and introduced into cells via retroviral infection or with lipofectamine2000 reagent according to the manufacturer’s protocol. shRNA that did not match any known human cDNA was used as a control. For TrkB overexpression, pcDNA3.1 plasmids containing the full length cDNA sequences for human TrkB was stably transfected into Hep2 cells.Western blotting was performed as previous reported , 37. BriTrkB: forward: 5-GGAAAAGCAAAAACCCTGTCTAGA-3;reverse: 5-TGTAGCATCACTTCCTGCCATT-3β-actin: forward: 5-TTGCCCCGAGGCTCTCTT-3;reverse: 5-AGTTGAAGGTGGTCTCGTGGAT-3.RT-PCR was performed as previous reported . BrieflyAfter a reverse transcription reactions at 55°C for 30 minutes, there was an initial 10-minute denaturation at 95°C followed by the 40 cycles run consists of a 15-second denaturation step at 94°C and an annealing step at 60°C for 30 seconds, and extension step at 68°C for 1 minute. The density of bands was determined by using Un-Scan-It gel Software  and statistical analysis was performed by utilizing either conventional Student’s t test. The PCR results were averaged with three independent experiments, which were used in the statistical analysis.5 cells in PBS per mouse) of the nude mice (n=10 in each). Immunohistochemistry of TrkB, cyclinD1, AKT, E-cadherin were also performed in the xenograft tumor tissue. The primary antibodies for TrkB , cyclinD1 , AKT , E-cadherin  were purchased from Santa Cruz Biotechnology . The procedures were similar with the part of immunohistochemistry. Briefly, the paraffin sections of xenograft laryngeal cancer were deparaffinized, rehydrated and immersed with 3% H2O2 and 10% goat serum. Subsequently, the sections were incubated overnight with primary antibodies, followed by incubation with HISTOFINE simple stain MAX-PO (R)  and visualization by DAB with hematoxylin-counterstain.Five-week-old male athymic nude mice (BALB/c nu/nu) were purchased from Chengdu Dashuo Biological Technology Co. Ltd and acclimated for two weeks. All animal procedures were approved by the Animal Care and Use Committee at animal experimental center of Southwest Medical University. The animal experiment was also approved by the institutional review board and the ethics committee of the Affiliated Hospital of Southwest Medical University (K2013167), Hep-2 cells transfected with TrkB-targeting shRNA or non-targeting control shRNA were subcutaneously implanted into flank regions  and were shown as the mean ± standard deviation (SD). Student’s t-test was used for the comparison of mean values between two groups. A value of P< 0.05 was considered significant.


Programmed death-1 receptor (PD-1) and its ligand (PD-L1) play an integral role in regulating the immune response against cancer. This study investigated the prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating immune cells (TILs) in the tumor microenvironment in Chinese patients with esophageal squamous cell carcinoma (ESCC). Archival formalin-fixed, paraffin-embedded ESCC samples from treatment-naïve patients with ESCC after surgery or by diagnostic endoscopic biopsy were collected between 2004 and 2014. Expression of PD-L1 in ESCC tumor specimens was assessed by immunohistochemistry (IHC), and the degree of TIL infiltration was evaluated by examining hematoxylin and eosin-stained (H&E) specimens. PD-L1+ as defined as ≥1% of tumor cell membranes showing ≥1+ intensity. In 428 patients, specimens from 341 (79.7%) were PD-L1+. In the definitive treatment group (patients who received curative esophagectomy or definitive [chemo-]radiation therapy), PD-L1 positivity was associated with a significantly shorter DFS and OS. In the palliative chemotherapy group exhibited, neither PFS nor OS correlated significantly with PD-L1 expression. PD-L1 expression was positively associated with TIL density. In 17 paired tumor tissues collected before and after treatment, an increase in PD-L1 expression was associated with disease progression, whereas a decrease in PD-L1 expression was associated with response to chemotherapy or disease control. So, PD-L1 expression was associated with a significantly worse prognosis in patients with ESCC. These observations suggest that PD-L1 may play a critical role in ESCC cancer progression and provide a rationale for developing PD-L1 inhibitors for treatment of a subset of ESCC patients. Esophageal cancer is one of the most aggressive and lethal malignancies among gastrointestinal cancers, ranking sixth in cancer-related death. It has two main subtypes: squamous-cell carcinoma (ESCC), which is mostly found in Asia, Africa, and South America, and adenocarcinoma (EAC), which is mostly found in North America and Europe . In ChinProgrammed cell death 1 (PD-1), a member of the CD28 family of co-stimulatory receptors, is a T cell immune-checkpoint involved in providing inhibitory signals in T-cell activation when engaged by its ligands: PD1 ligand 1 (PD-L1) and PD1 ligand 2 (PD-L2) . It is wTo explore the prognostic significance of PD-L1 expression and TILs for ESCC, we investigated the extent of PD-L1 expression and TILs in formalin-fixed samples from 428 Chinese ESCC patients, and analyzed the association between PD-L1 expression, TIL density and outcome.A total of 428 ESCC specimens, one from each patient, were obtained from patients admitted to the Affiliated 307 Hospital Cancer Center, Beijing, P. R. China during 2004 to 2014. Samples were collected either by surgery  or by endoscopic biopsy  from treatment-naïve patients. The median follow-up was 34.4 months (range 0.3-147.1 months) from the date of sample collection. The last follow-up date was December 31, 2015. At the time of last follow-up, 22.43% (n=96) of patients with ESCC were still alive.Treatment modalities depended on the stage and localization of ESCC. Patients with localized tumors underwent curative esophagectomy , while those with unresectable disease or who were medically unfit for surgery received definitive (chemo-)radiation . Patients with advanced metastatic disease received palliative chemotherapy , palliative R1/R2 resection , best supportive care , or chose not to receive any treatment .Tumor PD-L1 expression was assessed via IHC, using rabbit monoclonal antibody clone 73-10 proprietary of Merck KGaA. Table PD-L1 expression was found primarily on the membranes of the tumor cells Figure , and wasThree major patterns of PD-L1 expression were found in ESCC specimens: (i) a diffuse PD-L1 expression in the presence of TILs; (ii) regional expression of PD-L1 colocalized with TILs; and (iii) PD-L1 expression at the invasive front Figure . PD-L1 eth IUCC/AJCC staging system, 157 (36.7%) and 191 (44.6%) had stage 0-II and III disease, respectively. And the majority  of tumor specimens showed a moderate to high differentiated tumor. The clinicopathological features of patients are summarized in Table Among the 428 patients, the majority 335 (78.3%) were male and 247 (57.7%) were older than 60 years. According to the 7Statistical analyses using Spearman Rank Correlation indicated that PD-L1+ specimens were associated with aggressive clinicopathological features, which included deeper tumor invasion (p=0.037) and nodal metastasis (p=0.013). No significant association was found between PD-L1 expression and gender (p=0.545), age (p=0.943), level of differentiation (p=0.955), primary tumor location (p=0.231) or TNM stage (P=0.581).The correlation of PD-L1 expression with disease prognosis, as measured by disease free survival (DFS/PFS) and overall survival (OS), was analyzed separately in the definitive treatment cohort and palliative treatment cohorts.The definitive treatment cohort comprised 250 patients, including patients who received curative esophagectomy or definitive (chemo-)radiation therapy, and excluding those who had hospital mortality (n=16) and occurrence of other primary cancer (n=7). The DFS rates at 1, 3, and 5 years, respectively, were 79.5%, 53.9%, 32.3% for PD-L1– patients and 57.8%, 31.1%, 21.3% for PD-L1+ patients. Similarly, the OS rates at 1, 3, and 5 years, respectively, were 92.5%, 55.8%, 36.5% for PD-L1– patients and 84.5%, 36.3%, 29.8% for PD-L1+ patients. DFS and OS was significantly related to PD-L1 expression , PD-L1+ patients exhibited worse DFS and OS than PD-L1– patients Figure  & 4B. StPatients in the palliative treatment cohort exhibited no significant association between PD-L1 expression and PFS  or OS  .The correlation between TIL density and patient prognosis was examined. Similar to PD-L1 expression, there was no significant difference in TIL density between surgical and biopsy specimens (p=0.243), suggesting that TIL density was not affected by size of the specimens. In definitive treatment cohort, no significant correlation was observed between DFS/OS and TIL density  Figure  & 5B. HoExpression of PD-L1 and numbers of CD8+ and Foxp3+ cells was examined in 17 paired tumor specimens. This included 3 patients who did not receive any treatment between the times when paired tumor samples were harvested (mean duration 1.16 ± 0.28 months), 9 paired specimens collected before and after 2 cycles of chemotherapy (mean duration 1.23 ± 0.24 months), and 5 paired specimens collected before and after 4 or more cycles of chemotherapy (mean duration 4.97 ± 2.22 months).There was an increase in PD-L1 expression as disease progressed (accompanied by an increase in the depth of tumor invasion) in patients who did not receive any treatment , with a mean duration of 1.16 months between obtaining harvesting of the first and second tumor specimens. Representative changes in PD-L1 expression are shown in Figure No statistically significant change was found in CD8+ lymphocyte numbers levels between samples obtained before and after chemotherapy (p=0.151), including patients who received treated with 2 or 4 or more cycles chemotherapy between obtaining harvesting tumor specimens. Of the 14 patients with paired specimens who received chemotherapy, 10 showed an increase of CD8+ lymphocytes while 4 showed a decrease Figure . Of the Based on PD-L1 status and TIL density, ESCC specimens were classified into 4 groups. In this classification, PD-L1+ was defined as specimens with ≥1% expression on tumor cell membranes with ≥1+ intensity and a TIL score of 3 (high) was considered as TILs+. Type I was PD-L1+ and TILs+ (PD-L1+ with TILs driving adaptive immune resistance), type II was PD-L1– and TILs– (PD-L1–with no TILs indicating immune ignorance), type III was PD-L1+ and TILs– (PD-L1+ with no TILs indicating intrinsic induction), and type IV was PD-L1– and TILs+ (PD-L1– with TILs indicating the role of other suppressor(s) in promoting immune tolerance). The proportions of four types were 13.6% (58/428), 17.1% (73/428), 66.1% (283/428), and 3.3% (14/428), respectively . As a validation for our hypothesis that PD-L1 expression is associated with an aggressive tumor progression, we found that PD-L1 expression increased as disease progressed. In contrast, PD-L1 expression decreased between baseline and second tumor sampling obtained after 2 cycles of chemotherapy in those with satisfactory treatment responses. Interestingly, PD-L1 expression increased after 4 or more cycles of chemotherapy in patients with worse treatment responses. Recently, a study investigated changes of PD-L1 after neoadjuvant therapy in ESCC patients. Similarly, they found a decrease in PD-L1 expression after 2 cycles of neoadjuvant chemotherapy (NAC) and an increase after neoadjuvant concurrent chemoradiotherapy . HoweverWe also investigated the immunological impact of chemotherapy on CD8+ and Foxp3+ T cells in the ESCC tumor microenvironment. Among 14 patients who received chemotherapy, 10 exhibited an increase in CD8+ T cells. Increased levels of CD8+ T cells were associated with a better outcome, although the difference was not significant due to small number of available samples. This finding was also seen in several other studies –47, inclIn conclusion, PD-L1 is expressed on the cells of most (>70%) ESCC tumors, and is significantly associated with tumor aggressiveness and an enhanced risk for postoperative recurrence (shorter DFS and OS). As such, PD-L1 may represent a target for treatment of ESCC and is a potentially predictive biomarker for anti-PD-1/PD-L1 antibodies. The results presented in this study support the clinical development of PD-1/PD-L1 blockade as a treatment for ESCC. Further research investigating the effect of PD-L1/PD-1 blockade in ESCC is warranted.Archival formalin-fixed, paraffin-embedded ESCC samples were collected from patients diagnosed with primary ESCC after surgery or by endoscopic biopsy at the Affiliated 307 Hospital Cancer Center, Beijing, P. R. China between 2004 and 2014. Serial sections of 4 μm thickness were obtained from each specimen. Histopathologic data was obtained from pathology reports, and patient survival data was obtained from the patient's attending physician. The retrieval of tissue and clinical data were performed according to the regulations of the local institutional review boards and data safety laws with specific regard to ethical standards and patient confidentiality (Ethics No. ky-2016-2-12).Conventional clinicopathologic variables, including age, gender, level of differentiation, primary tumor location, tumor invasion depth (T), nodal metastasis (N), distant metastasis (M), TNM stage, and treatment received were collected from patients' attending physicians. Tumor stage was determined according to the 7th American Joint Committee on Cancer/International Union Against Cancer tumor-node-metastasis (TNM) classification system (2009).A subset of patients underwent right or left thoracotomy for curative resection by total or subtotal thoracic esophagectomy, in addition to regional lymphadenectomy; none underwent transhiatal esophagectomy. Regional lymph nodes included not only mediastinal but also perigastric nodes, thus regional lymphadenectomy represented at least a two-field lymphadenectomy. Esophageal reconstruction was performed using the stomach, colon, or jejunum. Postoperative treatment was recommended for patients with pathologically confirmed metastasis to the lymph nodes or deep tumor invasion (≥T3), and patients without severe postoperative complications. Postoperative treatment included adjuvant chemotherapy, radiotherapy or chemoradiotherapy. The first cycle of adjuvant chemotherapy was administered 4-5 weeks after surgery, and consisted of cisplatin and 5-FU or cisplatin and paclitaxel at standard doses. Adjuvant radiation therapy was administrated 4-5 weeks after surgery, and included external beam radiation with four fields of 6 MV photons to a total dose of 50.4-60 Gy using 1.8-2 Gy daily fractions in 5 weeks. Positioning of the fields and dosimetry were studied using a CT scan and 3D treatment plan. For adjuvant chemoradiotherapy, the radiation therapy program was the same as that described above, and chemotherapy regimens consisted of cisplatin or paclitaxel or 5-FU administered concurrently with radiation.Patients who were unfit for surgery due to the presence of comorbidities or other risk factors received definitive (chemo-)radiation at ≥50.4 Gy. For tumors of the cervical esophagus, high dose (60-66 Gy) definitive (chemo)-radiation therapy was prescribed. Patients with metastatic or unresectable esophageal cancer received palliative chemotherapy. Chemotherapy regimens consisted of cisplatin, taxanes (paclitaxel or docetaxel), fluorouracil  or others. All chemotherapeutic drugs were prescribed at standard doses. The clinical criteria for response were defined by RECIST 1.1.TM FLEX Target Retrieval Solutions (Dako), and then cooled to room temperature in TBST wash buffer for 5 min, then incubated with the primary antibodies. Antibody detection was visualized using DAB chromagen according to the manufacturer's instructions (Dako). Slides were counterstained with hematoxylin.Expression of PD-L1 in ESCC tumor specimens was assessed by IHC with an Autostainer Plus (Dako-Agilent Technologies), utilizing a proprietary PD-L1 antibody  at 1:1000 dilution. Human tonsil and muscle tissue served as positive and negative control tissue, respectively. Both CD8 and Foxp3 antibodies were purchased from Abcam and used at 1:200 dilution. Sections were baked for 60 min at 60°C in a dehydration oven and dewaxed and rehydrated using xylene and graded alcohol washes. Antigen retrieval and deparaffinization were carried out using the EnVisionPD-L1 expression on the membranes of tumor cells and TIL/histiocytes was scored blindly. Frequency of PD-L1+ cells and staining intensity were analyzed and every tumor specimen was scored according to the intensity of PD-L1 staining . In tumor cells, PD-L1+ by a 1% cut off was defined as specimen with ≥1% membranous expression on tumor cells with ≥1+ intensity, PD-L1+ by a 5% cut off was defined as specimens with ≥5% membranous expression on tumor cells with ≥1+ intensity, and PD-L1+ by a 25% cut off was defined as specimens with ≥25% membranous expression on tumor cells with ≥2+ intensity. Positive PD-L1 expression on lymphoid cells and stromal cells was defined as ≥1% positive cells with membranous staining at ≥1+ intensity. The degree of infiltration by TILs was evaluated from the H&E-stained specimens and scored from 0 to 3: 0 = none; 1 = rare; 2 = moderate and focal infiltration; and 3 = prominent and diffuse infiltration (high). Quantitative evaluation of CD8+ and Foxp3+ cells was carried out using 5 to 10 areas with the most abundant distribution within the tumor using an ocular grid at X200 magnification in a blinded manner. The average numbers of cells were used for statistical analysis.Disease-free survival (DFS) time was defined as the period from the date of surgery to confirmed tumor relapse date for relapsed patients or from the date of surgery to the date of last follow-up for nonrecurrent patients. Progression-free survival (PFS) time was defined as the period from the date of initiation of first-line chemotherapy to the date of tumor progress or last follow up for patients without tumor progression. All patients who did not experience the event of interest were censored at the last follow-up date.The distributions of patient and clinical characteristics between PD-L1+ and PD-L1- were compared using Chi-square test, Wilcoxon rank-sum test, or two-sample T test, when appropriate. The correlation between PD-L1 expression and other factors  was analyzed using Spearman's rank correlation. Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test. The stepwise Cox-regression model was used to identify factors significantly related to OS and DFS (PFS). P<0.05 (two-tailed) was considered to indicate statistical significance. All statistical analyses were performed using SAS 9.3.


Different anti-PD-1 and anti-PD-L1 antibodies bind different epitopes. However, whether the results from the SP142 and 22C3 immunochemistry (IHC) assays can be interchanged to determine patient eligibility for immunotherapy remains largely unknown. Histologic sections from 135 tumor samples were probed with both 22C3 and SP142 antibodies. The concordance of PD-L1 expression determined by the two assays was assessed. Additionally, we evaluated the association of PD-L1 expression detected by different assays with clinicopathological features and prognosis. In total, 105 (77.78%) of 135 samples evaluated by the 22C3-IHC platform produced the same results with the SP142-IHC platform . In addition, 69 (51.11%) of 135 samples evaluated by the SP142-IHC platform produced the same results with the 22C3-IHC platform . PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status, whereas that based on the SP142-IHC assay was correlated with smoking status, sex, and histology. Compared to the SP142-IHC assay, the 22C3-IHC assay usually resulted in an underestimation of PD-L1 expression in tumor cells and immune cells. Thus, the results from the two assays cannot be interchanged. Our data also suggest that the use of different reagents may account for inconsistencies in the literature regarding the association between PD-L1 expression and clinicopathological features. Tumor cells (TCs) can evade immune responses through the upregulation of PD-L1 and blockade of the PD-1/PD-L1 interaction via monoclonal antibodies and can produce a durable clinical response in patients with NSCLC. The anti-PD-1 antibody pembrolizumab and the anti-PD-L1 antibody atezolizumab are now approved by the US Food and Drug Administration (FDA) for treating patients with advanced NSCLC4. For example, the results from the phase II/III KEYNOTE-010 study showed that compared with docetaxel, pembrolizumab prolonged overall survival (OS) in previously treated advanced NSCLC patients with positive PD-L1 expression determined by immunohistochemistry (IHC) on at least 1% of TCs5. Data from patients with advanced NSCLC in a phase III study (KEYNOTE-024) demonstrated an objective response rate (ORR) with pembrolizumab in 44.8% of patients with at least 50% PD-L1-positive TCs compared with the ORR of 27.8% with chemotherapy. The median progression-free survival (PFS) was 10.3 months in the pembrolizumab group versus 6.0 months in the chemotherapy group6. Based on these data, pembrolizumab was approved for use in conjunction with a companion diagnostic, the Dako 22C3 PD-L1-IHC platform. In the phase II POPLAR study, previously treated NSCLC patients (in a second- or third-line setting) were stratified by PD-L1 expression on tumor-infiltrating immune cells (ICs) and TCs assessed by IHC with the SP142 antibody and were randomized to receive atezolizumab or docetaxel treatment. Improved efficacy with atezolizumab was observed with increasing PD-L1 expression. Additionally, in the subgroup of patients with the highest PD-L1 expression , the OS was 15.5 months and 11.1 months (hazard ratio (HR) 0.46, p = 0.070), the PFS was 7.8 months and 3.9 months (HR 0.57), and the ORR was 38% and 13% for atezolizumab and docetaxel, respectively. In contrast, patients with the lowest PD-L1 levels  did not appear to benefit from atezolizumab7.There is data indicating that the PD-L1 expression status might help guide therapy8, and various commercially available anti-PD-L1 antibodies have been used to determine PD-L1 expression in TCs and/or ICs9. This raises the following questions: Can different assays yield identical results for maximizing the therapeutic benefit and avoiding unnecessary toxicities? Is the use of different reagents one of the reasons for inconsistent results reporting the association between PD-L1 expression and clinicopathological features?Currently, clinical trials have utilized different PD-L1 assays and testing platformsTo better understand the differences in the results of two IHC assays for PD-L1, we assessed the conformance of PD-L1 expression between the 22C3-IHC and SP142-IHC assay results in surgically resected tumors from NSCLC patients. Moreover, we investigated the association between clinicopathologic characteristics and PD-L1 expression measured by the two assays.All samples were obtained with signed consent for the use of tissues under an approved protocol from the Ethical Committee of Fujian Cancer Hospital. All patients provided written informed consent for the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All experiments were performed in accordance with relevant guidelines and regulations.th TNM staging system10, and lung tumor histology was recategorized according to the 2015 World Health Organization (WHO) classification system for lung tumors11.We retrospectively collected 135 samples from patients with pathologically confirmed adenocarcinoma or squamous cell carcinoma at the Fujian Cancer Hospital in China. All surgeries were conducted between January 2008 and December 2010. These patients were not treated with PD-L1 axis therapies or targeted therapy. The pathological TNM stage was reassigned according to the 8Each tumor sample was pathologically examined using six 5-µm serial sections that were sent to two institutions for analysis as follows: three sections were sent to the University of Hong Kong for analysis with a 22C3-IHC on an Autostainer Link 48 with the murine 22C3 anti-human PD-L1 antibody  according to the manufacturer’s protocol – one section was stained with hematoxylin and eosin (HE); another section was stained for PD-L1; and a third section was used as the negative control. The other three sections were sent to Fujian Cancer Hospital for IHC analysis using the SP142 antibody on the Ventana Benchmark platform. Two board-certified pathologists (Chao Li and W Zhu) jointly evaluated all stained slides for PD-L1 membrane staining. All areas in each tissue section were observed for the appropriate evaluation of the expression of PD-L1 on TCs as an estimate of the percentage of TCs exhibiting partial or complete membranous staining and on ICs as an estimate of the percentage of the tumor area occupied by PD-L1-expressing ICs. Determination of PD-L1 expression was performed without considering any cut-off value, and the staining intensity was not part of this evaluation.Scoring was performed based on the standards for the 22C3-IHC and SP142-IHC assays. For the 22C3-IHC assay, PD-L1 expression was determined by using a Tumor Proportion Score (TPS), which is the percentage of viable TCs showing partial or complete membrane staining. PD-L1 expression was scored at three levels, namely, TPS < 1%, TPS 1 to 49% and TPS ≥ 50%. For the SP142-IHC assay, baseline PD-L1 expression was scored using the number of PD-L1 TCs as a percentage of total TCs, as follows: TC3 ≥ 50%, TC2 ≥ 5% and < 50%, TC1 ≥ 1% and < 5%, and TC0 < 1%. Additionally, tumor-infiltrating ICs were expressed as the percentage of the tumor area as follows: IC3 ≥ 10%, IC2 ≥ 5% and < 10%, IC1 ≥ 1% and < 5%, and IC0 < 1%. To compare the overall agreement of the two assays, we interchanged various cut-off values on each set of slides stained by the two assays.2 test. The OS of patients with PD-L1-positive and PD-L1-negative tumors was estimated using the Kaplan–Meier method, and differences were compared using the log-rank test. All tests were two-sided. Statistical significance was set at p < 0.05. Statistical analyses were performed using SPSS 21.0 software.The weighted kappa (κ) coefficient and McNemar-Bowker test were used to evaluate the agreement and inconsistency between the results using each assay. A κ coefficient of 0.75 or less indicated poor to fair agreement, and a value greater than 0.75 indicated almost perfect agreement. Additionally, clinicopathological characteristics of PD-L1-positive tumors according to the interpretation of the 22C3-IHC or the SP142-IHC assay standards were compared with those of PD-L1-negative tumors using the χA total of 135 patients were eligible for the study, and their characteristics are summarized in Table ). One sample demonstrated 1% PD-L1-positive TCs based on the SP142-IHC assay, but it was negative based on the 22C3-IHC assay. The variability in the staining pattern of ICs between samples was greater than that for TCs . In a total of 62 discordant samples, compared to the 22C3-IHC assay, the SP142-IHC assay resulted in the underestimation and overestimation of PD-L1 expression in ICs in 53 (39.2%) and 9 (6.7%) samples, respectively (p < 0.001). With the adoption of the 22C3-IHC assay scoring algorithm for TCs, 20 (14.8%), 25 (18.5%), and 90 (66.7%) of the 135 samples were classified into groups that were strongly positive, weakly positive, and negative for PD-L1, respectively. Moreover, 105 (77.78%) of 135 samples produced the same results with the SP142-IHC reagent assay . The PD-L1 levels on TCs as detected by the 22C3-IHC assay were underestimated by the SP142-IHC assay in 29 samples. Only one sample with negative PD-L1 expression based on the 22C3-IHC assay was misclassified as having weak positive PD-L1 expression based on the SP142-IHC assay  . In 60 (91.0%) of 66 discordant samples, PD-L1 status determined with the SP142-IHC assay was overestimated in the 22C3-IHC assay. In contrast, the PD-L1 status was underestimated by 22C3-IHC assay in six cases  , 14 (10.4%), 37 (27.4%) and 74 (54.8%) of 135 samples evaluated using the SP142-IHC the assay were classified into groups that were strongly positive, moderately positive, weakly positive, and negative for PD-L1 expression, respectively. However, only 69 (51.11%) of 135 samples produced the same results with the 22C3-IHC assay .We determined the association of clinicopathological characteristics with PD-L1 expression based on each assay. PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status. However, the results from the SP142-IHC assay were different, indicating that PD-L1 expression was significantly correlated not only with smoking status but also with sex and histology  indicated that the assay using the SP142 antibody differed from the others, as it detected significantly lower PD-L1 expression in TCs and ICs. The limitation of the study is that the authors used the SP142 antibody on a Ventana platform not identical to the platform now approved by the FDA14. In addition, the analysis was based on the average value as the gold standard to determine the sensitivity and specificity of various reagents. We used the standard for the 22C3-IHC as the gold standard with a 50% and 1% cut-off values and evaluated the sensitivity and specificity of PD-L1 labeling. We learned that the sensitivity with the SP142-IHC assay is poor but that the specificity is better. Thus, if the SP142-IHC assay results are negative, the 22C3-IHC assay results will also be negative. Because the details of antibodies are proprietary information, we can only speculate that the poor concordance of the results with the two antibodies may be due to different antibody affinities, limited specificity, or distinct target epitopes.The Blueprint study was designed to evaluate the agreement of results from four PD-L1 IHC assays, namely, Dako 28-8, Dako 22C3, 29 Ventana SP263, and Ventana SP142. Thirty-eight non-interventional NSCLC cases were enrolled into the study, and each tumor was probed for PD-L1 using all four assays then scored by three pathologists. For TC staining, Dako 22C3, Dako 28-8 and Ventana SP263 assays demonstrated similar analytical performance, whereas Ventana SP142-IHC assay consistently labeled fewer TCs. Similar to our results, the SP142-IHC assay was associated with significantly lower levels of PD-L1 detection than was the 22C3-IHC assayIt is important to note that we employed standardized platforms and reagents that are now approved by the FDA for this study and that this is currently the largest clinical study assessing the concordance between SP142-IHC and 22C3-IHC results in NSCLC.16. The reason for these discrepancies may be multifactorial17. Differences in distributions of histologic subtypes, antibodies and cut-off values may account for some of these discrepancies18. In our study, PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status. However, these results were different those obtained using the SP142-IHC assay, which indicated correlations of PD-L1 expression with sex, smoking status, and histology. Additionally, PD-L1 expression based on either IHC systems was not associated with patient prognosis. Our data represent the most direct evidence that demonstrates differences between two reagents and may partly explain the conflicting results regarding the correlation of PD-L1 expression and clinicopathological characteristics in other studies.The prognostic impact of PD-L1 protein expression and its association with clinicopathological features have not been conclusively elucidatedOur study has several limitations. First, all samples were obtained from patients who had not undergone PD1 or PD-L1 therapy. We did have access to therapeutic outcome data, and we could only evaluate the assay results by themselves without clinically meaningful comparisons of PD-L1 assays. Second, the vast majority of specimens were archived samples and not biopsies; these samples may not reflect the real situation in the clinic.In conclusion, we identified that the use of different reagents for testing PD-L1 expression can be misleading; the two reagents cannot be interchanged with each other. Our findings also suggest that different reagents may account for inconsistent associations between the expression of PD-L1 and clinicopathological features.


Visuospatial processing requires wide distribution or narrow focusing of attention to certain regions in space. This mechanism is described by the zoom lens model and predicts an inverse correlation between the efficiency of processing and the size of the attentional scope. Little is known, however, about the exact timing of the effects of attentional scaling on visual searching and whether or not additional processing phases are involved in this process.Electroencephalographic recordings were made while participants performed a visual search task under different attentional scaling conditions. Two concentric circles of different sizes, presented to the participants at the center of a screen modulated the attentional scopes, and search arrays were distributed in the space areas indicated by these concentric circles. To ensure consistent eccentricity of the search arrays across different conditions, we limited our studies to the neural responses evoked by the search arrays distributed in the overlapping region of different attentional scopes.Consistent with the prediction of the zoom lens model, our behavioral data showed that reaction times for target discrimination of search arrays decreased and the associated error rates also significantly decreased, with narrowing the attentional scope. Results of the event-related potential analysis showed that the target-elicited amplitude of lateral occipital N1, rather than posterior P1, which reflects the earliest visuospatial attentional processing, was sensitive to changes in the scaling of visuospatial attention, indicating that the modulation of the effect of changes in the spatial scale of attention on visual processing occurred after the delay period of P1. The N1 generator exhibited higher activity as the attentional scope narrowed, reflecting more intensive processing resources within the attentional focus. In contrast to N1, the amplitude of N2pc increased with the expansion of the attentional focus, suggesting that observers might further redistribute attentional resources according to the increased task difficulty.These findings provide electrophysiological evidence that the neural activity of the N1 generator is the earliest marker of the zoom lens effect of visual spatial attention. Furthermore, evidence from N2pc shows that there is also a redistribution of attentional resources after the action of the zoom lens mechanism, which allows for better perform of the search task in the context of low attentional resolution. On the basis of the timing of P1, N1, and N2pc, our findings provide compelling evidence that visuospatial attention processing in the zoom lens paradigm involves multi-stage dynamic processing. One of the most notable characteristics of the human attentional system is the limited amount of resources available to it. When performing visual search tasks, the amount of visual information pertinent to a given search situation often exceeds the maximal amount of information that can be processed by the attentional system at any given time. In this case, top-down attention control plays a key role in rationalizing the use of such limited resources for attention processing. Through this mechanism, an observer can choose to process a small number of spatial stimuli while ignoring information associated with other locations. This mechanism was described by the spotlight model, which assumes that attention can be shone over the attended area as a spotlight . InformaIn realistic situations, however, one may not be able to determine the location of a target, even when the target’s features are known in advance. At this point, a better strategy would be to adjust the scope of attention based on the current situation, aiming to include the possible location of the target. This attentional control mechanism is well captured by the zoom lens model . The zooBy recording and analyzing event-related potentials (ERPs) in high temporal resolution, In the current study, we further evaluated the multiple processing stages associated with the zoom lens mechanism. Observers were asked to search for a target in searching scopes that had different sizes. Different searching scopes appeared alternately. In this setting, some search arrays would be spread over the overlapping region of two searching scopes. We were only interested in these search arrays, since they belong to different attentional scopes but have perfectly matched physical properties (same eccentricity and number of distractors). Under this scenario, target-elicited neural activity could only be modulated by the different sizes of the attentional scopes, rather than by the physical properties of the search arrays.In addition, for the first time, we also examined the redistribution of the attentional resources driven by changes in the attentional scopes. The zoom lens model suggests that widening the attentional scope can reduce the density of processing resources in this region, and therefore can increase the difficulty of search tasks . HoweverData collection conformed to the Declaration of Helsinki and the ethics committee of Zhongshan hospital affiliated to Dalian university approved the research protocol . Verbal informed consent was obtained from all the study participants as agreed by the review board.Nineteen paid participants from the Liaoning Normal University community participated in this experiment. One participant was excluded from the analyses owing to poor performance (32% errors). Another two were excluded because of excessive alpha activity. The remaining sixteen participants  reported normal or corrected-to-normal vision, and had no known neurological or visual disorders.Stimuli were presented on an LCD monitor  at a viewing distance of 70 cm. In the experiment, a gray central fixation cross (0.26°) always appeared at the center of the monitor . The search array was a circular array of eight squares , arranged equidistantly. There were two different color squares, green  and blue . In each trial, the color of one square differed from that of the other squares, serving as a target, and the remaining seven squares served as distractors. The color of the target square was consistent within each block and was balanced within the observer. The target square was shown randomly at one of three possible locations on each side of the visual field . Each sqStimuli and procedure were controlled via E-Prime 2.0 routines . At the beginning of each trial, a central fixation cross was presented for 500 ms, followed by the concentric circles, which were displayed for 500–900 ms. Then, a search array was presented for 200 ms. Observers were instructed to respond as quickly and accurately as possible to press the number keys to the right of the keyboard using their right thumb and index finger. When the line within the target square was horizontal, the observers were asked to press the number key “1,” while when a vertical line appeared, the observers were asked to press the number key “2.” The concentric circles disappeared after the observers reacted, or disappeared automatically after 2,000 ms, and were replaced by a solid background, preparing the observers for the next trial.For each size of the attentional scope, search arrays were randomly distributed over one of the four virtual rings within a limited attentional scope (a detailed description was provided in the previous section). The overlapping region of the two attentional scopes was a virtual ring with a radius of 3.27°. In the current study, we were primarily interested in the search arrays distributed over this overlapping region. These search arrays had exactly the same physical properties (the eccentricity and the number of distractors), while being within the sizes of both attentional scopes. Search arrays that appeared outside of the overlapping region were not included in our analysis owing to their unmatched eccentricities.The entire experiment contained a total of 24 experimental blocks, with a total of 1,440 test trials. Each block contained 60 test trials, followed by a minimal break of 30 s. Within each block, 24 search arrays were distributed in the overlapping region, and 36 search arrays were distributed in other non-overlapping region. Therefore, for each size of the attentional scope, the number of search arrays distributed in the overlapping region was 288. Each size of the attentional scope lasted six continuous blocks and the order was well-balanced. To make the observers familiar with the task requirements, one training block with 12 trials was offered at the beginning of the experiment. The observers were only allowed to enter the formal experiment when their correct rate exceeded 75%. On the search task, the observers were asked to maintain a central fixation, while minimizing their head and eye movements.The ANT Neuro EEGO system was used to record electroencephalographic (EEG) signals using an array of 64 electrodes mounted using a cap with 10/20 montage. Horizontal electrooculograms, which recorded bipolar signals from the outer canthi of the eyes, were used to measure horizontal eye movements. Vertical electrooculogram, which recorded from the FPz site, was used to detect eye blinking. The CPz site was used as the online reference. Electrode impedances were kept below 5 kV with a sampling rate at 500 Hz for on-line recording.www.neuro.uestc.edu.cn/rest , while the statistical power was executed by G-Power 3.1 . The accuracy of the data also suggested that observers behaved worse for the larger attentional scope (90% ± 2%) compared with the smaller scope scenario (93% ± 1%), (t (15/2) = −2.77, p = 0.014, Cohen’s d = 0.70). These results were in line with the predictions of the zoom lens model, indicating that as the scope of attention narrowed, the observers were able to identify the feature of the target faster and more accurately.Trials with mean reaction time (RT) below or above two standard deviations were excluded . Analysis of RT data revealed faster responses to targets for the smaller attentional scope (424.06 ± 59.15 ms) compared with the larger one (444.82 ± 77.19 ms), (t (15/2) = 2.20, P1. p = 0.681) and the electrode sites  = 1.70, p = 0.21) were not significant. Besides, the interaction between the electrode sites and the attentional scope was also not significant  = 0.43, p = 0.54).N1. As shown in p = 0.01, ηp2 = 0.36). However, the main effect of the electrode sites  = 0.69, p = 0.43) and the interaction between the electrode sites and the attentional scope  = 3.20, p = 0.09) were not significant. Further examination of the main effects of the attentional scope showed that the targets for the smaller attentional scope (−8.43 ± 2.26 μV) elicited a more negative N1 compared with the larger attentional scope (−7.95 ± 2.48 μV), (t (15) = −2.921, p = 0.01, Cohen’s d = 0.73), indicating that as the size of the attentional scope decreased, the neural generators of P1 became more active.N2pc. Grand-average ERPs at the electrode sites PO7/PO8 as well as N2pc difference waves are shown in p = 0.005, Cohen’s d = 0.82).The present study investigated the different stages of attentional processing in the zoom lens paradigm. More specifically, we attempted to examine the effects of changes in the attentional scope on early visual evoked potentials (P1/N1) and N2pc, reflecting the redistribution of attentional resources. In agreement with previous studies , during The P1 component originating in the extrastriate cortex represents the earliest stage in the visual processing modulated by spatial attention. In the current study, we found that the amplitude of P1 did not change with the scaling of spatial attention, suggesting that the scale effect of visual spatial attention does not modulate the exogenous-like P1 component. Unlike the current results, previous studies have found that the amplitude of P1 was modulated by different sizes of attentional scopes. Different from P1, we found that the lateral occipital component N1 with a temporal lag could be sensitive to changes in the size of the attentional scope. Specifically, the amplitude of N1 increased with decreasing the size of the attentional scope. This is consistent with the findings of According to the zoom lens model, it is unlikely that the scale effect of visual spatial attention is driven by bottom-up factors; rather, it should be the result of a top-down modulation. Some recent studies have explored the involvement of higher brain regions in flexible modulation of the scale effect of spatial attention. In addition, we also explored the modulation of the late N2pc components by the scale effect of the attentional scope. We found that, contrary to the earlier occurring N1, the amplitude of N2pc increased with increasing the scale of spatial attention. N2pc is generally used as a neurological indicator of attentional spatial selection , and itsIt has been demonstrated that the amplitude of N2pc is also sensitive to the redistribution of attentional resources that is modulated by the task difficulty . In the It was shown previously that top-down modulation associated with the right frontal parietal network forms the neural basis of the zoom lens mechanism. In light of the above discussion, this modulation was likely to occur before the redistribution of attentional resources reflected by N2pc, approximately within the N1 delay period after the search arrays were presented. Based on this evidence, we speculated that the scaling effect of spatial attention during visual search may operate in at least three different stages. The first stage is the earliest visual spatial attention processing. At this stage, search arrays for different sizes of the attentional scope are firstly perceived with no difference. In the subsequent stages, the spatial gradient of the attention system is modulated by the zoom lens mechanism, as indicated by changes in the activity of the N1 generator. Before the final behavioral response, there is a redistribution of attentional resources. This process is reflected in the modulation of N2pc and is probably driven by reduced attentional resolution as the size of the attentional scope increases.In summary, utilizing high temporal-resolution ERP technology, we provided solid evidence to support the view that the modulation of the zoom lens effect on visual processing during search tasks involves multiple dynamic processing stages. Our results suggest that lateral occipital N1 might be the neurophysiological marker of the attentional scaling effect during visual search. In addition, there was also a redistribution of attentional resources after the action of the zoom lens mechanism, indicating that top-down control mechanisms are involved in the post-attentional scaling process.10.7717/peerj.4538/supp-1Supplemental Information 1Click here for additional data file.


Graphene nanoribbons (GNRs) are ultra-narrow strips of graphene that have the potential to be used in high-performance graphene-based semiconductor electronics. However, controlled growth of GNRs on dielectric substrates remains a challenge. Here, we report the successful growth of GNRs directly on hexagonal boron nitride substrates with smooth edges and controllable widths using chemical vapour deposition. The approach is based on a type of template growth that allows for the in-plane epitaxy of mono-layered GNRs in nano-trenches on hexagonal boron nitride with edges following a zigzag direction. The embedded GNR channels show excellent electronic properties, even at room temperature. Such in-plane hetero-integration of GNRs, which is compatible with integrated circuit processing, creates a gapped channel with a width of a few benzene rings, enabling the development of digital integrated circuitry based on GNRs. et al. have used a hexagonal boron nitride template to grow narrow, integrated graphene nanoribbons with small bandgaps.Graphene nanoribbons are promising candidates for 2D material electrical interconnects; however, the top-down fabrication of nanoribbons has remained a challenge. Here, Chen  Ideal graphene nanoribbons (GNRs) have been shown to exhibit extreme chirality dependence as metals or semiconductors24591113144 at room temperature and carrier mobility values of ∼750 cm2 V−1 s−1. For the narrowest GNRs, the band gaps extracted from the electrical transport data are >0.4 eV.Here, we demonstrate the successful growth of GNRs directly on h-BN substrates with smooth edges and controllable widths via templated growth using chemical vapour deposition (CVD). Transistors made with sub-10-nm GNRs demonstrate large on–off ratios of more than 10The nano-trenches exhibited predominant separation angles of 60° relative to each other with no obvious correlation to the direction of the gas flow, indicating anisotropic etching along the different crystallographic directions . SeparatThe existence of the giant Moiré pattern indicates that the graphene was highly crystalline and precisely aligned with the h-BN underneath. It was noticed that the Moiré pattern appeared to be stretched along the GNR, while it appeared relaxed laterally. This trend differs from regular hexagons with a periodicity of ∼14 nm, which have always been observed with well-aligned graphene domains on h-BN1617−1, which was attributed to the Raman-active LO phonon of h-BN (ref. −1) as well as a single-Lorentzian-shaped 2D-band , which were expected for monolayer graphene. Compared to that of the pristine graphene domain−1 in the G-band position indicates the existence of a tensile strain of ∼0.6% in the GNR20−1. At 1,617.5 cm−1 (D′-band), a tiny shoulder appears on the right side of the G band. Both the Raman D- and D′-bands may have primarily originated from the lattice distortions and disorder at the GNR/h-BN boundary. The 2D-band exhibits a characteristic of I(2D)/I(G)>1, also indicating the single-layer nature of the GNR, where I(2D) and I(G) represent the intensity values of the 2D and G-bands, respectively. In addition, the observed broadening of the full width at half-maximum for the Raman G- and 2D-bands may have been due to strain variations at the nanometre-scaleRaman measurements were carried out to investigate the structural and electronic properties of the nanoribbons. -BN ref. . For the-BN ref. . The Ram2 ; then, it gradually switched on, exhibiting a relatively high G=0.7 × e2 × h−1 at room temperature for Vgate=−40 V. The scattering mean free path was estimated to be ∼50 nm. The mobility of the GNR was estimated to be ∼765 cm2 V−1 s−1 at 300 K. Accurately measuring the off-state resistance of the narrowest GNR FET became challenging due to noise and set-up limitations. It was found that the conductance near the on-state exhibited an exponential relationship with inverse temperature from 200 to 300 K. This result indicates that a Schottky barrier dominated the conductance of the GNR FET because of the high work functions of Pd and Ni (refs Eg) of the narrow GNRs by fitting the conductance near the on-state kB is Boltzmann's constant and T is temperature. The extracted band gap value for the 5-nm GNR was 489.4±19.0 meV ∼α/(w+β) (w is in units of nm), where parameter α≈1.99 eV nm and parameter β=−1.28 nm.To investigate the electrical properties of the GNRs, field effect transistor (FET) devices were produced using p-type doped Si/SiOodel see , the valodel see . The fit Ni refs , 23. We e–e interactions. It is noted that our narrow GNRs with similar widths exhibited relatively larger band gaps than those reported in previous literature9In the GNR samples, the band gap exhibited a strong dependence on the width of the ribbons. In particular, the sub-10-nm GNRs exhibited an electronic band gap of ∼0.5 eV. It is important to understand the possible origins of the band gap. Normally, the electronic structure of GNRs is strongly dependent on the edge of the GNRs. For this study, it was challenging to characterize the exact geometry of the narrow ribbons obtained . However, the GNRs were generally parallel to the ZZ pattern of the h-BN and free of detectable edge roughness. Therefore, it is likely that most segments of the GNR edges followed a ZZ pattern. Previous studies in the literature predicted that GNRs with pristine ZZ edges would always be metallic because of their peculiar flat-band edge states localized near the Fermi level24264. Our results demonstrate that it is possible to resolve the fundamental gapless limitation of graphene, paving the way for the realization of graphene-based digital electronics that can operate at room temperature.The applicability of graphene for future digital devices is often questioned due to its intrinsic gapless nature. Nanoribbons offer a potential solution, but both the width and edges must be precisely controlled. In-plane graphene–BN hetero-structured films have been reported, however, only on metal surfaces313233342 flow (850:150 sccm) to remove the tape residue. After the heat cleaning process, a solution of NiCl2:H2O at a concentration of 0.01 mg ml−1 was spun at 1,800 r.p.m. for 100 s onto the substrate surface and then baked for 10 min at 80 °C on a hot plate to evaporate the solvent. This NiCl2-treated sample was then submitted to a two-step process under an Ar:H2 flow (850:150 sccm): annealing at 500 °C for 20 min, which resulted in Ni nano-particle formation, and etching at 1,200 °C for 60–180 min. The optimal etching pressure was found to be ∼150 Pa.To prepare h-BN samples for nano-particle-assisted etching, multiple cleaning steps were adapted to ensure the reliable production of the cuts. Before depositing the h-BN flake, quartz substrates were cleaned with acetone and isopropyl alcohol and then annealed at 600 °C for 30 min to remove organic contaminants. Subsequently, the h-BN was mechanically exfoliated using semiconductor-grade tape. Next, the h-BN samples were heated in a quartz tube at 500 °C for 15 min under an Ar:H2H2 flow and a mixture of silane and Ar (5% mole ratio of silane to Ar) were introduced into the system for GNR growth. The ratio of C2H2 to silane was ∼1:1. The pressure was maintained at 5 Pa during the growth process, and the growth time was ∼5 min. After growth, both the C2H2 and silane/Ar flow were turned off, and the system was cooled to room temperature with flowing Ar. The samples of GNRs on h-BN grown on a quartz surface were moved to a highly p-type doped silicon wafer with a 300-nm-thick SiO2 capping layer for electrical transport studies.Before graphene growth, etched h-BN flakes were cleaned separately in HCl solution, DI water and acetone. The substrate with the h-BN flakes was then loaded into a growth chamber. GNR growth was carried out in a low-pressure CVD furnace at 1,280 °C under an Ar flow of 10 sccm, corresponding to 15 Pa; the samples were then annealed for 5 min. Next, the Ar flow was turned off, and a Ck of the cantilever tips was in the range of 0.05–0.5 N m−1. The scan rate was set to 10–60 Hz to minimize any noise from thermal drift. The integral gain and set point were adjusted to be as low as possible during the measurement. Several hours of pre-scanning were carried out to warm up the scanner to obtain good stability during imaging. To ensure a highly accurate atomic-resolution image, scanners with a travel range of <10 μm along the x and y directions were used. Calibration at atomic resolution was performed with newly cleaved highly ordered pyrolytic graphite before measurement.Cleaned samples were characterized using one AFM , while atomic-resolution images were taken by another AFM  under ambient conditions. AFM measurements were acquired in contact mode to obtain height and friction images. SNL-10 AFM tips from Bruker, which possess a nominal tip radius of <10 nm, were used in all measurements. The use of friction contrast was necessary because this mode gives clear information about the super-structure and atomic lattice. For atomic-resolution scanning, the force constant Raman spectra were obtained with a commercially available confocal Raman instrument: model Alpha 300R from WITec. The Raman data were recorded using a laser wavelength of 488 nm 2. Next, the devices were annealed in a hydrogen atmosphere at 200 °C for 3 h to remove the resist residues and to reduce the contact resistance between the GNRs and metal electrodes before electrical measurements because the thickness of the h-BN flakes on the silicon wafer was ∼15 nm. Electrical transport measurements were carried out using a physical property measurement system  via a Keithley 4200 semiconductor characterization system.GNR devices were created by a standard electron beam lithographic technique with Ni or Pd as the source and drain contacts on p-doped silicon wafers with 300-nm-thick SiOThe authors declare that the main data supporting the findings of this study are available within the article and its How to cite this article: Chen, L. et al. Oriented graphene nanoribbons embedded in hexagonal boron nitride trenches. Nat. Commun.8, 14703 doi: 10.1038/ncomms14703 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary References.